TCR combination and application thereof

Abstract

The invention relates to the field of immunology, and particularly discloses a TCR combination and application thereof, the TCR combination is used for recognizing HBV antigen epitopes, the TCR combination comprises a plurality of separated TCRs, the plurality of separated TCRs form two TCR groups, and the two TCR groups comprise a first TCR group and a second TCR group; wherein the first TCR group specifically recognizes a first antigen epitope, and the amino acid sequence of the first antigen epitope is as shown in SEQ ID NO: 1; the first TCR group specifically recognizes a first antigen epitope, the second TCR group specifically recognizes a second antigen epitope, the amino acid sequence of the second antigen epitope is shown as SEQ ID NO: 2, the second antigen epitope respectively and correspondingly recognizes different antigen epitopes, the hepatitis B virus specific TCR and the epitope thereof are screened out, and an important means can be provided for treatment of TCR-T immune cells infected by hepatitis B virus.

Description

Technical Field

[0001] The present invention relates to the field of immunology, and particularly to a TCR combination and its application. Background Art

[0002] Research shows that in chronic hepatitis B virus (HBV) - infected patients, the levels of viral surface antigen and viral DNA in the body are inversely proportional to the proportion of hepatitis B virus - specific CD8+ T cells, suggesting the antiviral effect of virus - specific CD8+ T cells. Therefore, the isolation and identification of virus - specific CD8+ T cells play an important role in the prevention and treatment of chronic hepatitis B virus infection and hepatitis B - related liver cancer.

[0003] During the process of virus infection, antigen - presenting cells process viral antigens into antigenic short peptides, which combine with HLA molecules to form stable antigen - peptide - MHC complexes expressed on the cell membrane. Specific T cells bind to them through the T - cell receptor (TCR) on the surface. With the cooperation of co - stimulatory signals, a series of signal transduction and physiological functions are triggered to achieve the effect of clearing the virus. The TCR - T adoptive cell therapy mainly involves genetically modifying autologous cells of patients in vitro to introduce TCRs that specifically recognize hepatitis B virus antigens, and then re - infusing them into the patients' bodies, so as to achieve the purpose of efficiently clearing virus - infected cells. Transducing hepatitis B virus - specific TCRs onto the surface of healthy human T cells can endow normal T cells with reactivity to hepatitis B virus antigens.

[0004] Therefore, screening and identifying hepatitis B virus - specific TCRs and their epitopes can provide an important means for TCR - T immunotherapy for hepatitis B virus infection. Summary of the Invention

[0005] The main object of the present invention is to propose a TCR combination and its application, aiming to propose a TCR combination in which the TCRs in the combination can specifically recognize and bind to hepatitis B virus antigen epitopes, thereby providing an important basis for TCR - T immunotherapy for hepatitis B virus infection.

[0006] To achieve the above object, the present invention proposes a TCR combination for recognizing HBV antigen epitopes. The TCR combination includes a plurality of isolated TCRs, and the plurality of isolated TCRs form two TCR groups, namely a first TCR group and a second TCR group; wherein,

[0007] The first TCR group specifically recognizes a first antigen epitope, and the amino acid sequence of the first antigen epitope is as shown in SEQ ID NO: 1;

[0008] The second TCR group specifically recognizes a second antigen epitope, and the amino acid sequence of the second antigen epitope is as shown in SEQ ID NO: 2.

[0009] In some embodiments, the first TCR group includes a first TCR α-chain variable region, a first TCR β-chain variable region, a second TCR α-chain variable region, a second TCR β-chain variable region, a third TCR α-chain variable region, and a third TCR β-chain variable region. Among them, the first TCR α-chain variable region includes a first CDR1α, a first CDR2α, and a first CDR3α; the first TCR β-chain variable region includes a first CDR1β, a first CDR2β, and a first CDR3β; the second TCR α-chain variable region includes a second CDR1α, a second CDR2α, and a second CDR3α; the second TCR β-chain variable region includes a second CDR1β, a second CDR2β, and a second CDR3β; the third TCR α-chain variable region includes a third CDR1α, a third CDR2α, and a third CDR3α; the third TCR β-chain variable region includes a third CDR1β, a third CDR2β, and a third CDR3β. The amino acid sequence of the first CDR1α is as shown in SEQ ID NO:3; the amino acid sequence of the first CDR2α is as shown in SEQ ID NO:4; the amino acid sequence of the first CDR3α is as shown in SEQ ID NO:5; the amino acid sequence of the first CDR1β is as shown in SEQ ID NO:6; the amino acid sequence of the first CDR2β is as shown in SEQ ID NO:7; the amino acid sequence of the first CDR3β is as shown in SEQ ID NO:8; the amino acid sequence of the second CDR1α is as shown in SEQ ID NO:9; the amino acid sequence of the second CDR2α is as shown in SEQ ID NO:10; the amino acid sequence of the second CDR3α is as shown in SEQ ID NO:11; the amino acid sequence of the second CDR1β is as shown in SEQ ID NO:12; the amino acid sequence of the second CDR2β is as shown in SEQ ID NO:13; the amino acid sequence of the second CDR3β is as shown in SEQ ID NO:14; the amino acid sequence of the third CDR1α is as shown in SEQ ID NO:15; the amino acid sequence of the third CDR2α is as shown in SEQ ID NO:16; the amino acid sequence of the third CDR3α is as shown in SEQ ID NO:17; the amino acid sequence of the third CDR1β is as shown in SEQ ID NO:18; the amino acid sequence of the third CDR2β is as shown in SEQ ID NO:19; the amino acid sequence of the third CDR3β is as shown in SEQ ID NO:20; and / or,

[0010] The second TCR group includes a fourth TCR α-chain variable region and a fourth TCR β-chain variable region. The fourth TCR α-chain variable region includes a fourth CDR1α, a fourth CDR2α, and a fourth CDR3α. The fourth TCR β-chain variable region includes a fourth CDR1β, a fourth CDR2β, and a fourth CDR3β. The amino acid sequence of the fourth CDR1α is as shown in SEQ ID NO:21, the amino acid sequence of the fourth CDR2α is as shown in SEQ ID NO:22, the amino acid sequence of the fourth CDR3α is as shown in SEQ ID NO:23, the amino acid sequence of the fourth CDR1β is as shown in SEQ ID NO:24, the amino acid sequence of the fourth CDR2β is as shown in SEQ ID NO:25, and the amino acid sequence of the fourth CDR3β is as shown in SEQ ID NO:26.

