TCR combination for recognizing hepatitis B virus antigen and application

Abstract

The invention relates to the field of immunology, and particularly discloses a TCR combination for recognizing hepatitis B virus antigens and application, the TCR combination for recognizing hepatitis B virus antigens comprises a plurality of separated TCRs, the plurality of separated TCR combinations for recognizing hepatitis B virus antigens form four TCR groups, and the four TCR groups comprise a first TCR group, a second TCR group, a third TCR group and a fourth 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 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; the third TCR group specifically recognizes a third antigen epitope, and the amino acid sequence of the third antigen epitope is as shown in SEQ ID NO: 3; the fourth TCR group specifically recognizes a fourth antigen epitope, the amino acid sequence of the fourth antigen epitope is as shown in SEQ ID NO: 4, 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 application relates to the field of immunology, in particular to a TCR combination for recognizing hepatitis B virus antigen and application thereof. BACKGROUND

[0002] Studies have shown that the virus surface antigen and virus DNA levels in the body of chronic hepatitis B virus (HBV) infected persons are inversely proportional to the proportion of hepatitis B virus specific CD8+ T cells, which implies the antiviral effect of virus specific CD8+ T cells. Therefore, the isolation and identification of virus specific CD8+ T cells have important roles in the prevention and treatment of chronic hepatitis B virus infection and hepatitis B infection related liver cancer.

[0003] In the process of viral infection, antigen presenting cells process viral antigens into antigen short peptides and combine with HLA molecules to form stable antigen peptide-MHC complexes expressed on the cell membrane. Specific T cells combine with them through the surface T cell receptor (TCR), and under the coordination of co-stimulatory signals, a series of signal transduction and physiological functions are caused to achieve the effect of clearing viruses. The TCR-T adoptive cell therapy mainly converts the patient's autologous cells into specific TCRs that recognize hepatitis B virus antigens in vitro, and then returns them to the patient's body, so as to achieve the purpose of efficiently clearing virus infected cells. The hepatitis B virus specific TCR transduced to the surface of healthy human T cells can endow normal T cells with reactivity to hepatitis B virus antigens. Therefore, screening and identifying hepatitis B virus specific TCR and its epitope can provide an important means for TCR-T immune cell therapy of hepatitis B virus infection. SUMMARY

[0004] The main purpose of the present application is to provide a TCR combination for recognizing hepatitis B virus antigen and application thereof, aiming to provide a TCR combination for recognizing hepatitis B virus antigen. The TCR in the combination can specifically recognize and combine with the hepatitis B virus antigen epitope, thereby providing an important basis for TCR-T immune cell therapy of hepatitis B virus infection.

[0005] To achieve the above purpose, the present application provides a TCR combination for recognizing hepatitis B virus antigen, which is used for recognizing HBV antigen epitope. The TCR combination for recognizing hepatitis B virus antigen comprises a plurality of isolated TCRs. The plurality of isolated TCR combinations for recognizing hepatitis B virus antigen form four TCR groups, which comprise a first TCR group, a second TCR group, a third TCR group and a fourth TCR group. Wherein, The first TCR group specifically recognizes a first antigen epitope. The amino acid sequence of the first antigen epitope is shown in SEQ ID NO: 1. the second TCR group specifically recognizes a second antigenic epitope, an amino acid sequence of the second antigenic epitope is set forth in SEQ ID NO: 2; the third TCR group specifically recognizes a third antigenic epitope, an amino acid sequence of the third antigenic epitope is set forth in SEQ ID NO: 3; the fourth TCR group specifically recognizes a fourth antigenic epitope, an amino acid sequence of the fourth antigenic epitope is set forth in SEQ ID NO: 4.

[0006] In some embodiments, the first TCR group, comprising a first TCR a chain variable region and a first TCR β chain variable region, the first TCR a chain variable region comprising a first CDR1a, a first CDR2a and a first CDR3a, the first TCR β chain variable region comprising a first CDR1β, a first CDR2β and a first CDR3β, an amino acid sequence of the first CDR1a is set forth in SEQ ID NO: 5, an amino acid sequence of the first CDR2a is set forth in SEQ ID NO: 6, an amino acid sequence of the first CDR3a is set forth in SEQ ID NO: 7, an amino acid sequence of the first CDR1β is set forth in SEQ ID NO: 8, an amino acid sequence of the first CDR2β is set forth in SEQ ID NO: 9, and an amino acid sequence of the first CDR3β is set forth in SEQ ID NO: 10; and / or, the second TCR group, comprising a second TCR a chain variable region and a second TCR β chain variable region, the second TCR a chain variable region comprising a second CDR1a, a second CDR2a and a second CDR3a, the second TCR β chain variable region comprising a second CDR1β, a second CDR2β and a second CDR3β, an amino acid sequence of the second CDR1a is set forth in SEQ ID NO: 11, an amino acid sequence of the second CDR2a is set forth in SEQ ID NO: 12, an amino acid sequence of the second CDR3a is set forth in SEQ ID NO: 13, an amino acid sequence of the second CDR1β is set forth in SEQ ID NO: 14, an amino acid sequence of the second CDR2β is set forth in SEQ ID NO: 15, and an amino acid sequence of the second CDR3β is set forth in SEQ ID NO: 16; and / or, the third TCR group comprises a third TCR a chain variable region and a third TCR β chain variable region, the third TCR a chain variable region comprises a third CDR1a, a third CDR2a and a third CDR3a, the third TCR β chain variable region comprises a third CDR1β, a third CDR2β and a third CDR3β, the amino acid sequence of the third CDR1a is shown as SEQ ID NO: 17, the amino acid sequence of the third CDR2a is shown as SEQ ID NO: 18, the amino acid sequence of the third CDR3a is shown as SEQ ID NO: 19, the amino acid sequence of the third CDR1β is shown as SEQ ID NO: 20, the amino acid sequence of the third CDR2β is shown as SEQ ID NO: 21, and the amino acid sequence of the third CDR3β is shown as SEQ ID NO: 22; and / or, the fourth TCR group comprises a fourth TCR a chain variable region and a fourth TCR β chain variable region, the fourth TCR a chain variable region comprises a fourth CDR1a, a fourth CDR2a and a fourth CDR3a, the fourth TCR β chain variable region comprises a fourth CDR1β, a fourth CDR2β and a fourth CDR3β, the amino acid sequence of the fourth CDR1a is shown as SEQ ID NO: 23, the amino acid sequence of the fourth CDR2a is shown as SEQ ID NO: 24, the amino acid sequence of the fourth CDR3a is shown as SEQ ID NO: 25, the amino acid sequence of the fourth CDR1β is shown as SEQ ID NO: 26, the amino acid sequence of the fourth CDR2β is shown as SEQ ID NO: 27, and the amino acid sequence of the fourth CDR3β is shown as SEQ ID NO: 28.

[0007] In some embodiments, the plurality of isolated TCRs form four TCRs, wherein, each TCR comprises a TCR a chain variable region and a TCR β chain variable region, the amino acid sequences of the four TCR a chain variable regions are shown as SEQ ID NO: 29 to SEQ ID NO: 32, and the amino acid sequences of the four TCR β chain variable regions are shown as SEQ ID NO: 33 to SEQ ID NO: 36; and / or, each TCR comprises a TCR a chain and a TCR β chain, the amino acid sequences of the four TCR a chains are shown as SEQ ID NO: 37 to SEQ ID NO: 40, and the amino acid sequences of the four TCR β chains are shown as SEQ ID NO: 41 to SEQ ID NO: 44; and / or, each TCR comprises a TCR a mutant chain and a TCR β mutant chain, the amino acid of the four TCR a mutant chains and the amino acid of the four TCR β mutant chains.

