HBV (Hepatitis B Virus) specific T cell antigen receptor and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to an HBV C antigen specific T cell antigen receptor and application thereof, the HBV C antigen specific T cell antigen receptor comprises at least one of the following: a T cell antigen receptor TCR1, the T cell antigen receptor TCR1 comprises CDR1, CDR2 and CDR3 of an alpha chain and CDR1, CDR2 and CDR3 of a beta chain; the T cell antigen receptor TCR1 comprises CDR1, CDR2 and CDR3 of an alpha chain and CDR1, CDR2 and CDR3 of a beta chain, and the T cell antigen receptor TCR2 comprises CDR1, CDR2 and CDR3 of the alpha chain and CDR1, CDR2 and CDR3 of the beta chain; peripheral blood of a healthy volunteer after HBV acute infection and virus removal recovery is collected, HBVc1 specific CD8 cells in the peripheral blood are sorted, a TCR sequence of a full-human targeted HBVc1 antigen is obtained through primary screening with a single cell technology, corresponding TCR-T cells are constructed, and it is verified that the TCR-T cells have the capacity of specifically killing liver cancer cells at the in-vitro cytology level.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a HBV-specific T cell antigen receptor and a use thereof. Background Art

[0002] Immune cell therapy technology is the fastest growing area in the biomedical field recently. Most CAR-T and TCR-T use tumor-associated antigens as targets, but this often leads to the possibility of "off-target". For example, in the clinical treatment study of colorectal cancer TCR-T targeting carcinoma embryonic antigen (CEA), severe inflammatory bowel disease occurred and the study was terminated. Melanoma TCR-T treatment targeting MART-1, glycoprotein 100 (gp100) and melanomaassociated antigen-A3 (MAGE-A3) caused severe damage to normal tissues and severe neurotoxic reactions leading to the death of subjects, as well as the occurrence of cardiogenic shock and death. Screening safe and efficient tumor-specific antigens for TCR-T treatment can reduce the risk of "off-target". Infection with oncogenic viruses such as Epstein-Barr virus (EBV), high-risk human papilloma virus (HPV) subtypes, hepatitis B virus (HBV), hepatitis C virus (HCV) and human T-lymphotropic virus type-1 (HTLV-1) are the main pathogenic factors for some cancer patients. Oncogenic viral antigens are the main factors leading to tumorigenesis. These antigens are expressed in tumor tissues, and because the protein sequences encoded by the viral genome have very low similarity to human protein sequences, they are less affected by the central tolerance of lymphocytes. Therefore, oncogenic viral antigens have strong immune antigenicity and are ideal targets for TCR-T therapy.

[0003] Among the many environmental risk factors related to liver cancer, HBV and HCV infection are directly related to the occurrence of liver cancer. Up to 80% of liver cancer is attributed to HBV or HCV infection. HBV-specific CD8+T cells play a decisive role in controlling viral replication, as well as clearing the virus and clinical recovery of HBV infection. Adoptive transfer of HBV-specific TCR-modified T cells (TCR-T) for the treatment of chronic hepatitis B in mice has been preliminarily shown to have very good antiviral activity. In patients who have recovered from acute HBV infection, there are CD8 cells that can effectively clear HBV virus. Among them, HBVc1-specific CD8 cells are an important group of virus-specific cells in hepatitis B patients, and are positively correlated with the clearance of HBV virus. In particular, this group of cells is mainly present in patients in the recovery period of acute infection. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a T cell antigen receptor and its use.

[0005] To this end, the present invention provides the following technical solutions:

[0006] A T cell antigen receptor, characterized by comprising at least one of the following:

[0007] T cell antigen receptor TCR, CDR1, CDR2 and CDR3 of the α chain variable region and CDR1, CDR2 and CDR3 of the β chain variable region of the T cell antigen receptor TCR;

[0008] The CDR3 of the α chain variable region has an amino acid sequence as shown in SEQ ID NO.1, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0009] The CDR3 of the β chain variable region has an amino acid sequence as shown in SEQ ID NO.2, or has at least 90%, 95% or 99% similarity with the amino acid sequence, or the CDR3 of the β chain has an amino acid sequence as shown in SEQ ID NO.3, or has at least 90%, 95% or 99% similarity with the amino acid sequence.