[0011] In some embodiments, the isolated TCR combination forms four TCRs, wherein,

[0012] each TCR includes a TCR α-chain variable region and a TCR β-chain variable region. The amino acid sequences of the four TCR α-chain variable regions are as shown in SEQ ID NO:27 to SEQ ID NO:30, and the amino acid sequences of the four TCR β-chain variable regions are as shown in SEQ ID NO:31 to SEQ ID NO:34; and / or,

[0013] each TCR includes a TCR α-chain and a TCR β-chain. The amino acid sequences of the four TCR α-chains are as shown in SEQ ID NO:35 to SEQ ID NO:38, and the amino acid sequences of the four TCR β-chains are as shown in SEQ ID NO:39 to SEQ ID NO:42; and / or,

[0014] each TCR includes a TCR α-mutated chain and a TCR β-mutated chain. The amino acid sequences of the four TCR α-mutated chains are as shown in SEQ ID NO:43 to SEQ ID NO:46, and the amino acid sequences of the four TCR β-mutated chains are as shown in SEQ ID NO:47 to SEQ ID NO:50.

[0015] The present invention provides a nucleic acid molecule combination, which includes a plurality of nucleic acid molecules. The plurality of nucleic acid molecules form four TCRs, wherein,

[0016] each TCR further includes a TCR α-chain variable region and a TCR β-chain variable region. The nucleotide sequences of the four TCR α-chain variable regions are as shown in SEQ ID NO:51 to SEQ ID NO:54, and the nucleotide sequences of the four TCR β-chain variable regions are as shown in SEQ ID NO:55 to SEQ ID NO:58; and / or,

[0017] Each TCR also includes a TCR α chain and a TCR β chain. The nucleotide sequences of four said TCR α chains are shown as SEQ ID NO:59 to SEQ ID NO:62, and the nucleotide sequences of four said TCR β chains are shown as SEQ ID NO:63 to SEQ ID NO:66; and / or,

[0018] Each TCR also includes a TCR α mutant chain and a TCR β mutant chain. The nucleotide sequences of four said TCR α mutant chains are shown as SEQ ID NO:67 to SEQ ID NO:70, and the nucleotide sequences of four said TCR β mutant chains are shown as SEQ ID NO:71 to SEQ ID NO:74; and / or,

[0019] Each TCR also includes nucleotides with optimized codons for the α chain and nucleotides with optimized codons for the β chain. The nucleotide sequences of four said nucleotides with optimized codons for the α chain are shown as SEQ ID NO:75 to SEQ ID NO:78, and the nucleotide sequences of four said nucleotides with optimized codons for the β chain are shown as SEQ ID NO:79 to SEQ ID NO:82.

[0020] The present invention also provides an expression vector, which includes the nucleic acid molecules in the nucleic acid molecule combination as described above.

[0021] The present invention also provides a recombinant cell, which includes the nucleic acid molecule as described above or the expression vector as described above.

[0022] The present invention also provides a drug combination, which includes the TCR combination as described in any one of the above, or the isolated TCR as described above, or the nucleic acid molecule as described above, the expression vector as described above, and the recombinant cell as described above.

[0023] The present invention also provides the use of the TCR combination as described above, or the isolated TCR as described above, or the nucleic acid molecule as described above, the expression vector as described above, the recombinant cell as described above, or the drug composition as described above in the preparation of a drug for preventing and / or treating hepatitis B virus and / or related diseases caused by hepatitis B virus.

[0024] Multiple TCR groups in the TCR combination provided by the present invention can specifically recognize multiple antigenic epitopes respectively, and can endow normal cells with reactivity to hepatitis B virus antigens, providing a new solution for the prevention and treatment of hepatitis B virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other relevant accompanying drawings can also be obtained based on these drawings.

[0026] Figure 1 It is a diagram showing the identification results of the reactivity of hepatitis B virus-specific TCRs (C345-3 and C345-4) to hepatitis B virus proteins in Example 1;

[0027] Figure 2 It is a diagram showing the identification results of the reactivity of hepatitis B virus-specific TCRs (C345-7 and C345-8) to hepatitis B virus proteins in Example 1;

[0028] Figure 3 It is a schematic diagram showing the detection results of the polypeptide epitopes identified by TCR (C345-3) under the condition of homologous LCL cell presentation of epitopes in Example 1;

[0029] Figure 4 It is a schematic diagram showing the detection results of the polypeptide epitopes identified by TCR (C345-4) under the condition of homologous LCL cell presentation of epitopes in Example 1;

[0030] Figure 5 It is a schematic diagram showing the detection results of the polypeptide epitopes identified by TCR (C345-7) under the condition of homologous LCL cell presentation of epitopes in Example 1;

[0031] Figure 6 It is a schematic diagram showing the detection results of the polypeptide epitopes identified by TCR (C345-8) under the condition of homologous LCL cell presentation of epitopes in Example 1;

[0032] Figure 7 It is a diagram showing the identification of HLA molecules recognized by TCRs (C345-3, C345-4, C345-7, and C345-8) in Example 1;

[0033] Figure 8 It is a diagram showing the functional verification of TCR (C345-3) in Example 1;

[0034] Figure 9 It is a diagram showing the functional verification of TCR (C345-4) in Example 1;

[0035] Figure 10 It is a diagram showing the functional verification of TCR (C345-7) in Example 1;

[0036] Figure 11 It is a diagram showing the functional verification of TCR (C345-8) in Example 1.

[0037] The realization, functional features and advantages of the present invention will be further described in conjunction with the embodiments and with reference to the accompanying drawings. Specific Embodiments

[0038] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part rather than all of the embodiments of the present invention.

[0039] It should be noted that for those conditions not specified in the embodiments, they shall be carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase. In addition, the meaning of "and / or" appearing throughout the text includes three parallel solutions. Taking "A and / or B" as an example, it includes solution A, or solution B, or a solution where A and B are satisfied simultaneously. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of those of ordinary skill in the art to implement. When the combination of technical solutions results in contradictions or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention.

[0040] Research has shown that the levels of viral surface antigen and viral DNA in the bodies of chronic hepatitis B virus (HBV) infected individuals are inversely proportional to the proportion of hepatitis B virus-specific CD8+ T cells, suggesting the antiviral effect of virus-specific CD8+ T cells. Therefore, screening and identifying hepatitis B virus-specific TCRs and their epitopes play an important role in the prevention and treatment of chronic hepatitis B virus infection and hepatitis B virus-related liver cancer.

[0041] In view of this, the present invention proposes a TCR combination and its application for identifying HBV antigenic epitopes. The TCR combination includes a plurality of isolated TCRs, and the plurality of isolated TCRs form two TCR groups, namely a first TCR group and a second TCR group; wherein,

[0042] The first TCR group specifically recognizes a first antigenic epitope, and the amino acid sequence of the first antigenic epitope is as shown in SEQ ID NO: 1;

[0043] The second TCR group specifically recognizes a second antigenic epitope, and the amino acid sequence of the second antigenic epitope is as shown in SEQ ID NO: 2.

[0044] In view of the above two antigenic epitopes, the present invention obtains a TCR combination capable of specifically recognizing the HBV virus. The TCRs in this combination can specifically recognize and target and bind to the hepatitis B virus antigenic epitopes, thus providing an important basis for TCR-T immunotherapy for hepatitis B virus infection.