[0008] The present application provides a nucleic acid molecule encoding any one of the TCR combinations recognizing hepatitis B virus antigen as claimed in any one of the above, the nucleic acid molecule corresponding to encoding any one of the TCR combinations recognizing hepatitis B virus antigen comprises a plurality of, wherein, each TCR comprises a TCR alpha chain variable region and a TCR beta chain variable region, the nucleotide sequences of the four TCR alpha chain variable regions are as shown in SEQ ID NO: 53 to SEQ ID NO: 56, and the nucleotide sequences of the four TCR beta chain variable regions are as shown in SEQ ID NO: 57 to SEQ ID NO: 60; and / or, each TCR comprises a TCR alpha chain and a TCR beta chain, the nucleotide sequences of the four TCR alpha chains are as shown in SEQ ID NO: 61 to SEQ ID NO: 64, and the nucleotide sequences of the four TCR beta chains are as shown in SEQ ID NO: 65 to SEQ ID NO: 68; and / or, each TCR comprises a TCR alpha mutant chain and a TCR beta mutant chain, the nucleotide sequences of the four TCR alpha mutant chains are as shown in SEQ ID NO: 69 to SEQ ID NO: 72, and the nucleotide sequences of the four TCR beta mutant chains are as shown in SEQ ID NO: 73 to SEQ ID NO: 76; and / or, each TCR comprises alpha chain codon-optimized nucleotides and beta chain codon-optimized nucleotides, the nucleotide sequences of the four alpha chain codon-optimized nucleotides are as shown in SEQ ID NO: 77 to SEQ ID NO: 80, and the nucleotide sequences of the four beta chain codon-optimized nucleotides are as shown in SEQ ID NO: 81 to SEQ ID NO: 84.

[0009] The present application also provides an expression vector comprising the nucleic acid molecule as described above.

[0010] The present application also provides a recombinant cell comprising the nucleic acid molecule as described above or the expression vector as described above.

[0011] The present application also provides a pharmaceutical combination comprising the TCR combination recognizing hepatitis B virus antigen as claimed in any one of the above or the isolated TCR as claimed above or the nucleic acid molecule as claimed above, the expression vector as claimed above, the recombinant cell as claimed above.

[0012] The application also provides use of the TCR combination for recognizing hepatitis B virus antigen, the isolated TCR, the nucleic acid molecule, the expression vector, the recombinant cell or the pharmaceutical composition in the preparation of a medicine for preventing and / or treating hepatitis B virus and / or diseases caused by hepatitis B virus.

[0013] The TCR combination for recognizing hepatitis B virus antigen provided by the application can specifically recognize multiple antigen epitopes, and can endow normal cells with reactivity to hepatitis B virus antigen, thereby providing a new solution for the prevention and treatment of hepatitis B virus. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description only illustrate some embodiments of the application, and other related drawings can be obtained by those skilled in the art without any creative effort.

[0015] Figure 1 Figure for identification results of hepatitis B virus specific TCR (C337-2, C341-7) in Example 1; Figure 2 Figure for identification results of hepatitis B virus specific TCR (C342-6, C345-5) in Example 1; Figure 3 Figure for detection results of polypeptide epitopes identified by TCR (C337-2) in Example 1 under the condition of epitope presentation by homologous LCL cells; Figure 4 Figure for detection results of polypeptide epitopes identified by TCR (C341-7) in Example 1 under the condition of epitope presentation by homologous LCL cells; Figure 5 Figure for detection results of polypeptide epitopes identified by TCR (C342-6) in Example 1 under the condition of epitope presentation by homologous LCL cells; Figure 6 Figure for detection results of polypeptide epitopes identified by TCR (C345-5) in Example 1 under the condition of epitope presentation by homologous LCL cells; Figure 7 Figure for identification of HLA molecules recognized by TCR (C337-2) in Example 1; Figure 8 Figure for identification of HLA molecules recognized by TCR (C341-7) in Example 1; Figure 9Figure for identifying the HLA molecule recognized by the TCR (C342-6) in Example 1; Figure 10 Figure for identifying the HLA molecule recognized by the TCR (C345-5) in Example 1; Figure 11 Figure for functional verification of the TCR (C337-2) in Example 1; Figure 12 Figure for functional verification of the TCR (C341-7) in Example 1; Figure 13 Figure for functional verification of the TCR (C342-6) in Example 1; Figure 14 Figure for functional verification of the TCR (C345-5) in Example 1.

[0016] The purposes, functional features and advantages of the present application will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION

[0017] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments.

[0018] It should be noted that, in the embodiments, the specific conditions not mentioned are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments used are not marked with the manufacturers, and are all the conventional products that can be obtained by market purchase. In addition, the meaning of "and / or" appearing in the whole text includes three parallel solutions, for example, "A and / or B" includes the solution of A, or the solution of B, or the solution of A and B at the same time. In addition, the technical solutions of each embodiment can be combined with each other, but it must be based on the premise that the ordinary skilled in the art can realize it, when the combination of technical solutions appears contradictory or unachievable, it should be considered that the combination of technical solutions does not exist, and is not within the protection scope required by the present application. Based on the embodiments in the present application, all other embodiments obtained by the ordinary skilled in the art without creative labor are within the protection scope of the present application.

[0019] In the process of viral infection, antigen presenting cells process viral antigens into short antigen peptides, and combine with HLA molecules to form stable antigen peptide-MHC complexes expressed on the cell membrane, and specific T cells bind to them through the surface T cell receptor (TCR), under the synergistic effect of costimulatory signals, causing a series of signal transduction and physiological functions, and achieving the effect of clearing viruses. The TCR-T adoptive cell therapy mainly converts the patient's autologous cells into TCRs that specifically recognize hepatitis B virus antigens in vitro, and then reinfuses them into the patient's body, thereby achieving the purpose of efficiently clearing virus-infected cells. The hepatitis B virus-specific TCR transduced to the surface of healthy human T cells can endow normal T cells with reactivity to hepatitis B virus antigens, therefore, screening and identifying hepatitis B virus-specific TCRs and their epitopes can provide an important means for TCR-T immune cell therapy for hepatitis B virus infection.

[0020] In view of this, the present application provides a TCR combination for recognizing hepatitis B virus antigens and application thereof, which is used for recognizing HBV antigen epitopes, and the TCR combination for recognizing hepatitis B virus antigens comprises a plurality of isolated TCRs, the plurality of isolated TCR combinations for recognizing hepatitis B virus antigens form four TCR groups, and the four TCR groups comprise a first TCR group, a second TCR group, a third TCR group and a fourth TCR group. The first TCR group specifically recognizes a first antigen epitope, and the amino acid sequence of the first antigen epitope is shown as SEQ ID NO: 1. The second TCR group specifically recognizes a second antigen epitope, and the amino acid sequence of the second antigen epitope is shown as SEQ ID NO: 2. The third TCR group specifically recognizes a third antigen epitope, and the amino acid sequence of the third antigen epitope is shown as SEQ ID NO: 3. The fourth TCR group specifically recognizes a fourth antigen epitope, and the amino acid sequence of the fourth antigen epitope is shown as SEQ ID NO: 4.

[0021] The present application aims at the above four antigen epitopes, and obtains a TCR combination for recognizing hepatitis B virus antigens that can specifically recognize HBV virus. The TCRs in the combination can specifically recognize and target bind to hepatitis B virus antigen epitopes, thereby providing an important basis for TCR-T immune cell therapy for hepatitis B virus infection.