[0010] Optionally, at least one of the following is also included:

[0011] The CDR1 of the α chain variable region has an amino acid sequence as shown in SEQ ID NO.4, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0012] The CDR2 of the α chain variable region has the amino acid sequence shown in SEQ ID NO.5, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0013] The CDR1 of the beta chain variable region has an amino acid sequence as shown in SEQ ID NO.6, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0014] The CDR2 of the beta chain variable region has an amino acid sequence as shown in SEQ ID NO.7, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0015] The FR1 of the α chain variable region has the amino acid sequence shown in SEQ ID NO.8, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0016] The FR2 of the α chain variable region has the amino acid sequence shown in SEQ ID NO.9, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0017] The FR3 of the α chain variable region has the amino acid sequence shown in SEQ ID NO.10, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0018] The FR1 of the beta chain variable region has the amino acid sequence shown in SEQ ID NO.11, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0019] The FR2 of the beta chain variable region has an amino acid sequence as shown in SEQ ID NO. 12, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0020] The FR3 of the beta chain variable region has an amino acid sequence as shown in SEQ ID NO. 13, or has at least 90%, 95% or 99% similarity to the amino acid sequence;

[0021] Optionally, the variable region of the α chain of the T cell antigen receptor TCR has an amino acid sequence shown in SEQ ID NO.14, and the variable region of the β chain has an amino acid sequence shown in any one of SEQ ID NOs.15 to 16;

[0022] Optionally, the constant region of the α chain of the T cell antigen receptor TCR has the amino acid sequence shown in SEQ ID NO.17, and the constant region of the β chain has the amino acid sequence shown in SEQ ID NO.18;.

[0023] A nucleic acid molecule encoding the T cell antigen receptor; optionally, the nucleic acid molecule is as shown in any one of SEQ ID NO.19-20; optionally, the nucleic acid molecule is mRNA, and the mRNA can be translated into the T cell antigen receptor according to claim 1.

[0024] A recombinant plasmid comprises the nucleic acid molecule.

[0025] A host cell, comprising the nucleic acid molecule or the recombinant plasmid.

[0026] Optionally, the host cells are co-transfected with the recombinant plasmid and the lentiviral packaging plasmid.

[0027] A recombinant virus, the genome of which carries the nucleic acid molecule.

[0028] A T cell receptor engineered T cell targeting HBV, wherein the T cell receptor engineered T cell comprises the nucleic acid molecule or the T cell antigen receptor.

[0029] Use of the T cell antigen receptor, the nucleic acid molecule, the recombinant plasmid, the host cell, the recombinant virus, and the HBV-targeting T cell receptor engineered T cell in the preparation of drugs for hepatitis B virus-related diseases.

[0030] Optionally, the hepatitis B virus-related diseases include liver cancer and acute and chronic viral infections.

[0031] The technical solution of the present invention has the following advantages:

[0032] 1. A T cell antigen receptor provided by the present invention comprises at least one of the following: a T cell antigen receptor TCR, wherein the CDR1, CDR2 and CDR3 of the α chain variable region and the CDR1, CDR2 and CDR3 of the β chain variable region of the T cell antigen receptor TCR; the CDR3 of the α chain variable region has the amino acid sequence shown in SEQ ID NO.1, or has at least 90%, 95% or 99% similarity with the amino acid sequence; the CDR3 of the β chain variable region has the amino acid sequence shown in SEQ ID NO.2, or has at least 90%, 95% or 99% similarity with the amino acid sequence, or the CDR3 of the β chain has the amino acid sequence shown in SEQ ID NO.3, or has at least 90%, 95% or 99% similarity with the amino acid sequence; the present invention has found that the HLA-A11 restricted high affinity TCR from healthy volunteers has better clinical safety, and the specimens used are from patients who have recovered from acute HBV infection. After HBV virus infection, patients rely on their own CD8 The CTL immune response clears the virus, and the presence of CD8 cells in the body that can effectively clear the HBV virus is helpful for the screening of related high-affinity TCR clones, effectively avoiding the shortcomings of clinical HBV chronic infection samples that cannot obtain high-affinity TCR clones and the potential risk that animal-derived TCRs may cause human immune rejection. High-affinity TCRs derived from volunteers who have recovered from acute infection theoretically have better clinical safety and clinical effectiveness. Therefore, peripheral blood was collected from healthy volunteers who had cleared the virus after acute HBV infection, and HBVc1-specific CD8 cells in the peripheral blood were sorted. The 10× single-cell technology was used to preliminarily screen and obtain the fully human TCR sequence targeting the HBVc1 antigen, and the corresponding TCR-T cells were constructed. The ability to specifically kill liver cancer cells was verified at the in vitro cytological level. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 This is the screening process of fully humanized HBV antigen targeting TCR clones in Example 1 of the present invention;