[0045] Specifically, the amino acid sequence of the first antigenic epitope is shown in SEQ ID NO:1, specifically represented as: LWFHISCLTF-HLA-A*24:02; the amino acid sequence of the second antigenic epitope is shown in SEQ ID NO:2, specifically represented as: PFMPLLPIFF-HLA-A*24:02.

[0046] It should be noted that taking LWFHISCLTF-HLA-A*24:02 as an example, among them, LWFHISCLTF is the peptide segment of the antigenic epitope, and HLA-A*24:02 is the subtype of the HLA molecule. HLA-A belongs to a category of the HLA family.

[0047] Furthermore, it should be noted that the T cell receptor (TCR) is a membrane protein on the surface of T cells, which can recognize the antigen short peptides on the surface of the corresponding target cells. The human leukocyte antigen HLA, usually called the MHC molecule, is a class of genes on the short arm of chromosome 6. Each person's HLA is very different and is an important molecular marker for identifying human leukocytes. In the immune system, the binding of the antigen short peptide-specific TCR to the peptide-major histocompatibility antigen (pMHC) complex triggers the direct physical contact between the T cell and the antigen-presenting cell (APC). Then, other cell membrane surface molecules of both the T cell and the APC interact with each other, causing a series of subsequent cell signal transmissions and other physiological reactions, so that T cells with different antigen specificities exert immune effects on their target cells.

[0048] pMHC (Peptide-Major Histocompatibility Complex) and TCR (T-cell Receptor) are important components in the immune system.

[0049] pMHC is a complex composed of an antigen peptide and a major histocompatibility complex (MHC) molecule. MHC molecules are a class of membrane molecules, and their main function is to present antigen peptides to T cells.

[0050] It should be noted that each TCR consists of a TCRα chain and a TCRβ chain, and at least the variable region is included in the TCRα chain or the TCRβ chain. The variable region is the main function that can specifically bind to antigens. The region that performs this function is called the complementarity-determining region (CDR). CDR refers to three sequences in the variable region of an antibody, namely CDR1, CDR3, and CDR3, which together form the site for binding antigens, determine specificity, are responsible for recognizing and binding antigens, and thus exert an immune effect.

[0051] In some embodiments, the first TCR group includes a first TCR α-chain variable region, a first TCR β-chain variable region, a second TCR α-chain variable region, a second TCR β-chain variable region, a third TCR α-chain variable region, and a third TCR β-chain variable region. Among them, the first TCR α-chain variable region includes a first CDR1α, a first CDR2α, and a first CDR3α, the first TCR β-chain variable region includes a first CDR1β, a first CDR2β, and a first CDR3β, the second TCR α-chain variable region includes a second CDR1α, a second CDR2α, and a second CDR3α, the second TCR β-chain variable region includes a second CDR1β, a second CDR2β, and a second CDR3β, the third TCR α-chain variable region includes a third CDR1α, a third CDR2α, and a third CDR3α, the third TCR β-chain variable region includes a third CDR1β, a third CDR2β, and a third CDR3β. The amino acid sequence of the first CDR1α is shown in SEQ ID NO:3, the amino acid sequence of the first CDR2α is shown in SEQ ID NO:4, the amino acid sequence of the first CDR3α is shown in SEQ ID NO:5, the amino acid sequence of the first CDR1β is shown in SEQ ID NO:6, the amino acid sequence of the first CDR2β is shown in SEQ ID NO:7, the amino acid sequence of the first CDR3β is shown in SEQ ID NO:8, the amino acid sequence of the second CDR1α is shown in SEQ IDNO:9, the amino acid sequence of the second CDR2α is shown in SEQ ID NO:10, the amino acid sequence of the second CDR3α is shown in SEQ ID NO:11, the amino acid sequence of the second CDR1β is shown in SEQ ID NO:12, the amino acid sequence of the second CDR2β is shown in SEQ ID NO:13, the amino acid sequence of the second CDR3β is shown in SEQ ID NO:14, the amino acid sequence of the third CDR1α is shown in SEQ ID NO:15, the amino acid sequence of the third CDR2α is shown in SEQ IDNO:16, the amino acid sequence of the third CDR3α is shown in SEQ ID NO:17, the amino acid sequence of the third CDR1β is shown in SEQ ID NO:18, the amino acid sequence of the third CDR2β is shown in SEQ ID NO:19, and the amino acid sequence of the third CDR3β is shown in SEQ ID NO:20;

[0052] The second TCR group includes a fourth TCR α-chain variable region and a fourth TCR β-chain variable region. The fourth TCR α-chain variable region includes a fourth CDR1α, a fourth CDR2α, and a fourth CDR3α. The fourth TCR β-chain variable region includes a fourth CDR1β, a fourth CDR2β, and a fourth CDR3β. The amino acid sequence of the fourth CDR1α is shown in SEQ ID NO:21. The amino acid sequence of the fourth CDR2α is shown in SEQ ID NO:22. The amino acid sequence of the fourth CDR3α is shown in SEQ ID NO:23. The amino acid sequence of the fourth CDR1β is shown in SEQ ID NO:24. The amino acid sequence of the fourth CDR2β is shown in SEQ ID NO:25. The amino acid sequence of the fourth CDR3β is shown in SEQ ID NO:26.

[0053] Specifically, the hepatitis B virus-specific TCRs identified in the present invention include C345-3, C345-4, C345-7, and C345-8. Among them, C345-3, C345-4, and C345-7 can bind to the LWFHISCLTF-HLA-A*24:02 complex (the first antigen epitope), and C345-8 can bind to the PFMPLLPIFF-HLA-A*24:02 complex (the second antigen epitope). Moreover, after these TCRs transduce primary T cells, they can endow normal cells with specific responses to hepatitis B virus antigens, providing a new strategy for the prevention and treatment of hepatitis B virus infection. The specific sequences of the CDRs in the α-chain variable region and β-chain variable region of the above TCRs are shown in Table 1:

[0054] Table 1

[0055]

[0056] In the present invention, by analyzing the single-cell sequencing data of liver biopsy samples from patients with chronic hepatitis B virus infection, several TCR clones were selected, synthesized in vitro, and inserted into a lentiviral vector. A T cell line stably expressing TCR was constructed through lentiviral infection. After stimulation with hepatitis B virus polypeptides, hepatitis B virus-specific TCRs were identified by detecting the activation level of the cells, and the antigen peptides and HLA molecules they recognized, namely the two antigen epitopes described in the text, were clarified.