[0022] Specifically, the amino acid sequence of the first antigen epitope is shown as SEQ ID NO: 1, and is specifically represented as: FGRETVLEY-HLA-B*46:01; the amino acid sequence of the second antigen epitope is shown as SEQ ID NO: 2, and is specifically represented as: FAVPNLQSL-HLA-C*08:22; the amino acid sequence of the third antigen epitope is shown as SEQ ID NO: 3, and is specifically represented as: RETVLEYLV-HLA-B*13:02; and the amino acid sequence of the fourth antigen epitope is shown as SEQ ID NO: 4, and is specifically represented as: GRETVLEYL-HLA-B*40:01.

[0023] It should be noted that, taking FGRETVLEY-HLA-B*46:01 as an example, FGRETVLEY is the peptide segment of the antigen epitope, and HLA-B*46:01 is a subtype of the HLA molecule, and HLA-B belongs to a category of HLA family.

[0024] It should be further noted that the T cell receptor (TCR) is a membrane protein on the surface of T cells, which can recognize the antigen short peptide on the surface of the corresponding target cell, and the human leukocyte antigen HLA, commonly known as the MHC molecule, is a class of genes on the short arm of chromosome 6, and the HLA of each person is different, which is an important molecular marker for recognizing human leukocytes. In the immune system, the specific TCR of the antigen short peptide binds to the short peptide-major histocompatibility antigen (pMHC) complex to initiate direct physical contact between T cells and antigen-presenting cells (APC), and then other cell membrane surface molecules of T cells and APC interact to cause a series of subsequent cell signal transmission and other physiological reactions, so that different antigen-specific T cells exert immune effects on their target cells.

[0025] pMHC (Peptide-Major Histocompatibility Complex) and TCR (T-cell Receptor) are important components in the immune system.

[0026] 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 display antigen peptides to T cells.

[0027] It should be noted that each TCR includes a TCRa chain and a TCRP chain, and at least one of the TCRa chain or the TCRP chain includes a variable region, the variable region is the main function of being able to specifically bind to an antigen, the region performing the function is called a complementarity determining region (CDR), and the CDR refers to three sequences in the variable region of an antibody, which are CDR1, CDR3 and CDR3, which together constitute a part of the antigen binding site, determine specificity, and are responsible for recognizing and binding to an antigen, thereby exerting an immune effect.

[0028] In some embodiments, the first TCR group includes a first TCRa chain variable region and a first TCRP chain variable region, the first TCRa chain variable region includes a first CDR1a, a first CDR2a and a first CDR3a, the first TCRP chain variable region includes a first CDR1P, a first CDR2P and a first CDR3P, the amino acid sequence of the first CDR1a is shown as SEQ ID NO: 5, the amino acid sequence of the first CDR2a is shown as SEQ ID NO: 6, the amino acid sequence of the first CDR3a is shown as SEQ ID NO: 7, the amino acid sequence of the first CDR1P is shown as SEQ ID NO: 8, the amino acid sequence of the first CDR2P is shown as SEQ ID NO: 9, and the amino acid sequence of the first CDR3P is shown as SEQ ID NO: 10.

[0029] The second TCR group includes a second TCRa chain variable region and a second TCRP chain variable region, the second TCRa chain variable region includes a second CDR1a, a second CDR2a and a second CDR3a, the second TCRP chain variable region includes a second CDR1P, a second CDR2P and a second CDR3P, the amino acid sequence of the second CDR1a is shown as SEQ ID NO: 11, the amino acid sequence of the second CDR2a is shown as SEQ ID NO: 12, the amino acid sequence of the second CDR3a is shown as SEQ ID NO: 13, the amino acid sequence of the second CDR1P is shown as SEQ ID NO: 14, the amino acid sequence of the second CDR2P is shown as SEQ ID NO: 15, and the amino acid sequence of the second CDR3P is shown as SEQ ID NO: 16.

[0030] the third CDR1a has the amino acid sequence of SEQ ID NO: 17, the third CDR2a has the amino acid sequence of SEQ ID NO: 18, the third CDR3a has the amino acid sequence of SEQ ID NO: 19, the third CDR1b has the amino acid sequence of SEQ ID NO: 20, the third CDR2b has the amino acid sequence of SEQ ID NO: 21, and the third CDR3b has the amino acid sequence of SEQ ID NO: 22.

[0031] the fourth CDR1a has the amino acid sequence of SEQ ID NO: 23, the fourth CDR2a has the amino acid sequence of SEQ ID NO: 24, the fourth CDR3a has the amino acid sequence of SEQ ID NO: 25, the fourth CDR1b has the amino acid sequence of SEQ ID NO: 26, the fourth CDR2b has the amino acid sequence of SEQ ID NO: 27, and the fourth CDR3b has the amino acid sequence of SEQ ID NO: 28.

[0032] Specifically, the identified hepatitis B virus specific TCRs in the application include four groups of TCRs, C337-2, C341-7, C342-6 and C345-5, wherein, C337-2 can bind to FGRETVLEY-HLA-B*46:01 complex (first antigen epitope), C341-7 can bind to FAVPNLQSL-HLA-C*08:22 complex (second antigen epitope), C342-6 can bind to RETVLEYLV-HLA-B*13:02 complex (third antigen epitope), and C345-5 can bind to GRETVLEYL-HLA-B*40:01 complex (fourth antigen epitope), and the TCRs can endow normal cells with specific response to hepatitis B virus antigen after being transduced into primary T cells, thereby providing a new strategy for the prevention and treatment of hepatitis B virus infection. The specific sequences of CDRs in the variable region of alpha chain and the variable region of beta chain of the above-mentioned TCRs are shown in Table 1, wherein the first TCR group (C337-2) includes a first TCR alpha chain variable region and a first TCR beta chain variable region, the first TCR alpha chain variable region includes a first CDR1 alpha, a first CDR2 alpha and a first CDR3 alpha, and the first TCR beta chain variable region includes a first CDR1 beta, a first CDR2 beta and a first CDR3 beta, and the second TCR group (C341-7) includes a second TCR alpha chain variable region and a second TCR beta chain variable region, the second TCR alpha chain variable region includes a second CDR1 alpha, a second CDR2 alpha and a second CDR3 alpha, and the second TCR beta chain variable region includes a second CDR1 beta, a second CDR2 beta and a second CDR3 beta, the third TCR group (C342-6) includes a third TCR alpha chain variable region and a third TCR beta chain variable region, the third TCR alpha chain variable region includes a third CDR1 alpha, a third CDR2 alpha and a third CDR3 alpha, and the third TCR beta chain variable region includes a third CDR1 beta, a third CDR2 beta and a third CDR3 beta, and the fourth TCR group (C345-5) includes a fourth TCR alpha chain variable region and a fourth TCR beta chain variable region, the fourth TCR alpha chain variable region includes a fourth CDR1 alpha, a fourth CDR2 alpha and a fourth CDR3 alpha, and the fourth TCR beta chain variable region includes a fourth CDR1 beta, a fourth CDR2 beta and a fourth CDR3 beta. The application analyzes the single cell sequencing data of liver puncture samples of patients with chronic hepatitis B virus infection, selects a number of TCR clones for in vitro synthesis and insertion into a lentivirus vector, constructs a T cell line stably expressing TCR by lentivirus infection, stimulates with hepatitis B virus polypeptide, identifies hepatitis B virus specific TCR by detecting the activation level of cells, and identifies the antigen peptide and HLA molecule recognized by the TCR, i.e. the four antigen epitopes described in the application. The specific sequences of CDRs in the variable region of alpha chain and the variable region of beta chain of the above-mentioned TCRs are shown in Table 1, i.e. SEQ ID NO: 5-28 as shown in Table 1. Table 1

[0033] In some embodiments, the four TCRs are αβ heterodimers, which comprise the constant region of the α chain TRAC*01 and the constant region of the β chain TRBC*02 of the TCR, the sequences of the constant region of the TCR can be found in the public database of International Immunogenetics Information System (IMGT), such as the constant region sequence of the α chain of the TCR molecule is “TRAC*01”, the constant region sequence of the β chain of the TCR molecule is “TRBC1*01” or “TRBC2*01”, wherein, an artificial disulfide bond is introduced between the α chain and the β chain constant region of the TCR of the application, the threonine at position 48 of TRAC*01 and the serine at position 57 of TRBC*02 are mutated into cysteine; in addition, the cysteine at position 75 of TRBC*02 is mutated into alanine.