[0035] Figure 2 It is the verification of HBVc1 TCR specificity and affinity in Example 2 of the present invention;

[0036] Figure 3This is the TCR-T cell construction and vector optimization design in Example 2 of the present invention; Figure A is the flow cytometry result; Figure B is the statistics of Figure A;

[0037] Figure 4 This is the result that the TCR-T cells in Example 3 of the present invention can effectively kill liver cancer tumor cells. DETAILED DESCRIPTION

[0038] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0039] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0040] The sequences involved in the following examples are shown in the table below:

[0041] Table 1. Sequence

[0042]

[0043]

[0044] Example 1

[0045] (1) Screening of fully human TCR clones targeting HBV antigens

[0046] Blood samples were collected from 5 volunteers who met the requirements (peripheral blood after recovery from acute HBV infection). Tetramer-positive and CD8-positive T cells (i.e., antigen-specific cytotoxic T cells CTL) were flow-sorted. The TCR-α and TCR-β sequences of each cell were obtained by single-cell TCR sequencing, and the corresponding TCR sequences were synthesized ( Figure 1 ).

[0047] Human T cell isolation and culture

[0048] 1) Take 10 mL of sterile human blood in a heparin anticoagulant tube and transfer it to a sterile centrifuge tube;

[0049] 2) Add an equal volume of sterile PBS buffer to dilute and mix well;

[0050] 3) Add human peripheral blood lymphocyte separation solution to the bottom of a sterile 50 mL centrifuge tube, and add the diluted peripheral blood and human peripheral blood lymphocyte separation solution to the upper layer of the separation solution in a ratio of 1:1. Note that when adding peripheral blood, add it slowly along the wall of the tube so that the peripheral blood and separation solution are clearly separated;

[0051] 4) Centrifuge at 800g for 30 minutes at room temperature. After centrifugation, the centrifuge tube is divided into four layers from top to bottom: plasma layer / diluent layer; mononuclear / mononuclear cell layer; transparent separation liquid layer and red blood cell layer;

[0052] 5) Insert the pipette into the mononuclear cell layer, gently suck out the cell layer, and transfer it to a new centrifuge tube;

[0053] 6) Add 5 mL of saline to wash, repeat two to three times, centrifuge at 500 g for 5 min, and discard the supernatant;

[0054] 7) Prepare T cell culture medium: Add IL-2 cytokine (Beijing Shuanglu Pharmaceutical Co., Ltd.) to Takara GT-T551 Culture Medium (final concentration 100U / mL), use DMSO or Takara GT-T551 to dissolve HBV epitope peptide (GenScript, SEQ ID No. 25) at a concentration of 10μg / mL. On the second and fourth days of cell culture, add filtered sterile HBV mixed epitope peptides at a final concentration of 1μg / mL;

[0055] 8) The cell culture cycle was 15 to 18 days. During the culture process, the cells were observed every 2-3 days, the medium was replaced by half, and the live cell density, cell suspension volume and cell viability were recorded using a cell counter. On the 18th day, T cells were stained with human CD8 antibody (purchased from Biolegend) and HBV epitope tetramer (Tetramer) (purchased from MBL) for flow cytometry.