[0057] In some embodiments, the plurality of isolated TCRs form four TCRs, wherein,

[0058] Each TCR comprises a variable region of TCR α chain and a variable region of TCR β chain. The amino acids of the four TCR α chain variable regions specifically include the amino acids of C345-3 α chain variable region, the amino acids of C345-4 α chain variable region, the amino acids of C345-7 α chain variable region, and the amino acids of C345-8 α chain variable region. The specific sequences of the amino acids of the four TCR α chain variable regions are as shown in SEQ ID NO:27 to SEQ ID NO:30. Similarly, the specific sequences of the amino acids of the four TCR β chain variable regions are as shown in SEQ ID NO:31 to SEQ ID NO:34; and / or,

[0059] Each TCR comprises a TCR α chain and a TCR β chain. The amino acids of the four TCR α chains specifically include the amino acids of C345-3 α chain, the amino acids of C345-4 α chain, the amino acids of C345-7 α chain, and the amino acids of C345-8 α chain. The specific sequences are as shown in SEQ ID NO:35 to SEQ ID NO:38. Similarly, the specific sequences of the amino acids of the four TCR β chains are as shown in SEQ ID NO:39 to SEQ ID NO:42; and / or,

[0060] Each TCR comprises a TCR α mutant chain and a TCR β mutant chain. The amino acids of the four TCR α mutant chains specifically include the amino acids of C345-3 α mutant chain, the amino acids of C345-4 α mutant chain, the amino acids of C345-7 α mutant chain, and the amino acids of C345-8 α mutant chain. The specific sequences are as shown in SEQ ID NO:43 to SEQ ID NO:46. Similarly, the amino acid sequences of the four TCR β mutant chains are as shown in SEQ ID NO:47 to SEQ ID NO:50.

[0061] The present invention also provides a nucleic acid molecule combination, which comprises a plurality of nucleic acid molecules. The plurality of nucleic acid molecules form four TCRs, wherein,

[0062] Each TCR further comprises a variable region of TCR α chain and a variable region of TCR β chain. The nucleotides of the four TCR α chain variable regions specifically include the nucleotides of C345-3 α chain variable region, the nucleotides of C345-4 α chain variable region, the nucleotides of C345-7 α chain variable region, and the nucleotides of C345-8 α chain variable region. The specific sequences are as shown in SEQ ID NO:51 to SEQ ID NO:54. Similarly, the nucleotides of the four TCR β chain variable regions specifically include the nucleotides of C345-3 β chain variable region, the nucleotides of C345-4 β chain variable region, the nucleotides of C345-7 β chain variable region, and the nucleotides of C345-8 β chain variable region. The specific sequences are as shown in SEQ ID NO:55 to SEQ ID NO:58; and / or,

[0063] Each TCR also includes a TCR α chain and a TCR β chain. The nucleotides of the four TCR α chains specifically include the nucleotides of the C345-3α chain, the nucleotides of the C345-4α chain, the nucleotides of the C345-7α chain, and the nucleotides of the C345-8α chain. Their specific sequences are shown in SEQ ID NO:59 to SEQ ID NO:62. Similarly, the nucleotide sequences of the four TCR β chains are shown in SEQ ID NO:63 to SEQ ID NO:66; and / or,

[0064] Each TCR also includes a TCR α mutant chain and a TCR β mutant chain. The nucleotides of the four TCR α mutant chains specifically include the nucleotides of the C345-3α mutant chain, the nucleotides of the C345-4α mutant chain, the nucleotides of the C345-7α mutant chain, and the nucleotides of the C345-8α mutant chain. Their specific sequences are shown in SEQ ID NO:67 to SEQ ID NO:70. Similarly, the nucleotide sequences of the four TCR β mutant chains are shown in SEQ ID NO:71 to SEQ ID NO:74; and / or,

[0065] Each TCR also includes nucleotides with codon-optimized α chains and nucleotides with codon-optimized β chains. The nucleotides of the four codon-optimized α chains specifically include the nucleotides of the C345-3α chain with codon optimization, the nucleotides of the C345-4α chain with codon optimization, the nucleotides of the C345-7α chain with codon optimization, and the nucleotides of the C345-8α chain with codon optimization. Their specific sequences are shown in SEQ ID NO:75 to SEQ ID NO:78. Similarly, the nucleotide sequences of the four codon-optimized β chains are shown in SEQ ID NO:79 to SEQ ID NO:82.

[0066] Specifically, the TCR can be modified with various mutations that do not change the affinity and dissociation rate of the TCR for the target antigen. These mutations are to prevent the introduced TCR from mismatching with the endogenous autologous TCR. These mutations are made on the nucleotide sequence with codon optimization.

[0067] Furthermore, each TCR includes a TCRα mutant chain and a TCRβ mutant chain. The four TCRs are C345-3, C345-4, C345-7, and C345-8. Among them, the 183rd amino acid of the α-chain amino acid sequence of C345-3 is mutated from T to C, and the corresponding α-chain nucleotide is mutated from ACT at positions 547, 548, and 549 to TGC; the 489th amino acid of the β-chain amino acid sequence of C345-3 is mutated from S to C, and the 507th amino acid is mutated from C to A. The corresponding β-chain nucleotide is mutated from TCC at positions 1465, 1466, and 1467 to TGC, and from TGC at positions 1519, 1520, and 1521 to GCA.

[0068] The 177th amino acid of the α-chain amino acid sequence of C345-4 is mutated from T to C, and the corresponding α-chain nucleotide is mutated from ACC at positions 529, 530, and 531 to TGC; the 494th amino acid of the β-chain amino acid sequence of C345-4 is mutated from S to C, and the 512th amino acid is mutated from C to A. The corresponding β-chain nucleotide is mutated from AGT at positions 1480, 1481, and 1482 to TGC, and from TGC at positions 1534, 1535, and 1536 to GCA.

[0069] The 183rd amino acid of the α-chain amino acid sequence of C345-7 is mutated from T to C, and the corresponding α-chain nucleotide is mutated from ACC at positions 547, 548, and 549 to TGC; the 489th amino acid of the β-chain amino acid sequence of C345-7 is mutated from S to C, and the 507th amino acid is mutated from C to A. The corresponding β-chain nucleotide is mutated from TCG at positions 1465, 1466, and 1467 to TGC, and from TGC at positions 1519, 1520, and 1521 to GCA.

[0070] The 183rd amino acid of the α-chain amino acid sequence of C345-8 is mutated from T to C, and the corresponding α-chain nucleotide is mutated from ACC at positions 547, 548, and 549 to TGC; the 489th amino acid of the β-chain amino acid sequence of C345-8 is mutated from S to C, and the 507th amino acid is mutated from C to A. The corresponding β-chain nucleotide is mutated from TCG at positions 1465, 1466, and 1467 to TGC, and from TGC at positions 1519, 1520, and 1521 to GCA.

[0071] The present invention also provides an expression vector. The expression vector includes the nucleic acid molecule as described above. The vector is any molecule or composition capable of carrying the nucleic acid sequence into a suitable host cell, in which the synthesis of the encoded protein can occur. The vector includes plasmids, phagemids, or particles for gene therapy.

[0072] The present invention also provides a recombinant cell, which comprises the nucleic acid molecule or the expression vector as described above, and the recombinant cell contains a nucleic acid molecule encoding the TCR as described above or a vector comprising the nucleic acid molecule.