[0034] It should be noted that the sequences shown as SEQ ID NO: 1 to SEQ ID NO: 28 above are not shown in the sequence listing because the length of the gene sequence is not long, and the mutant sequence can be recorded in the specification in text form, and all other sequences are shown in the sequence listing, the name of the sequence corresponds to the sequence number, which may not be consistent, for example, the amino acid sequence of the TCR α chain variable region of C337-2 is shown as SEQ ID NO: 29, but the sequence number in the sequence listing is 1, and the like.

[0035] In some embodiments, the plurality of isolated TCRs form four TCRs, wherein, Each TCR comprises 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 shown as SEQ ID NO: 29 to SEQ ID NO: 32, specifically, the amino acid sequences of the TCR α chain variable regions of C337-2, C341-7, C342-6 and C345-5 are shown as SEQ ID NO: 29 to SEQ ID NO: 32 in turn, the amino acid sequences of the four TCR β chain variable regions are shown as SEQ ID NO: 33 to SEQ ID NO: 36; specifically, the amino acid sequences of the TCR β chain variable regions of C337-2, C341-7, C342-6 and C345-5 are shown as SEQ ID NO: 33 to SEQ ID NO: 36 in turn.

[0036] Each of the TCRs comprises a TCR a chain and a TCR β chain, the amino acid sequences of the four TCR a chains are shown as SEQ ID NO: 37 to SEQ ID NO: 40, specifically, the amino acid sequences of the TCR a chains of C337-2, C341-7, C342-6 and C345-5 are shown as SEQ ID NO: 37 to SEQ ID NO: 40 in turn, the amino acid sequences of the four TCR β chains are shown as SEQ ID NO: 41 to SEQ ID NO: 44, specifically, the amino acid sequences of the TCR β chains of C337-2, C341-7, C342-6 and C345-5 are shown as SEQ ID NO: 41 to SEQ ID NO: 44 in turn.

[0037] Each of the TCRs comprises a TCR a chain and a TCR β chain, the amino acid sequences of the four TCR a chains are shown as SEQ ID NO: 37 to SEQ ID NO: 40, specifically, the amino acid sequences of the TCR a chains of C337-2, C341-7, C342-6 and C345-5 are shown as SEQ ID NO: 37 to SEQ ID NO: 40 in turn, the amino acid sequences of the four TCR β chains are shown as SEQ ID NO: 41 to SEQ ID NO: 44, specifically, the amino acid sequences of the TCR β chains of C337-2, C341-7, C342-6 and C345-5 are shown as SEQ ID NO: 41 to SEQ ID NO: 44 in turn.

[0038] The present application also provides a nucleic acid molecule encoding any of the TCRs in the TCR combination for recognizing the hepatitis B virus antigen as described in any of the above, the nucleic acid molecule corresponding to the nucleic acid molecule encoding any of the TCRs in the TCR combination for recognizing the hepatitis B virus antigen comprises a plurality of, wherein, Each of the TCRs comprises a TCR a chain and a TCR β chain, the amino acid sequences of the four TCR a chains are shown as SEQ ID NO: 37 to SEQ ID NO: 40, specifically, the amino acid sequences of the TCR a chains of C337-2, C341-7, C342-6 and C345-5 are shown as SEQ ID NO: 37 to SEQ ID NO: 40 in turn, the amino acid sequences of the four TCR β chains are shown as SEQ ID NO: 41 to SEQ ID NO: 44, specifically, the amino acid sequences of the TCR β chains of C337-2, C341-7, C342-6 and C345-5 are shown as SEQ ID NO: 41 to SEQ ID NO: 44 in turn.

[0039] The nucleotide sequences of the four TCR β chain variable regions are shown as SEQ ID NO: 57 to SEQ ID NO: 60, specifically, the nucleotide sequences of the TCR β chain variable regions of C337-2, C341-7, C342-6 and C345-5 are shown as SEQ ID NO: 57 to SEQ ID NO: 60 in turn.

[0040] Each TCR comprises a TCR a chain and a TCR β chain, the nucleotide sequence of four of the TCR a chains is shown as SEQ ID NO: 61 to SEQ ID NO: 64, in particular, the nucleotide sequence of the TCR a chain of C337-2, C341-7, C342-6 and C345-5 is shown as SEQ ID NO: 61 to SEQ ID NO: 64. The nucleotide sequence of four of the TCR β chains is shown as SEQ ID NO: 65 to SEQ ID NO: 68, in particular, the nucleotide sequence of the TCR β chain of C337-2, C341-7, C342-6 and C345-5 is shown as SEQ ID NO: 65 to SEQ ID NO: 68.

[0041] Each TCR comprises a TCR a mutant chain and a TCR β mutant chain, the nucleotide sequence of four of the TCR a mutant chains is shown as SEQ ID NO: 69 to SEQ ID NO: 72, in particular, the nucleotide sequence of the TCR a mutant chain of C337-2, C341-7, C342-6 and C345-5 is shown as SEQ ID NO: 69 to SEQ ID NO: 72. The nucleotide sequence of four of the TCR β mutant chains is shown as SEQ ID NO: 73 to SEQ ID NO: 76, in particular, the nucleotide sequence of the TCR β mutant chain of C337-2, C342-6 and C345-5 is shown as SEQ ID NO: 73 to SEQ ID NO: 76.

[0042] Each TCR comprises a TCR a mutant chain and a TCR β mutant chain, the nucleotide sequence of four of the TCR a mutant chains is shown as SEQ ID NO: 69 to SEQ ID NO: 72, in particular, the nucleotide sequence of the TCR a mutant chain of C337-2, C341-7, C342-6 and C345-5 is shown as SEQ ID NO: 69 to SEQ ID NO: 72. The nucleotide sequence of four of the TCR β mutant chains is shown as SEQ ID NO: 73 to SEQ ID NO: 76, in particular, the nucleotide sequence of the TCR β mutant chain of C337-2, C342-6 and C345-5 is shown as SEQ ID NO: 73 to SEQ ID NO: 76.

[0043] The nucleotide sequence of four of the β chain codon-optimized nucleotides is shown as SEQ ID NO: 81 to SEQ ID NO: 84, in particular, the β chain codon-optimized nucleotide sequence of C337-2, C341-7, C342-6 and C345-5 is shown as SEQ ID NO: 81 to SEQ ID NO: 84.

[0044] In particular, the TCR can be modified with various mutations that do not alter the affinity and dissociation rate of the TCR to the target antigen, and these mutations are to prevent the mispairing of the introduced TCRs with the endogenous autologous TCRs. These mutations are made on the codon-optimized nucleotide sequences, and the specific mutation sequences are modified as follows: Further, each TCR comprises a TCRa mutant chain and a TCRp mutant chain, and the four TCRs are C337-2, C341-7, C342-6 and C345-5 respectively, wherein, the amino acid at position 182 of the a chain amino acid sequence of C337-2 is mutated from T to C, and the corresponding a chain nucleotide is mutated from ACC at positions 544, 545, 546 to TGC; the amino acid at position 485 of the b chain amino acid sequence of C337-2 is mutated from S to C, and the amino acid at position 503 is mutated from C to A, and the corresponding b chain nucleotide is mutated from TCC at positions 1453, 1454, 1455 to TGC, and from TGC at positions 1507, 1508, 1509 to GCA.