[0056] Staining of cell membrane proteins

[0057] 1) Transfer immune cells to flow cytometry tubes, approximately 10 6 cells, added 2 mL PBS for washing, centrifuged at 400 × g for 5 min at 4 °C, and discarded the supernatant;

[0058] 2) Resuspend with 100 μL PBS buffer and stain extracellular proteins. Add CD16 / CD32 blocking antibody (purchased from Biolegend) to reduce nonspecific staining of cells. Incubate at room temperature in the dark for 15 min, add 2 mL PBS for washing, centrifuge at 400 × g for 5 min at 4°C, and discard the supernatant;

[0059] 3) Resuspend the cells with 100 μL PBS buffer, add the corresponding flow cytometry antibodies, for example, use CD45 antibody (purchased from Biolegend) to mark all immune cells, use CD3 / CD8 antibody (purchased from Biolegend) to mark CD8+ T cells, and use Tetramer (purchased from MBL) to mark antigen-specific T cells. The extracellular antibody incubation conditions are 4°C in the dark for 30 minutes;

[0060] 4) Add 2 mL of PBS buffer to the flow tube, centrifuge at 400 × g for 5 min at 4°C, and discard the supernatant;

[0061] 5) Add 300 μL PBS and 5 μL 7-AAD nucleic acid dead dye (purchased from Biolegend) to the flow tube, incubate at room temperature in the dark for 10 min, and then directly test on the machine.

[0062] Single-cell sequencing and bioinformatics analysis

[0063] 1) CD8-positive and Tetramer-positive single cells in the sample were sorted out by flow cytometry, and then 10×Genomics single-cell transcriptome and TCR sequencing were performed;

[0064] 2) Cell quality inspection: The prepared single cell suspension of the sample is inspected and counted, and the cell survival rate is required to reach more than 80%. The cells that pass the quality inspection are prepared into a suspension with a concentration of 700-1200 cells / uL for subsequent machine operation;

[0065] 3) Using the 10x Genomics ChromiumTM system, the prepared sample suspension, gel beads (GelBead), reagent premix and oil were added to their respective channels, and a "double cross" cross system was formed through the microfluidic channel, and finally a micro-reaction system wrapped in oil droplets - single-cell gel bead emulsion (GEMs) was formed for single-cell separation;

[0066] 4) Collect GEMs, lyse cells in GEMs to obtain mRNA, and reverse transcribe in a PCR instrument to obtain labeled cDNA;

[0067] 5) GEMs were broken into oil, cDNA was enriched by magnetic beads, PCR amplified and quality tested, and the cDNA was used for library construction after passing the quality test;

[0068] 6) Library construction and quantification: Using the 5' end capture method, the cDNA that has passed the quality inspection is fragmented, end-repaired, polyadenylic acid is added, and sequencing adapters are connected. After fragment screening and quality inspection, the sequencing library is obtained;

[0069] 7) Sequence the constructed library using Illumina Hiseq or NovaSeq platform in PE150 sequencing mode, and perform further analysis after obtaining the data.

[0070] 8) A high-frequency cloned TCR was obtained, the amino acid sequences of CDR1, CDR2, and CDR3 of the α chain and β chain variable regions are shown in Table 1, the FR1, FR2, and FR3 of the α chain and β chain variable regions are shown in Table 1, the variable region of the α chain of the T cell antigen receptor TCR is the amino acid sequence shown in SEQ ID NO.14, and the variable region of the β chain is the amino acid sequence shown in any one of SEQ ID NOs.15 to 16.

[0071] Example 2 Preparation of TCR-T cells

[0072] 1. Construction of plasmid vector expressing HBV-specific TCR

[0073] The α and β variable region sequences of the two HBV-specific TCRs obtained in Example 1 were fused with the human α constant region and β constant region respectively (the constant region of the α chain of the T cell antigen receptor TCR is the amino acid sequence shown in SEQ ID NO.17, and the constant region of the β chain of the T cell antigen receptor TCR is the amino acid sequence shown in SEQ ID NO.18), and the TCRα and TCRβ chains were connected with the 2A sequence of the internal self-cleaving porcine teschovirus (Porcine teschovirus) (the nucleotide sequence is shown in SEQ ID NO.21, and the amino acid sequence is shown in SEQ ID NO.22). The nucleotide sequence of the complete TCR is shown in SEQ ID No.19 and SEQ ID No.20.