[0073] The present invention also provides a drug combination, which comprises the TCR combination as described in any one of the above, or the isolated TCR as described above, or the nucleic acid molecule as described above, the expression vector as described above, the recombinant cell as described above. In addition to the active ingredients, the drug combination of the present invention may further comprise additional ingredients and be mixed with an acceptable carrier for use as a pharmaceutical composition for treating or alleviating diseases.

[0074] The present invention also provides the use of the TCR combination as described above, the isolated TCR as described above, the nucleic acid molecule as described above, the expression vector as described above, the recombinant cell as described above, or the pharmaceutical composition as described above in the preparation of a drug for preventing and / or treating hepatitis B virus and / or related diseases caused by hepatitis B virus.

[0075] Multiple TCR groups in the TCR combination provided by the present invention can specifically recognize multiple antigenic epitopes respectively, and can endow normal cells with reactivity to hepatitis B virus antigens, providing a new solution for the prevention and treatment of hepatitis B virus.

[0076] The technical solutions of the present invention will be further described in detail below in conjunction with specific examples and drawings. It should be understood that the following examples are only used to explain the present invention and are not intended to limit the present invention.

[0077] This study was approved by the Ethics Committee of the Third People's Hospital of Shenzhen (approval number: 2021-210), and the participants provided written informed consent for sample collection and subsequent analysis.

[0078] Experimental materials:

[0079] pHR-SFFV-IRES-EGFP: Addgene, 46911;

[0080] pMD2.G; Addgene, 12259;

[0081] pSPAX2: Addgene, 12260.

[0082] Example 1: Obtaining hepatitis B virus-specific TCR and lentivirus packaging

[0083] The present invention collects liver biopsy tissue samples from patients with chronic hepatitis B virus, enzymatically digests the tissue samples taken during surgery with collagenase and DNase, reacts at 37 degrees for half an hour, filters to obtain a cell suspension, and after centrifugation, resuspends the cells in DPBS containing 2% fetal bovine serum. A single cell is prepared using an instrument from 10X Genomics, and then PCR and library construction operations are performed according to the company's instructions. The constructed single cell library is subjected to second-generation sequencing, and the nucleotide and amino acid sequence information of TCR is obtained by analyzing the single cell sequencing results. The amino acid sequence of the variable region of the α chain of TCR is the sequence shown in SEQ ID NO.27 - 30, the nucleotide sequence of the variable region of the α chain is the sequence shown in SEQ ID NO.51 - 54, and the amino acid sequence of the α chain is the sequence shown in SEQ ID NO.35 - 38; the amino acid sequence of the variable region of the β chain of TCR is the sequence shown in SEQ ID NO.31 - 34, the nucleotide sequence of the variable region of the β chain is the sequence shown in SEQ ID NO.55 - 58, and the amino acid sequence of the β chain is the sequence shown in SEQ ID NO.39 - 42. The analysis results show that the amino acid sequences of CDR1, CDR2, and CDR3 of the α and β chains of TCR are in sequence:

[0084]

[0085] According to the sequencing information, the full-length sequences of the α and β chains of TCR (C345 - 3, C345 - 4, C345 - 7, and C345 - 8) are spliced. The full-length nucleotide sequence of the α chain is the sequence shown in SEQ ID NO.59 - 62, and the full-length nucleotide sequence of the β chain is the sequence shown in SEQ ID NO.63 - 66. The full-length nucleic acid sequence of the α chain of the spliced TCR and the full-length nucleic acid sequence of the β chain are ligated through T2A to form a TCRα - T2A - TCRβ nucleic acid fragment, and then the spliced TCRα - T2A - TCRβ nucleic acid fragment is codon-optimized and cloned into the lentiviral expression vector pHR - SFFV - IRES - EGFP by Nanjing GenScript Biotech Corporation to obtain the lentiviral vector pHR - SFFV - TCR - IRES - EGFP containing the TCR sequence of the present invention. The full-length nucleotide sequence of the α chain of the codon-optimized TCR is the sequence shown in SEQ ID NO.75 - 78, and the full-length nucleotide sequence of the β chain of the codon-optimized TCR is the sequence shown in SEQ ID NO.79 - 82. Using the second-generation lentiviral packaging system, three plasmids (pHR - SFFV - TCR - IRES - EGFP, and two plasmids pMD2.G and pSPAX2 containing other components necessary for constructing infectious but non-replicating lentiviral particles) are co-transfected into 293T cells to prepare lentivirus and concentrate the virus. The specific steps are described taking the lentivirus packaging experiment in a 175 cm 2 culture flask as an example:

[0086] Take 15×10 6 293T cells were cultured in a 175 cm 2 culture flask overnight at 37°C in a 5% CO2 incubator. When the cells reached approximately 60% confluence in the culture flask, transfection was performed; 1 hour before transfection, the cells were changed to fresh medium, and 30 mL of fresh complete DMEM medium was added; the plasmid dosage for each 175 cm 2 culture flask was as follows: 10 μg of pMD2.G, 30 μg of pSPAX2, and 40 μg of pHR-SFFV-C152-1-IRES-EGFP. The ratio of the transfection reagent PEI to the plasmid was 4:1, i.e., 320 μg; Solution A was prepared by adding the above three plasmids to 750 μL of serum-free DEME and vortexing well; Solution B was prepared by adding the corresponding PEI to 750 μL of serum-free DEME and vortexing well; Solution A and Solution B were mixed and vortexed thoroughly, then allowed to stand at room temperature for 20 minutes, and then added dropwise to the prepared 293T cells. The culture flask was gently shaken to mix the medium, and the cells were cultured at 37°C in a 5% CO2 incubator; 72 hours after transfection, the culture supernatant was collected and centrifuged (500 g, 4°C, 5 minutes). The centrifuged supernatant was filtered through a 0.45 μm filter, and finally 5×PEG8000 was added to concentrate the virus, and the mixture was mixed well at 4°C for 2 hours and then allowed to stand overnight at 4°C; after standing overnight, the mixture was centrifuged (4000 g, 4°C, 20 minutes), and the supernatant was completely discarded. The precipitate was dissolved in 200 μL of PBS, and the prepared lentivirus sample was stored at -80°C.

[0087] As a control, lentivirus packaging of empty pHR-SFFV-IRES-EGFP was performed simultaneously, and the packaging steps were the same as those of the lentivirus packaging of pHR-SFFV-TCR-IRES-EGFP described above.

[0088] Example 2: Specific identification of TCR and screening of hepatitis B virus antigen peptides

[0089] In the present invention, the T cell line Jurkat 76 (J76) with knocked-out TCR was modified by transducing the CD8 molecule and inserting a luciferase reporter gene downstream of NFAT, so that the specific activation level of the T cell line could be characterized by detecting the fluorescence signal after specific activation, which was beneficial for high-throughput identification of the specificity of TCR. This cell line was abbreviated as J76.8 cells. The TCRs (C345-3, C345-4, C345-7, and C345-8) of the present invention were expressed on the surface of the J76.8 cell line by lentivirus infection, and this cell was abbreviated as J76.8-TCR cells. The specific method was as follows: 1 mL of the lentivirus supernatant of the TCR packaged in Example 1 was used to infect the cell line before adding PEG8000 for concentration; the J76.8 cells were counted, and 1×10 5Put one J76.8 cell into one well of a 24-well plate; add 1 mL of the lentiviral supernatant of TCR, and centrifuge for infection (700 g, 32 °C, 1.5 hours); after centrifugation, supplement 1 mL of 1640 complete medium, and culture at 37 °C in 5% CO2 for 3 days; when the GFP positive rate and the CD3+ TCRαβ+ double positive rate detected by flow cytometry are above 90%, the construction of J76.8-TCR cells is successful.