[0045] The amino acid at position 179 of the a chain amino acid sequence of C346-6 is mutated from T to C, and the corresponding a chain nucleotide is mutated from ACC at positions 535, 536, 537 to TGC; the amino acid at position 485 of the b chain amino acid sequence of C346-6 is mutated from S to C, and the amino acid at position 503 is mutated from C to A, and the corresponding b chain nucleotide is mutated from TCC at positions 1453, 1454, 1455 to TGC, and from TGC at positions 1507, 1508, 1509 to GCA.

[0046] The amino acid at position 172 of the a chain amino acid sequence of C346-7 is mutated from T to C, and the corresponding a chain nucleotide is mutated from ACC at positions 514, 515, 516 to TGC; the amino acid at position 480 of the b chain amino acid sequence of C346-7 is mutated from S to C, and the amino acid at position 498 is mutated from C to A, and the corresponding b chain nucleotide is mutated from TCG at positions 1438, 1439, 1440 to TGC, and from TGC at positions 1492, 1493, 1494 to GCA.

[0047] The amino acid at position 176 of the a chain amino acid sequence of C346-10 is mutated from T to C, and the corresponding a chain nucleotide is mutated from ACC at positions 526, 527, 528 to TGC; the amino acid at position 480 of the b chain amino acid sequence of C346-10 is mutated from S to C, and the amino acid at position 498 is mutated from C to A, and the corresponding b chain nucleotide is mutated from TCC at positions 1438, 1439, 1440 to TGC, and from TGC at positions 1492, 1493, 1494 to GCA.

[0048] The amino acid at position 179 of the alpha chain amino acid sequence of C342-3 is mutated from T to C, and the corresponding alpha chain nucleotide is mutated from ACC at positions 535, 536, 537 to TGC; the amino acid at position 483 of the beta chain amino acid sequence of C342-3 is mutated from S to C, and the amino acid at position 501 is mutated from C to A, and the corresponding beta chain nucleotide is mutated from TCT at positions 1447, 1448, 1449 to TGC, and from TGT at positions 1501, 1502, 1503 to GCA.

[0049] The present application also provides an expression vector comprising the nucleic acid molecule as described above, which 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, and the vector comprises a plasmid, a phagemid or a particle for gene therapy.

[0050] The present application also provides a recombinant cell comprising the nucleic acid molecule as described above or the expression vector as described above, which comprises a nucleic acid molecule encoding the TCR as described above or a vector comprising the nucleic acid molecule.

[0051] The present application also provides a pharmaceutical combination comprising the TCR combination recognizing the hepatitis B virus antigen 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, and the pharmaceutical combination of the present application can further comprise additional components in addition to the active ingredient, and is mixed with an acceptable carrier for a pharmaceutical composition for treating or alleviating diseases.

[0052] The present application also provides the use of the TCR combination recognizing the hepatitis B virus antigen 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 combination as described above in the preparation of a medicament for preventing and / or treating hepatitis B virus and / or related diseases caused by hepatitis B virus.

[0053] The TCR combination recognizing the hepatitis B virus antigen provided by the present application can specifically recognize a plurality of antigen epitopes, respectively, and can confer normal cells with reactivity to hepatitis B virus antigens, thereby providing a new solution for the prevention and treatment of hepatitis B virus.

[0054] The technical solutions of the present application will be further described in detail below in combination with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present application and do not limit the present application.

[0055] The present study was approved by the Ethics Committee of the Third People's Hospital of Shenzhen (Approval No.: 2021-210), and the participants provided written informed consent for sample collection and subsequent analysis.

[0056] Experimental materials: pHR-SFFV-IRES-EGFP: Addgene, 46911; pMD2.G; Addgene, 12259; pSPAX2: Addgene, 12260; Jurkat 76: Alost, Cat. No. ELT-200100; cdbNFAT from promega 9PIE848; LCL cell preparation method: B95-8 cells were cultured for two weeks, and EBV virus supernatant was collected and filtered with a sterile disposable 0.45 μm filter. One million PBMC cells were infected with 1 ml of virus supernatant, and then the cells were plated in a 24-well plate for culture, with the addition of 0.4 μg / ml of cyclosporine in complete RPMI-1640 medium. The fresh medium was replaced half a week, and the cell population was obtained until the appearance of the clustered cell population.

[0057] Example 1: Acquisition and lentivirus packaging of hepatitis B virus specific TCR The present application collects liver biopsy samples of chronic hepatitis B virus patients, and enzymatically digests the surgical tissue samples with collagenase and DNase at 37 degrees for half an hour. Cell suspension is obtained by filtration, and after centrifugation, the cells are resuspended in DPBS containing 2% fetal bovine serum. Single cells are prepared using the instrument of 10X genomics company, and then PCR and library construction operations are performed according to the company's instructions. The single cell library is sequenced by next-generation sequencing, and the nucleotide and amino acid sequence information of TCR is obtained by analyzing the single cell sequencing results. The analysis results show that the amino acid sequences of CDR1, CDR2 and CDR3 of the alpha and beta chains of TCR are shown in Table 1 above in order.

[0058] According to the sequencing information, the full-length sequences of the alpha chain and the beta chain of the TCR (C337-2, C341-7, C342-6 and C345-5) are spliced, the full-length nucleotide sequence of the alpha chain is the sequence as shown in SEQ ID NO: 61 to SEQ ID NO: 64, and the full-length nucleotide sequence of the beta chain is the sequence as shown in SEQ ID NO: 65 to SEQ ID NO: 68. The spliced full-length nucleic acid sequence of the alpha chain and the full-length nucleic acid sequence of the beta chain of the TCR are connected by T2A to form a TCR alpha-T2A-TCR beta nucleic acid fragment, and then the spliced TCR alpha-T2A-TCR beta nucleic acid fragment is codon-optimized by Nanjing Kingsriver Biotechnology Co., Ltd. and cloned into a lentiviral expression vector pHR-SFFV-IRES-EGFP to obtain a lentiviral vector pHR-SFFV-TCR-IRES-EGFP containing the TCR sequence of the application. The full-length nucleotide sequence of the alpha chain of the codon-optimized TCR is the sequence as shown in SEQ ID NO: 77 to SEQ ID NO: 80, and the full-length nucleotide sequence of the beta chain of the codon-optimized TCR is the sequence as shown in SEQ ID NO: 81 to SEQ ID NO: 84. Using a second-generation lentivirus packaging system, 293T cells are transfected with three plasmids (pHR-SFFV-TCR-IRES-EGFP, two plasmids pMD2.G and pSPAX2 containing other components necessary for constructing infectious but non-replicative lentivirus particles) to prepare lentivirus and concentrate the virus. The specific steps are as follows: 175cm 2 Culture flask packaging lentivirus test is described as follows: Take 15×10 6 cm 2 Culture flask, 37℃, 5% CO2 incubator overnight, when the cells grow to about 60% density of the culture flask, transfection; 1 hour before transfection, add 30 mL of fresh DMEM complete medium; each 175cm 2The amount of plasmid for culture bottle is: 10 μg pMD2.G, 30 μg pSPAX2, 40 μg pHR-SFFV-C152-1-IRES-EGFP, and the ratio of transfection reagent PEI to plasmid is 4:1, that is, 320 μg; the A liquid is prepared by adding the above three kinds of plasmids in 750 μL serum-free DEME, and vortexing uniformly; the B liquid is prepared by adding the corresponding PEI in 750 μL serum-free DEME, and vortexing uniformly; the A liquid and the B liquid are mixed and vortexed uniformly, and then are placed at room temperature for 20 minutes, and then are added dropwise into the prepared 293T cells, and the culture bottle is gently shaken to mix the medium, and then is cultured at 37°C under 5% CO2; after 72 hours of transfection, the supernatant of the culture medium is collected, and then is centrifuged (500g, 4°C, 5 minutes); the supernatant after centrifugation is filtered through a 0.45 μm filter, and then 5×PEG8000 is added to concentrate the virus, and then is mixed uniformly at 4°C for 2 hours, and then is placed at 4°C overnight; after overnight placement, centrifugation is performed (4000g, 4°C, 20 minutes), the supernatant is completely discarded, the precipitate is dissolved in 200 μL PBS, and then the dissolved lentivirus sample is frozen at -80°C.