[0074] After codon optimization of TCRα / β, the exogenous TCR gene was synthesized by gene synthesis. Furthermore, the obtained TCR gene sequence was successfully inserted into the retroviral vector MSCV-IRES-NGFR (addgene, Plasmid#27489) between the restriction endonucleases XhoI and EcoRI by molecular cloning, while other sequences remained unchanged, to obtain the recombinant plasmid MSCV-TCR-NGFR.

[0075] 2. TCR transduction of Jurkat cells to verify the affinity of TCR for restricted epitopes

[0076] The TCR was successfully expressed on the surface of Jurkat cells by electroporation of Jurkat cells. Tetramer antigen-specific staining and flow cytometry were used to evaluate whether the TCR clones obtained by screening could specifically recognize HBV antigens and the strength of the related affinity. The results showed that both pairs of TCRs obtained by screening could specifically recognize the corresponding HBV antigens and had good affinity ( Figure 2 ).

[0077] Transduce TCR into Jurkat cells by electroporation (Lonza 4D-Nucleofector):

[0078] (1) Take 100 μl Human T Cell Nucleofector Solution (purchased from Lonza) and preheat to room temperature;

[0079] (2) Add 2 ml / well of RPMI 1640 Medium (purchased from Gibcal) containing 10% FBS into a 6-well plate and preheat in an incubator for more than 30 min;

[0080] (3) Take 1×10 7 Jurkat cells were centrifuged at 200 g for 10 min, the supernatant was removed, the cells were collected, and washed twice with 1640 medium without antibiotics;

[0081] (4) Resuspend the cells with 100 μl of room temperature Human T Cell Nucleofector Solution;

[0082] (5) Add recombinant plasmid MSCV-TCR-NGFR, 20 μg, and mix well;

[0083] (6) Transfer the specimen into the electroporation cup and cover it with the blue lid;

[0084] (7) Select program T-020, insert the electroporation cuvette, and start the electroporation program;

[0085] (8) Immediately after electroporation, remove the electroporation cup, add 500 μl of preheated RPMI1640 Medium containing 10% FBS, and then transfer all the cell suspension into a 6-well plate;

[0086] (9) After incubating the transfected cells in a 37°C, 5% CO2 incubator for 4-12 hours, the cells were centrifuged at 200g for 8 minutes, and fresh culture medium was replaced to continue culturing for 24-48 hours. Tetramer staining was detected by flow cytometry.

[0087] 3. Construction of TCR-T cells by lentiviral infection

[0088] First, the natural TCR is composed of an α chain and a β chain. When constructing TCR-T, the TCR sequence is usually connected through the 2A sequence. There is no clear conclusion on whether the α chain or the β chain in front is conducive to the expression and assembly of TCR. To this end, we constructed two TCR connections, HC-TCR-a2b and HC-TCR-b2a. HC-TCR-a2b (HC means the use of α chain and β chain containing human constant region) means the α chain is in front and the β chain is in the back, that is, from N-terminus to C-terminus, the amino acid sequence is the complete amino acid sequence of α chain, P2A amino acid sequence, and the complete amino acid sequence of β chain. HC-TCR-b2a means the α chain is in the back and the β chain is in the front, that is, from N-terminus to C-terminus, the amino acid sequence is the complete amino acid sequence of β chain, P2A amino acid sequence, and the complete amino acid sequence of α chain. Secondly, since normal T cells have a pair of endogenous TCR sequences, when constructing TCR-T, the transduced TCR sequence will pair with the endogenous TCR, resulting in a reduction in specific TCR and an increase in off-target toxicity. To avoid the above problems, we replaced the constant region of the fully human TCR with the mouse TCR constant region: MC-TCR (MC means the use of α and β chains containing the mouse constant region) to enhance the probability of correct pairing of the transduced TCR. Lentiviruses were packaged separately, T cells were infected to construct the corresponding TCR-T cells, and tetramer antigen-specific staining was used to evaluate the expression of specific TCR on the surface of TCR-T cells.