[0090] The present invention simultaneously constructs lymphoblastoid cell lines (LCLs) homologous to TCR (C345-3, C345-4, C345-7, and C345-8) as antigen-presenting cells for specific identification and antigen peptide screening. The specific method is as follows: Culture B95-8 cells in 1640 medium with 2% FBS low-concentration serum for two weeks to produce the supernatant containing EBV virus; collect the supernatant containing EBV virus, centrifuge (500 g, 4 °C, 5 minutes) to remove cell debris, and then filter through a 0.45 μm sterile filter; resuscitate the peripheral mononuclear cells (PBMCs) from the patient with the source of TCR C152-1, and according to the standard of 1×10 6 cells / mL of virus supernatant, take the corresponding volume of EBV supernatant to resuspend the cells, and infect at 37 °C for 2 hours; centrifuge the infected cells (350 g, 4 °C, 5 minutes), remove the supernatant, and resuspend the cells at a density of 2×10 5 cells / mL with 1640 complete medium (containing 0.4 μg / mL cyclosporine A); resuspend the cells and culture them in a 48-well plate at a standard of 1 mL / well, culture at 37 °C in 5% CO2 for three weeks, change the medium by half amount every week, and supplement half of the fresh 1640 complete medium (containing 0.4 μg / mL cyclosporine A); after three weeks, the cells start to agglomerate and the lymphoblastoid cell line (LCL) is formed.

[0091] Co-incubate the constructed J76.8-TCR (C345-3, C345-4, C345-7, and C345-8) cells and LCL cells, and detect the fluorescence signal of J76.8-TCR cells under the stimulation of hepatitis B virus protein polypeptide to identify the specificity of TCR and the recognized antigen peptide. The specific method is as follows: Add 1×10 5 J76.8-TCR cells and 1×10 5A total of 100 homologous LCL cells were added with a hepatitis B virus peptide library at a concentration of 3.5 μg / mL (each polypeptide consisted of 15 amino acids, with 4 amino acids overlapping between each polypeptide and the previous one. The peptide library used covered the entire genome of the hepatitis B virus and was synthesized by Nanjing Genscript Biotech Co., Ltd.). The total volume in each well was 200 μL. Meanwhile, an unstimulated control group was set up. The cells were stimulated in an incubator at 37°C with 5% CO2 for 6 hours; then centrifuged at 600 g for 3 minutes, and the supernatant was discarded. Each well was resuspended with 50 μL of PBS and transferred to a 96-well white plate. Then 50 μL of luciferase detection substrate (a product of Novizan) was added, and the mixture was reacted for 3 minutes. The chemiluminescence was detected using an enzyme-labeling instrument.

[0092] The specific screening process was as follows: First, the peptide libraries of each hepatitis B virus protein were subjected to primary screening. Then, the large peptide libraries were subdivided into small peptide libraries containing 10 single peptides for further screening. Subsequently, the small peptide libraries that could activate TCR were further divided into single peptides for screening. Finally, the positive 15-amino acid single peptides were sequentially split into 9-amino acid single peptides for screening. Ultimately, the specific antigenic epitopes of TCR were confirmed as shown in SEQ ID NO.1-2.

[0093] The results were as Figure 1 and 2 shown. After the co-incubation experiment of J76.8-TCR and LCL, the hepatitis B virus proteins recognized by these TCRs were confirmed using the upregulated expression of CD69 as a marker. The abscissa represents the synthesized polypeptide libraries constituting the hepatitis B virus antigens (polymerase protein PLM, surface protein LS, core protein Core, and X protein). Among them, NS is the non-specific control; PLM1 and PLM2 are sub-libraries of PLM; anti-CD3 is antibody stimulation (also a positive control); PMA is a protein kinase A activator (also a positive control); the ordinate represents the proportion of the upregulated expression of CD69 molecules, which are activated by TCR-receiving signal cells, and this proportion of molecule expression is used as an index of the activation intensity of T cells under different stimulations. Among them, DMSO treatment was used as the negative control (NS), and PMA / ionomycin and Anti-CD3 antibody were used as positive controls. By comparing the reactivity of different TCRs under the same antigen stimulation, TCRs with higher reactivity to hepatitis B virus proteins could be screened out. The above four TCRs included 3 TCRs specific for the Core protein and 1 TCR specific for the S protein.

[0094] Then, Luciferase Assay was used to prove that J76.8-TCR cells were significantly activated under the stimulation of the hepatitis B virus peptide library. Further experiments proved that the epitopes causing the activation of these J76.8-TCR cells were polypeptides derived from the hepatitis B virus core protein and surface protein (Core32-10-1 and LS96-10-2), as Figures 3 - 6As shown, C345-3, C345-7 and C345-4 recognize LWFHISCLTF on the core protein (Core) of hepatitis B virus. C345-8 TCR recognizes the antigen peptide PFMPLLPIFF on the surface protein (HBsAg) of hepatitis B virus. Specifically, taking C345-3 as an example, a peptide library of Core protein was synthesized. First, a small peptide library was mixed in groups of ten polypeptides for experimental screening to confirm that the target peptide was present in Core31-40; then the individual polypeptides Core31 to Core40 were stimulated in turn to determine that the Core32 polypeptide had a signal, indicating that the epitope was present in this polypeptide; finally, based on the Core32 polypeptide (15 amino acids in length), polypeptides with a length of 9 amino acids or 10 amino acids covering this polypeptide were designed, and the polypeptide with the strongest stimulation signal was selected to confirm the final epitope Core32-10-1 (LWFHISCLTF), as Figure 3 shown.

[0095] Example 3: Identification of HLA Molecules Recognized by TCR

[0096] In Example 2, it has been identified that the TCRs (C345-3, C345-4, C345-7 and C345-8) of the present invention recognize hepatitis B virus antigen peptides, and the LCLs homologous to the TCRs include 6 HLA class I molecule types. It is necessary to further clarify the HLA molecule types that present the antigen peptides. To achieve this goal, in this Example 3, homologous LCL cells were co-incubated with J76.8-TCR cells, and the above antigen peptides were added for stimulation. Similarly, the activation level of cells was indicated by detecting fluorescence signals to identify the HLA molecule types that bind to the antigen peptides.