[0059] As a control, the packaging of lentivirus of empty pHR-SFFV-IRES-EGFP is simultaneously carried out, and the packaging steps are the same as those of the lentivirus packaging steps of pHR-SFFV-TCR-IRES-EGFP described above.

[0060] Example 2: Specificity identification of TCR and hepatitis B virus antigen peptide screening The present application transforms the T cell line Jurkat 76 (J76) which has knocked out TCR by transducing CD8 molecules and inserting luciferase reporter genes downstream of NFAT, so that the T cell line can characterize the specific activation level of the cell by detecting the fluorescence signal after being specifically activated, which is beneficial to high-throughput identification of the specificity of TCR, and the cell line is simply referred to as J76.8 cell. The TCR (C337-2, C341-7, C342-6 and C345-5) of the present application is expressed on the surface of the J76.8 cell line by the method of lentivirus infection, and the cell is simply referred to as J76.8-TCR cell. The specific method is as follows: 1 mL of the lentivirus supernatant of the TCR packaged in Example 1 is taken before PEG8000 is added for concentration; the J76.8 cells are counted, and 1×10 5 J76.8 cells are put into one hole of a 24-well plate; 1 mL of the lentivirus supernatant of the TCR is added, and centrifugal infection is carried out (700g, 32°C, 1.5 hours); after centrifugation, 1 mL of 1640 complete culture medium is supplemented, and then the culture is carried out at 37°C under 5% CO2 for 3 days; the flow cytometry is used to detect the GFP positive rate and the CD3+TCRαβ+double positive rate, and the J76.8-TCR cell construction is successful when the rate is more than 90%.

[0061] The present application also constructs a lymphoblastoid cell line (LCL) homologous to the TCR (C337-2, C341-7, C342-6 and C345-5) as an antigen presenting cell for specific identification and antigen peptide screening. The specific method is as follows: B95-8 cells are cultured in 2% FBS low concentration serum 1640 culture medium for two weeks to produce EBV virus-containing supernatant; the EBV virus-containing supernatant is centrifuged (500g, 4°C, 5min) to remove cell debris, and then filtered through a 0.45μm sterile filter; the patient's peripheral mononuclear cells (PBMC) are recovered, and the corresponding volume of EBV supernatant is taken to resuspend the cells according to the standard of 1×10 6 cells / mL virus supernatant, and the cells are infected at 37°C for 2 hours; the infected cells are centrifuged (350g, 4°C, 5min) to remove the supernatant, and the cells are resuspended at a density of 2×10 5 cells / mL with 1640 complete culture medium (containing 0.4μg / mL cyclosporine A); the resuspended cells are cultured in a 48-well plate at a standard of 1mL / well, and cultured at 37°C, 5% CO2 for three weeks, with half of the liquid being replaced every week and half of the fresh 1640 complete culture medium (containing 0.4μg / mL cyclosporine A) being supplemented; after three weeks, the cells start to aggregate, and the lymphoblastoid cell line (LCL) is formed.

[0062] The constructed J76.8-TCR (C337-2, C341-7, C342-6 and C345-5) cells and LCL cells are co-incubated, and the fluorescence signal of the J76.8-TCR cells is detected under the stimulation of hepatitis B virus protein polypeptides to identify the specificity of the TCR and the recognized antigen peptide. The specific method is as follows: 1×10 5 J76.8-TCR cells and 1×10 5 LCL cells are added to each well of a 96-well plate, 3.5μg / mL hepatitis B virus peptide library (15 amino acids as one polypeptide, each polypeptide has 11 amino acids overlapping with the previous polypeptide, and the peptide library covers the whole hepatitis B virus genome, synthesized by Nanjing Kingsway Biotech Co., Ltd.) is added, the total volume of each well is 200μL, and a non-stimulation control group is set, and the cells are stimulated in a 37°C, 5% CO2 incubator for 6 hours; centrifuged at 600g for 3min, the supernatant was discarded, and each well was resuspended with 50μL PBS and transferred to a 96-well white plate, 50μL luciferase detection substrate (Novozyme product) was added, mixed well, reacted for 3min, and the chemiluminescence was detected by an enzyme marker. The specific screening process is as follows: first, the peptide library of each protein of hepatitis B virus is subjected to preliminary screening, then the large protein peptide library is subdivided into small peptide libraries containing 10 single peptides for further screening, then the small peptide library capable of activating TCR is further subdivided into single peptides for screening, finally the positive fifteen amino acid size single peptide is sequentially split into nine amino acid size single peptides for screening, and finally the specific antigen epitope of TCR is confirmed, such as SEQ ID NO. 1-4.

[0063] The results are shown in Figure 1 and 2 After the J76.8-TCR and LCL co-incubation experiment, the CD69 up-regulated expression marker is used to confirm the hepatitis B virus protein recognized by the TCR, and the abscissa represents the synthesized polypeptide library constituting the hepatitis B virus antigen (polymerase protein PLM, surface protein LS, core protein Core and X protein), wherein NS is the non-stimulation group control (negative control); PLM1 and PLM2 are sub-libraries of PLM; anti-CD3 is antibody stimulation (also as a positive control); PMA is a protein kinase A activator (also as a positive control); the ordinate represents the proportion of TCR signal receiving cell activation up-regulated expression of CD69 molecules, which is used as an index of the activation intensity of T cells under different stimulation. Among them, DMSO treatment is used as a negative control (NS), PMA / ionomycin and Anti-CD3 antibody are used as positive controls, and the reactivity of different TCRs under the same antigen stimulation is compared to screen TCRs with higher reactivity to hepatitis B virus proteins. The above C337-2, C342-6 and C345-5 are all Core protein specific TCRs, and C341-7 is a polymerase protein PLM specific TCR.

[0064] Then, the Luciferase Assay is used to prove that the J76.8-TCR cells are significantly activated under the stimulation of the hepatitis B virus protein peptide library, and further experiments prove the epitopes that cause the activation of these J76.8-TCR cells. Specifically, taking C337-2 as an example, as shown in Figure 3 The abscissa indicates different experimental treatment conditions related to the presented peptide segment or stimulation method; NS (Negative Control): negative control group, without adding any peptide segment or stimulating factor, as a baseline control. Core: represents the whole protein of hepatitis B virus core antigen (HBcAg), Anti-CD3 is an anti-CD3 antibody, which is used for non-specific activation of T cells, and is usually used as a positive control. PMA is a strong cell activator, similar to anti-CD3, which is used to evaluate the overall activity of T cells, and is often used as another positive control. The ordinate represents the fold change of expression or response. The peptide library of Core protein, Figure 3 In the first group of the abscissa,Figure 1 The peptide library screened by C337-2 is divided into 10 groups, and smaller peptide libraries of Core1-10, Core10-20, Core20-30, Core30-40 and Core40-50 are obtained; Core30-40 with strong specificity screened is further divided into Figure 3 2 figures in Figure 3 3 figures in Figures 4-6 It can be concluded that the peptide segment of Core34-4 is the key epitope presented by C337-2 antigen, and the remaining Figure 1 and 2 The peptide segments screened by different TCRs in

[0065] It is concluded that C341-7 TCR recognizes FAVPNLQSL antigen peptide on the polymerase protein of hepatitis B virus, and other remaining TCRs recognize FGRETVLEY (C337-2), RETVLEYLV (C342-6) and GRETVLEYL (C345-5) antigen peptides on the core protein of hepatitis B virus.