[0089] (1) Lentiviral vector construction

[0090] Construction of lentiviral plasmid vector expressing HBV-specific TCR: The small DNA fragment between the restriction endonucleases EcoRI and BamHI of the lentiviral packaging vector pCDH-MSCV-MCS-IRES-GFP (System Biosciences, No.: CD731B-1) was replaced with a TCR DNA fragment (the nucleotide sequence of the complete TCR1 is as shown in SEQ ID No. 19, and the constant region of the above-mentioned fully human TCR is replaced with the mouse TCR constant region, the mouse TCRα constant region nucleotide sequence is as shown in SEQ ID No. 23, and the TCRβ constant region nucleotide sequence is as shown in SEQ ID No. 24) to obtain the pCDH-MSCV-TCR-GFP plasmid.

[0091] (2) Lentiviral packaging

[0092] 1) Cell preparation: Select HEK-293FT cells with good growth status, and when the cell density reaches 70%, subculture them into 10mm large dishes at a ratio of 1:3, and add 10mL of fresh DMEM complete culture medium. When the cell density reaches 80%, add 3mL of fresh DMEM complete culture medium and continue to culture for 1-2 hours to allow the cells to fully expand. Then discard the supernatant, leaving only 1mL of culture medium, and add 4mL of newly prepared virus-specific culture medium, and continue to culture for 2 hours.

[0093] 2) Preparation of plasmid transfection complex: Take the 10mm cell culture dish culture system as an example: Take two clean 1.5mL EP tubes, add 500μL Opti-MEM solution (purchased from Gibcal), add 42μg PEI (purchased from Merck) to tube ①, add the third-generation virus packaging system (PLP1 3.6μg, PLP2 3.6μg, PLP-VSVG 1.8μg) (commercially available) and TCR expression plasmid 5μg to tube ②, mix them well, and let them stand at room temperature for 15min. Then add the complex in tube ① to tube ②, mix well, and let them stand at room temperature for 20min. The plasmid transfection complex is ready.

[0094] 3) Lentivirus packaging: Add the prepared transfection complex to HKE-293FT cells, shake gently and evenly, culture at 37°C, 5% CO2 for 4-5 hours, add 4mL of virus packaging special culture medium, continue to culture for 2 days, collect the cell supernatant, and keep 1mL of old supernatant. Add 8mL of fresh virus packaging special culture medium, continue to culture for 1 day, collect the cell supernatant again, mix the two supernatants and centrifuge at 400g×5min, discard the cell pellet, filter the supernatant with a 0.45μm filter to remove cell debris, and obtain the supernatant rich in lentivirus (if the unconcentrated virus supernatant is used directly for infection, the 2-day virus supernatant collected for the first time is preferred).

[0095] 4) Lentivirus Concentration

[0096] Transfer the lentiviral supernatant to an ultracentrifuge tube, weigh it with a balance, and centrifuge it at 4°C × 20,000g × 90min. After the centrifugation, white virus particles can be seen precipitating. Carefully discard the supernatant and add an appropriate amount of serum-free 1640 culture medium in proportion to achieve 100-fold concentration (for example, if there is 40mL of virus supernatant before centrifugation, 400μL of culture medium needs to be added to the precipitate). Tighten the cap of the centrifuge tube, place it on a shaker at 4°C to shake and dissolve for 24 hours, collect the virus concentrate, and store it at -80°C for later use.