[0097] The results are as Figure 7 shown. Only when co-incubated with homologous LCL (Lymphoblastoid cell lines, an immortalized cell line prepared from patient-derived cells as an antigen-presenting cell) and K562 cells expressing HLA-A*24:02, J76.8-TCR cells will be activated under the stimulation of antigen peptides. The abscissa indicates the treatment with the above-identified epitope peptides and irrelevant control peptides, and the ordinate represents the multiple increase value of the downstream fluorescence signal after TCR activation compared with the DMSO treatment without polypeptides, suggesting specific activation. The antigen peptides recognized by these four TCRs are presented by HLA-A*24:02, as Figure 7 shown.

[0098] Example 4: Mutation of TCR Constant Region

[0099] To verify whether the exogenous transduction of TCR (C345-3, C345-4, C345-7, and C345-8) into T cells can endow normal cells with the function of secreting inflammatory cytokines and cytotoxic cytokines, the present invention needs to further transduce TCR into primary T cells for detection. To reduce the mismatch rate between the exogenous TCR and the TCR molecule of the cell itself during the transduction of primary T cells and improve the stability of the TCR molecule, an artificial disulfide bond was introduced between the constant regions of the α-chain and β-chain of the TCR molecule in this example. Specifically, the 48th threonine of the constant region TRAC*01 of the α-chain of TCR was mutated to cysteine, and the 57th serine of the constant region TRBC*02 of the β-chain of TCR was mutated to cysteine; in addition, the 75th cysteine of the constant region TRBC*02 of the β-chain of TCR was mutated to alanine to prevent the cysteine at this site from mismatching with the 48th cysteine of the mutated constant region TRAC*01 of the α-chain to form a disulfide bond, thereby ensuring the formation of an artificial disulfide bond between the 48th cysteine of the mutated constant region TRAC*01 of the α-chain and the 57th cysteine of the constant region TRBC*02 of the β-chain, and reducing the mismatch rate of TCR transduction into primary T cells. After site-directed mutagenesis, the full-length nucleotide sequence of the α-chain of TCR is the sequence shown in SEQ ID NO.67-70, the full-length amino acid sequence of the α-chain is the sequence shown in SEQ ID NO43-46, the full-length nucleotide sequence of the β-chain of TCR is the sequence shown in SEQ ID NO.71-74, and the full-length amino acid sequence of the β-chain is the sequence shown in SEQID NO.47-50.

[0100] Example 5: Transduction of TCR into primary T cells

[0101] In order to prove that the TCR has functional activity after recognizing the antigen epitope in the present invention, in this example, the method of lentivirus infection was used to transduce TCR onto the surface of primary T cells, and the transduction efficiency of TCR was identified by detecting the GFP expression carried by the lentiviral vector. The specific infection method is as follows: Take PBMC for resuscitation, centrifuge (400g, 4°C, 5 minutes), and count; according to the standard of 1×10 6 cells / mL of culture medium, add the corresponding volume of human serum medium to culture the cells in a suitable well plate; add 10 μL / mL of anti-CD3 + anti-CD28 activation mixture (product of Miltenyi Biotec) and 50 U / mL of IL-2 (product of PeproTech) to activate T cells; collect the primary T cells 24 hours after activation, centrifuge (400g, 4°C, 5 minutes), and resuspend and count with human serum medium; add 2.5×10 5Cells were divided into 48-well plates according to the standard of a single cell, and the packaged lentivirus of point-mutated TCR (345-3, 345-4, 345-7, and 345-8) was added to each well (the lentivirus packaging method was the same as in Example 1). Correspondingly, the packaged lentivirus of the blank vector was used as the control group. The volume of each well was supplemented to 500 μL with human serum medium, and centrifugal infection was carried out (1000 g, 32 °C, centrifugation for 1 hour). After centrifugation, 500 μL of human serum medium was added to each well, 50 U / mL IL-2 was added, and the cells were cultured at 37 °C in 5% CO2 for 3 days.

[0102] Example 6: Functional detection of TCR

[0103] To verify whether the 4 TCRs of the present invention can activate the transduced T cells by hepatitis B antigen peptides and produce corresponding inflammatory cytokines and cytotoxic cytokines after transducing primary T cells. In this example, TCRs were transduced into primary T cells by the method of Example 5, and then co-incubated with homologous LCLs. Under the stimulation of hepatitis B virus antigen peptides, the levels of cytokines were detected by flow cytometry.

[0104] The specific test method was as follows: 5×10 5 primary T cells transduced with TCR and 5×10 5 homologous LCL cells were added to each well of a 96-well plate, 0.01 μg / mL coronavirus antigen peptide, 1 μg / mL anti-CD28 antibody (product of Biolegend), and 3 μL / well of CD107a-PE / Cy7 flow antibody (product of Biolegend) were added. The total volume of each well was 200 μL. At the same time, an irrelevant peptide stimulation control group and a DMSO control group with equal amounts mixed were set up. After stimulation in a 37 °C, 5% CO2 incubator for 2 hours, a protein transport blocker (product of Biolegend) was added to block the secretion of cytokines after stimulation, and the cells were continued to be cultured for 18 hours. After centrifugation at 600 g for 3 minutes, the supernatant was discarded, and surface antibody staining was carried out. Staining was carried out at 4 °C in the dark for 30 minutes, and washed twice with PBS. After discarding the supernatant, 100 μL of permeabilization solution (product of BD) was added to each well, and the cells were permeabilized for 20 minutes, and washed twice with the supporting permeabilization wash solution. 50 μL of intracellular flow antibody prepared with the permeabilization wash solution was added to each well, and staining was carried out at 4 °C in the dark for 30 minutes, and washed twice with the permeabilization wash solution. The cells were resuspended in 200 μL of 2% paraformaldehyde, and detected by flow cytometry. Flow antibodies: CD3-BUV805, CD8-BUV737, CD4-BB700, Viability-eFluor506, Perforin-BV421, CD107a-PE / Cy7, TNF-PE-CF574, IFN-γ-APC, Granzyme-PE, IL-2-APC / Cy7.

[0105] The test results were as Figures 8 - 11As shown, in the figure, NS represents no stimulation, Unrelated peptide represents stimulation with an unrelated peptide, and specific peptide represents stimulation with a specific peptide. The specific peptides are the peptide segments screened above, such as Core32-10-1 and LS96-10-2 peptide segments. Compared with the non-stimulation control group and the unrelated antigen peptide control group, after stimulation with the corresponding antigen peptides, both enhanced the production of inflammatory cytokines IFN-γ and TNFα in T cells transducing the TCR of the present invention and increased the expression of the cytotoxic cytokine CD107a, demonstrating that these TCRs have functional activity and can endow normal T cells with reactivity to hepatitis B virus antigens, and are expected to become a new immunocyte therapy strategy for hepatitis B virus infection.

[0106] The above are only the preferred embodiments of the present invention and do not limit the patent scope of the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A TCR combination for identifying HBV epitopes, characterized in that, The TCR combination includes a plurality of isolated TCRs, and the plurality of isolated TCRs form two TCR groups, and the two TCR groups include a first TCR group and a second TCR group; wherein, The first TCR group specifically recognizes a first epitope, and the amino acid sequence of the first epitope is shown in SEQ ID NO: 1; The second TCR group specifically recognizes a second epitope, and the amino acid sequence of the second epitope is shown in SEQ ID NO:

2.