[0066] Example 3: Identification of HLA molecules recognized by TCR In Example 2, the TCRs (C337-2, C341-7, C342-6 and C345-5) of the present application have been identified to recognize hepatitis B virus antigen peptides, and the LCL homologous to the TCR includes 6 HLA class I molecule types, and the HLA molecule type presenting the antigen peptide needs to be further clarified. To achieve this purpose, the homologous LCL cells and J76.8-TCR cells are co-incubated in this Example 3, and the above-mentioned antigen peptides are added for stimulation, and the HLA molecule type combined with the antigen peptide is identified by detecting the fluorescence signal indicating the level of cell activation.

[0067] The results are shown in Figures 7-10 Only when co-incubated with homologous LCL (lymphoblastoid cell lines, human lymphoblast cell lines, are immortal cell lines prepared from patient-derived cells that present antigens) and K562 cells expressing specific HLA, J76.8-TCR cells are activated under the condition of antigen peptide stimulation, Figures 7-10 In the figure, the abscissa represents the antigen epitopes (hepatitis B antigen peptides) identified by the above-mentioned different TCRs; the ordinate represents the increase in luciferase expression after the activation of J76.8-TCR cells, and the signal value increased by antigen peptide treatment is obtained by subtracting the control treatment, which indicates specific activation. The HLA molecule identification figure recognized by these TCRs is as shown in Figures 7-10B*46:01 (C337-2), C*08:22 (C341-7), B*13:02 (C342-6) and B*40:01 (C345-5), respectively. Among them, the C*08:22 (C341-7) has the highest Fold Change value. Figure 7 For example, the chart shows the Fold Change of expression or response of different cell lines (K562, K562-B*46:01 and LCL18) under two experimental conditions of Core1 and Core34-4. The chart shows that the response or expression of the three cell lines does not change much under the irrelevant control peptide Core1 condition; while under the target peptide Core34-4 condition, the response of LCL18 cell line and K562-B*46:01 is significantly higher than K562, showing a very high Fold Change value. This indicates that under the Core34-4 condition, B*46:01 is responsible for presenting the Core34-4 target peptide and activating the TCR signal. The rest of the charts are similar.

[0068] Example 4: Mutation of TCR constant region To verify whether the exogenous TCR (C337-2, C341-7, C342-6 and C345-5) transduced T cells can confer the function of secreting inflammatory cytokines and killer cytokines on normal cells, the present application needs to further transduce the TCR into primary T cells for detection. To reduce the mismatch rate between the exogenous TCR and the TCR molecules of the cells themselves when transducing primary T cells and improve the stability of the TCR molecules, this embodiment introduces an artificial disulfide bond between the alpha chain constant region and the beta chain constant region of the TCR molecule. Specifically, the 48th threonine in the alpha chain constant region TRAC*01 of the TCR is mutated to cysteine, and the 57th serine in the beta chain constant region TRBC*02 of the TCR is mutated to cysteine; in addition, the 75th cysteine in the beta chain constant region TRBC*02 of the TCR is mutated to alanine to reduce the mismatch rate between the exogenous TCR and the endogenous TCR when the TCR transduces primary T cells.

[0069] Example 5: TCR transduction of primary T cells To prove that the TCR has functional activity after recognizing the antigen epitope, this embodiment uses the method of lentivirus infection to transduce the TCR to the surface of primary T cells, and detects the transduction efficiency of the TCR by staining the GFP expression carried by the lentivirus vector. The infection method is as follows: take PBMC, centrifuge (400g, 4°C, 5 minutes), count; according to 1x10 6The standard of 2.5x10 cells / mL of culture medium was added with the corresponding volume of human serum medium to culture the cells in the appropriate well plate; 10 μL / mL anti-CD3+anti-CD28 activation mixture (a product of Meitainyi Company) and 50 U / mL IL-2 (a product of Pepromax Company) were added for T cell activation; the primary T cells activated for 24 hours were collected, centrifuged (400 g, 4°C, 5 minutes), and resuspended with human serum medium for counting; the cells were divided into 48 well plates at the standard of 2.5x10 cells per well, and the packaged point-mutated TCR (C337-2, C341-7, C342-6 and C345-5) lentivirus (the lentivirus packaging method was the same as in Example 1) was added to each well, and the packaged blank vector lentivirus was correspondingly used as a control group; each well was supplemented with human serum medium to a volume of 500 μL, and centrifuged for infection (1000 g, 32°C, centrifuged 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 culture was carried out at 37°C, 5% CO2 for 3 days. 5 The standard of 2.5x10 cells / mL of culture medium was added with the corresponding volume of human serum medium to culture the cells in the appropriate well plate; 10 μL / mL anti-CD3+anti-CD28 activation mixture (a product of Meitainyi Company) and 50 U / mL IL-2 (a product of Pepromax Company) were added for T cell activation; the primary T cells activated for 24 hours were collected, centrifuged (400 g, 4°C, 5 minutes), and resuspended with human serum medium for counting; the cells were divided into 48 well plates at the standard of 2.5x10 cells per well, and the packaged point-mutated TCR (C337-2, C341-7, C342-6 and C345-5) lentivirus (the lentivirus packaging method was the same as in Example 1) was added to each well, and the packaged blank vector lentivirus was correspondingly used as a control group; each well was supplemented with human serum medium to a volume of 500 μL, and centrifuged for infection (1000 g, 32°C, centrifuged 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 culture was carried out at 37°C, 5% CO2 for 3 days.

[0070] Example 6: Functional detection of TCR In order to verify whether the four TCRs of the application can make the transduced T cells be activated by hepatitis B antigen peptides and produce corresponding inflammatory cytokines and killing cytokines after being transduced into primary T cells, the TCRs were transduced into primary T cells by the method of Example 5, and then were co-incubated with the homologous LCL; under the stimulation of hepatitis B virus antigen peptides, the levels of cytokines were detected by flow cytometry.

[0071] The specific test method was as follows: 5x10 5 cells of TCR-transduced primary T cells and 5x10 5One homologous LCL cell line was incubated with 1 μg / mL antigenic peptide, 1 μg / mL anti-CD28 antibody (Biolegend), and 3 μL / well of CD107a-PE / Cy7 flow cytometry antibody (Biolegend), for a total volume of 200 μL per well. An irrelevant peptide stimulation control group and a control group containing an equal volume of DMSO were also included. After stimulation at 37°C and 5% CO2 for 2 hours, a protein transport inhibitor (Biolegend) was added to block post-stimulation cytokine secretion, and the cells were cultured for another 18 hours. The cells were centrifuged at 600g for 3 minutes, the supernatant was discarded, and surface antibody staining was performed at 4°C in the dark for 30 minutes, followed by washing twice with PBS. The supernatant was discarded, and 100 μL of permeabilization buffer (BD) was added to each well. Permeabilization was performed for 20 minutes, followed by washing twice with the accompanying permeabilization wash buffer. 50 μL of intracellular flow cytometry antibody prepared with permeabilization wash buffer was added to each well, and the cells were stained at 4°C in the dark for 30 minutes, followed by washing twice with the permeabilization wash buffer. A total volume of 200 μL of permeabilization buffer was then added to each well. Cells were resuspended in 2% paraformaldehyde and analyzed by flow cytometry. Flow cytometry 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.