[0097] 5) Lentiviral infection

[0098] PBMCs from peripheral blood of healthy donors were separated by Ficoll density gradient centrifugation and resuspended in Takara GT-T551 medium. T cells were purified and isolated by magnetic beads, activated with CD3 / CD28 magnetic beads, and resuspended in Takara T551 medium containing 2.5% human autologous serum and 30 IU / mL IL-2 for three days. On the third day, cells were collected, centrifuged at 200g × 3min, washed twice with PBS buffer, and counted. Sufficient amount of lentiviral supernatant was added according to the amount of cells (per 1 × 10 6 2 mL of lentiviral supernatant or 20 μL of concentrate was added to each cell), and then polybrene (purchased from Sigma-Aldrich) was added to maintain its concentration at 10 μg / mL. After continuous infection for 2-3 days, TCR-T cells expressing the target gene were obtained. HBV Tetramer (purchased from MBL) was used to stain the TCR-expressing T cells 24 hours later. The staining results are shown in Figure 3 ,like Figure 3 As shown in Figure A, HC-TCR-a2b (the TCR in both HC-TCR-a2b and HC-TCR-b2a in the figure selects TCR1) is a better connection method, such as Figure 3 As shown in Figures A and B (the MC-TCR in the figure is MC-TCR-a2b), replacing the constant region of the fully human TCR with the mouse TCR constant region can significantly reduce the mismatch with the endogenous TCR and effectively increase the expression of specific TCR on the cell surface. Compared with the control group, the Tetramer positivity rate of T cells electroporated with MC-TCR was 20.9%, and the TCR-T can be used for subsequent experiments.

[0099] Example 3

[0100] 1. TCR-T cells can specifically kill liver cancer cells

[0101] In order to verify whether HBV TCR-transduced TCR-T cells can kill tumor cells, we used HepG2.2.15 (HLA) cells (commercially available) as target cells and TCR-T as effector cells. The effector cells and target cells were mixed and incubated for cytotoxicity detection. The specific method is as follows:

[0102] LDH method to detect T cell killing ability

[0103] Select HepG2.2.15 (A11 + HLA) and TCR-T (including TCR1 and TCR2, both of which contain mouse constant regions in Experimental Example 2) cells with a good growth state, and co-culture them in an incubator at 37°C and 5% CO2 for 5 hours at an effector-target ratio (E / T) of 1:1, collect the cell supernatant, and operate according to the instructions provided by the LDH detection kit (purchased from Promega). The details are as follows:

[0104] 1) HepG2.2.15 (HLA) tumor cells in the logarithmic growth phase were digested and counted, and resuspended in an appropriate volume of cell culture medium to a concentration of 4 × 10 4 pcs / 100μL;

[0105] 2) Tumor cells and T cells of the corresponding species were inoculated in a 96-well plate, and 2×10 4 Tumor cells were added, and then TCR-T cells were added in different proportions, with three replicate wells set for each proportion. At the same time, blank wells and maximum release control wells were laid according to the LDH reagent instructions;

[0106] 3) Continue to culture in a 37°C CO2 incubator for 5 hours and then take out;

[0107] 4) Take a 96-well plate and centrifuge at 250g for 4 minutes; then transfer 50μL of supernatant to a new 96-well plate, add 50μL of substrate to each well, and incubate at room temperature and away from light for 30 minutes; then add 50μL of stop solution to each well, and use a microplate reader (wavelength 490nm) to read the value, which is the experimental group value. Add 50μl of labeled target cells and 150μl of 2% TritonX-100 to the maximum release control well.

[0108] 5) Use an ELISA reader to detect the OD490 nm and OD630 nm values. The supernatant of tumor cells cultured alone was used as the background control. Normalized LDH release = [supernatant of TCR-T cells co-cultured with HepG2.2.15 (HLA) (OD490-OD630) - supernatant of HepG2.2.15 (HLA) cultured alone (OD490-OD630)] / [supernatant of the maximum release control well (OD490-OD630) - supernatant of HepG2.2.15 (HLA) cultured alone (OD490-OD630)].

[0109] The results are as follows Figure 4In the experiment with HepG2.2.15 (HLA) as the target cell, the lysis rate of the target cells in the control group (vector) was at a lower level. In contrast, TCR-T exerted a good killing effector function, and more than 40% of the cells were lysed and died during co-culture. The results showed that the two pairs of TCR-T we screened had a good ability to kill liver cancer cells ( Figure 4 ).

[0110] In summary, the present invention isolated and identified two pairs of HBVc1-specific TCR sequences from healthy human bodies, verified the TCR antigen specificity and affinity in vitro, and used in vitro target cell killing experiments to prove that the constructed TCR-T has good activity in killing target cells.