2. The TCR combination according to claim 1, wherein The first TCR group includes a first TCR α-chain variable region, a first TCR β-chain variable region, a second TCR α-chain variable region, a second TCR β-chain variable region, a third TCR α-chain variable region, and a third TCR β-chain variable region. Among them, the first TCR α-chain variable region includes a first CDR1α, a first CDR2α, and a first CDR3α; the first TCR β-chain variable region includes a first CDR1β, a first CDR2β, and a first CDR3β; the second TCR α-chain variable region includes a second CDR1α, a second CDR2α, and a second CDR3α; the second TCR β-chain variable region includes a second CDR1β, a second CDR2β, and a second CDR3β; the third TCR α-chain variable region includes a third CDR1α, a third CDR2α, and a third CDR3α; the third TCR β-chain variable region includes a third CDR1β, a third CDR2β, and a third CDR3β. The amino acid sequence of the first CDR1α is as shown in SEQ ID NO:3; the amino acid sequence of the first CDR2α is as shown in SEQ ID NO:4; the amino acid sequence of the first CDR3α is as shown in SEQ ID NO:5; the amino acid sequence of the first CDR1β is as shown in SEQ ID NO:6; the amino acid sequence of the first CDR2β is as shown in SEQ ID NO:7; the amino acid sequence of the first CDR3β is as shown in SEQ ID NO:8; the amino acid sequence of the second CDR1α is as shown in SEQ ID NO:9; the amino acid sequence of the second CDR2α is as shown in SEQ ID NO:10; the amino acid sequence of the second CDR3α is as shown in SEQ ID NO:11; the amino acid sequence of the second CDR1β is as shown in SEQ ID NO:12; the amino acid sequence of the second CDR2β is as shown in SEQ ID NO:13; the amino acid sequence of the second CDR3β is as shown in SEQ ID NO:14; the amino acid sequence of the third CDR1α is as shown in SEQ ID NO:15; the amino acid sequence of the third CDR2α is as shown in SEQ ID NO:16; the amino acid sequence of the third CDR3α is as shown in SEQ ID NO:17; the amino acid sequence of the third CDR1β is as shown in SEQ ID NO:18; the amino acid sequence of the third CDR2β is as shown in SEQ ID NO:19; the amino acid sequence of the third CDR3β is as shown in SEQ ID NO:20; and / or, The second TCR group includes a fourth TCR α-chain variable region and a fourth TCR β-chain variable region. The fourth TCR α-chain variable region includes a fourth CDR1α, a fourth CDR2α, and a fourth CDR3α. The fourth TCR β-chain variable region includes a fourth CDR1β, a fourth CDR2β, and a fourth CDR3β. The amino acid sequence of the fourth CDR1α is as shown in SEQ ID NO:

21. The amino acid sequence of the fourth CDR2α is as shown in SEQ ID NO:

22. The amino acid sequence of the fourth CDR3α is as shown in SEQ ID NO:

23. The amino acid sequence of the fourth CDR1β is as shown in SEQ ID NO:

24. The amino acid sequence of the fourth CDR2β is as shown in SEQ ID NO:

25. The amino acid sequence of the fourth CDR3β is as shown in SEQ ID NO:

26.

3. The TCR combination according to claim 1, characterized in that The multiple isolated TCRs form four TCRs, wherein, each TCR includes a TCR α-chain variable region and a TCR β-chain variable region. The amino acid sequences of the four TCR α-chain variable regions are as shown in SEQ ID NO:27 to SEQ ID NO:

30. The amino acid sequences of the four TCR β-chain variable regions are as shown in SEQ ID NO:31 to SEQ ID NO:34; and / or, each TCR includes a TCR α-chain and a TCR β-chain. The amino acid sequences of the four TCR α-chains are as shown in SEQ ID NO:35 to SEQ ID NO:

38. The amino acid sequences of the four TCR β-chains are as shown in SEQ ID NO:39 to SEQ ID NO:42; and / or, each TCR includes a TCR α-mutant chain and a TCR β-mutant chain. The amino acid sequences of the four TCR α-mutant chains are as shown in SEQ ID NO:43 to SEQ ID NO:

46. The amino acid sequences of the four TCR β-mutant chains are as shown in SEQ ID NO:47 to SEQ ID NO:

50.

4. A nucleic acid molecule combination, characterized in that, The nucleic acid molecule combination includes multiple nucleic acid molecules. The multiple nucleic acid molecules form four TCRs, wherein, each TCR further includes a TCR α-chain variable region and a TCR β-chain variable region. The nucleotide sequences of the four TCR α-chain variable regions are as shown in SEQ ID NO:51 to SEQ ID NO:

54. The nucleotide sequences of the four TCR β-chain variable regions are as shown in SEQ ID NO:55 to SEQ ID NO:58; and / or, each TCR further includes a TCR α-chain and a TCR β-chain. The nucleotide sequences of the four TCR α-chains are as shown in SEQ ID NO:59 to SEQ ID NO:

62. The nucleotide sequences of the four TCR β-chains are as shown in SEQ ID NO:63 to SEQ ID NO:66; and / or, Each TCR also includes a TCRα mutant chain and a TCRβ mutant chain. The nucleotide sequences of the four TCRα mutant chains are shown in SEQ ID NO: 67 to SEQ ID NO: 70, and the nucleotide sequences of the four TCRβ mutant chains are shown in SEQ ID NO: 71 to SEQ ID NO: 74; and / or, Each TCR also includes nucleotides with optimized codons for the α chain and nucleotides with optimized codons for the β chain. The nucleotide sequences of the four nucleotides with optimized codons for the α chain are shown in SEQ ID NO: 75 to SEQ ID NO: 78, and the nucleotide sequences of the four nucleotides with optimized codons for the β chain are shown in SEQ ID NO: 79 to SEQ ID NO:

82.

5. An expression vector, characterized in that, The expression vector includes the nucleic acid molecules in the nucleic acid molecule combination as claimed in claim 4.

6. A recombinant cell, characterized in that, The recombinant cell includes the nucleic acid molecules in the nucleic acid molecule combination as claimed in claim 4 or the expression vector as claimed in claim 5.

7. A drug combination, characterized in that, Includes the nucleic acid molecules in the nucleic acid molecule combination as claimed in claim 4, or the expression vector as claimed in claim 5, or the recombinant cell as claimed in claim 6.

8. Use of a nucleic acid molecule in the nucleic acid molecule combination as claimed in claim 4, or the expression vector as claimed in claim 5, or the recombinant cell as claimed in claim 6, or the pharmaceutical composition as claimed in claim 7 in the preparation of a drug for preventing and / or treating hepatitis B virus and / or related diseases caused by hepatitis B virus.

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