[0111] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A T cell antigen receptor, characterized in that Include at least one of the following: T cell antigen receptor TCR, CDR1, CDR2 and CDR3 of the α chain variable region and CDR1, CDR2 and CDR3 of the β chain variable region of the T cell antigen receptor TCR; The CDR3 of the α chain variable region has an amino acid sequence as shown in SEQ ID NO.1, or has at least 90%, 95% or 99% similarity to the amino acid sequence; The CDR3 of the β chain variable region has an amino acid sequence as shown in SEQ ID NO.2, or has at least 90%, 95% or 99% similarity with the amino acid sequence, or the CDR3 of the β chain has an amino acid sequence as shown in SEQ ID NO.3, or has at least 90%, 95% or 99% similarity with the amino acid sequence.

2. The T cell antigen receptor according to claim 1, characterized in that Also includes at least one of the following: The CDR1 of the α chain variable region has an amino acid sequence as shown in SEQ ID NO.4, or has at least 90%, 95% or 99% similarity to the amino acid sequence; The CDR2 of the α chain variable region has the amino acid sequence shown in SEQ ID NO.5, or has at least 90%, 95% or 99% similarity to the amino acid sequence; The CDR1 of the beta chain variable region has an amino acid sequence as shown in SEQ ID NO.6, or has at least 90%, 95% or 99% similarity to the amino acid sequence; The CDR2 of the beta chain variable region has an amino acid sequence as shown in SEQ ID NO.7, or has at least 90%, 95% or 99% similarity to the amino acid sequence; The FR1 of the α chain variable region has the amino acid sequence shown in SEQ ID NO.8, or has at least 90%, 95% or 99% similarity to the amino acid sequence; The FR2 of the α chain variable region has the amino acid sequence shown in SEQ ID NO.9, or has at least 90%, 95% or 99% similarity to the amino acid sequence; The FR3 of the α chain variable region has the amino acid sequence shown in SEQ ID NO.10, or has at least 90%, 95% or 99% similarity to the amino acid sequence; The FR1 of the beta chain variable region has the amino acid sequence shown in SEQ ID NO.11, or has at least 90%, 95% or 99% similarity to the amino acid sequence; The FR2 of the beta chain variable region has an amino acid sequence as shown in SEQ ID NO. 12, or has at least 90%, 95% or 99% similarity to the amino acid sequence; The FR3 of the beta chain variable region has an amino acid sequence as shown in SEQ ID NO. 13, or has at least 90%, 95% or 99% similarity to the amino acid sequence; Optionally, the variable region of the α chain of the T cell antigen receptor TCR has an amino acid sequence shown in SEQ ID NO.14, and the variable region of the β chain has an amino acid sequence shown in any one of SEQ ID NOs.15 to 16; Optionally, the constant region of the α chain of the T cell antigen receptor TCR has the amino acid sequence shown in SEQ ID NO.17, and the constant region of the β chain has the amino acid sequence shown in SEQ ID NO.

18.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the T cell antigen receptor according to any one of claims 1-2; optionally, the nucleic acid molecule is shown in any one of SEQ ID NO.19-20; optionally, the nucleic acid molecule is mRNA, and the mRNA can be translated into the T cell antigen receptor according to claim 1.

4. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the nucleic acid molecule of claim 3.

5. A host cell, characterized in that The host cell comprises the nucleic acid molecule according to claim 3 or the recombinant plasmid according to claim 4.

6. The host cell according to claim 5, characterized in that The host cell is co-transfected with the recombinant plasmid according to claim 4 and a lentiviral packaging plasmid.

7. A recombinant virus, characterized in that The genome of the recombinant virus carries the nucleic acid molecule of claim 3.

8. A T cell receptor engineered T cell targeting HBV, characterized in that: The T cell receptor engineered T cell comprises the nucleic acid molecule of claim 3, or the T cell antigen receptor of any one of claims 1-2.

9. Use of the T cell antigen receptor according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the recombinant plasmid according to claim 5, the host cell according to claim 6, the recombinant virus according to claim 7, and the T cell receptor engineered T cell targeting HBV according to claim 8 in the preparation of drugs for hepatitis B virus-related diseases.

10. The use according to claim 9, characterized in that The hepatitis B virus-related diseases include liver cancer and acute and chronic viral infections.

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