[0072] Test results are as follows Figures 11-14 As shown in the figure, NS represents unstimulated, Unrelated peptide represents unrelated peptide stimulation, and specific peptide stimulation represents specific peptide stimulation. The specific peptides are the peptides selected above, such as Core34-4 and PLM101-4 peptides. Compared with the unstimulated control group and the unrelated antigen peptide control group, stimulation with the corresponding antigen peptides all resulted in enhanced production of inflammatory cytokines IFN-γ and TNFα and increased expression of cytotoxic cytokine CD107a in T cells transducing the TCR of this invention. This demonstrates that these TCRs have functional activity and can confer responsiveness to hepatitis B virus antigens to normal T cells, which is expected to become a new immunocellular therapy strategy for hepatitis B virus infection.

[0073] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of patent protection of the present invention.

Claims

1. A TCR combination for identifying an antigen of hepatitis B virus, for identifying an HBV antigen epitope, characterized in that, The TCR combination recognizing the hepatitis B virus antigen comprises a plurality of isolated TCRs, and the plurality of isolated TCRs form four TCR groups, including a first TCR group, a second TCR group, a third TCR group, and a fourth TCR group; wherein, the first TCR group specifically recognizes a first antigen epitope, and the amino acid sequence of the first antigen epitope is shown as SEQ ID NO: 1; the second TCR group specifically recognizes a second antigen epitope, and the amino acid sequence of the second antigen epitope is shown as SEQ ID NO: 2; the third TCR group specifically recognizes a third antigen epitope, and the amino acid sequence of the third antigen epitope is shown as SEQ ID NO: 3; the fourth TCR group specifically recognizes a fourth antigen epitope, and the amino acid sequence of the fourth antigen epitope is shown as SEQ ID NO:

4.

2. The TCR combination recognizing the hepatitis B virus antigen according to claim 1, wherein the first TCR group comprises a first TCR α chain variable region and a first TCR β chain variable region, the first TCR α chain variable region comprises a first CDR1α, a first CDR2α, and a first CDR3α, the first TCR β chain variable region comprises a first CDR1β, a first CDR2β, and a first CDR3β, the amino acid sequence of the first CDR1α is shown as SEQ ID NO: 5, the amino acid sequence of the first CDR2α is shown as SEQ ID NO: 6, the amino acid sequence of the first CDR3α is shown as SEQ ID NO: 7, the amino acid sequence of the first CDR1β is shown as SEQ ID NO: 8, the amino acid sequence of the first CDR2β is shown as SEQ ID NO: 9, and the amino acid sequence of the first CDR3β is shown as SEQ ID NO: 10; and / or the second TCR group comprises a second TCR α chain variable region and a second TCR β chain variable region, the second TCR α chain variable region comprises a second CDR1α, a second CDR2α, and a second CDR3α, the second TCR β chain variable region comprises a second CDR1β, a second CDR2β, and a second CDR3β, the amino acid sequence of the second CDR1α is shown as SEQ ID NO: 11, the amino acid sequence of the second CDR2α is shown as SEQ ID NO: 12, the amino acid sequence of the second CDR3α is shown as SEQ ID NO: 13, the amino acid sequence of the second CDR1β is shown as SEQ ID NO: 14, the amino acid sequence of the second CDR2β is shown as SEQ ID NO: 15, and the amino acid sequence of the second CDR3β is shown as SEQ ID NO: 16; and / or the third TCR group comprises a third TCR α chain variable region and a third TCR β chain variable region, the third TCR α chain variable region comprises a third CDR1α, a third CDR2α, and a third CDR3α, the third TCR β chain variable region comprises a third CDR1β, a third CDR2β, and a third CDR3β, the amino acid sequence of the third CDR1α is shown as SEQ ID NO: 17, the amino acid sequence of the third CDR2α is shown as SEQ ID NO: 18, the amino acid sequence of the third CDR3α is shown as SEQ ID NO: 19, the amino acid sequence of the third CDR1β is shown as SEQ ID NO: 20, the amino acid sequence of the third CDR2β is shown as SEQ ID NO: 21, and the amino acid sequence of the third CDR3β is shown as SEQ ID NO: 22; and / or the fourth TCR group comprises a fourth TCR α chain variable region and a fourth TCR β chain variable region, the fourth TCR α chain variable region comprises a fourth CDR1α, a fourth CDR2α, and a fourth CDR3α, the fourth TCR β chain variable region comprises a fourth CDR1β, a fourth CDR2β, and a fourth CDR3β, the amino acid sequence of the fourth CDR1α is shown as SEQ ID NO: 23, the amino acid sequence of the fourth CDR2α is shown as SEQ ID NO: 24, the amino acid sequence of the fourth CDR3α is shown as SEQ ID NO: 25, the amino acid sequence of the fourth CDR1β is shown as SEQ ID NO: 26, the amino acid sequence of the fourth CDR2β is shown as SEQ ID NO: 27, and the amino acid sequence of the fourth CDR3β is shown as SEQ ID NO:

28. the third TCR group comprises a third TCR alpha chain variable region and a third TCR beta chain variable region, the third TCR alpha chain variable region comprises a third CDR1a, a third CDR2a and a third CDR3a, the third TCR beta chain variable region comprises a third CDR1b, a third CDR2b and a third CDR3b, the amino acid sequence of the third CDR1a is shown as SEQ ID NO: 17, the amino acid sequence of the third CDR2a is shown as SEQ ID NO: 18, the amino acid sequence of the third CDR3a is shown as SEQ ID NO: 19, the amino acid sequence of the third CDR1b is shown as SEQ ID NO: 20, the amino acid sequence of the third CDR2b is shown as SEQ ID NO: 21, and the amino acid sequence of the third CDR3b is shown as SEQ ID NO: 22; and / or, the fourth TCR group comprises a fourth TCR alpha chain variable region and a fourth TCR beta chain variable region, the fourth TCR alpha chain variable region comprises a fourth CDR1a, a fourth CDR2a and a fourth CDR3a, the fourth TCR beta chain variable region comprises a fourth CDR1b, a fourth CDR2b and a fourth CDR3b, the amino acid sequence of the fourth CDR1a is shown as SEQ ID NO: 23, the amino acid sequence of the fourth CDR2a is shown as SEQ ID NO: 24, the amino acid sequence of the fourth CDR3a is shown as SEQ ID NO: 25, the amino acid sequence of the fourth CDR1b is shown as SEQ ID NO: 26, the amino acid sequence of the fourth CDR2b is shown as SEQ ID NO: 27, and the amino acid sequence of the fourth CDR3b is shown as SEQ ID NO:

28.

3. The TCR combination of claim 1, wherein the TCR combination recognizes an antigen of hepatitis B virus. the plurality of isolated TCRs form four TCRs, wherein, each TCR comprises a TCR alpha chain variable region and a TCR beta chain variable region, the amino acid sequences of the four TCR alpha chain variable regions are shown as SEQ ID NO: 29 to SEQ ID NO: 32, and the amino acid sequences of the four TCR beta chain variable regions are shown as SEQ ID NO: 33 to SEQ ID NO: 36; and / or, each TCR comprises a TCR alpha chain and a TCR beta chain, the amino acid sequences of the four TCR alpha chains are shown as SEQ ID NO: 37 to SEQ ID NO: 40, and the amino acid sequences of the four TCR beta chains are shown as SEQ ID NO: 41 to SEQ ID NO: 44; and / or, each TCR comprises a TCR alpha mutant chain and a TCR beta mutant chain, the amino acid sequences of the four TCR alpha mutant chains are shown as SEQ ID NO: 45 to SEQ ID NO: 48, and the amino acid sequences of the four TCR beta mutant chains are shown as SEQ ID NO: 49 to SEQ ID NO:

52.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes any one of the TCRs in the TCR combination for recognizing the hepatitis B virus antigen according to any one of claims 1-3, and the nucleic acid molecule corresponding to the nucleic acid molecule encoding any one of the TCRs in the TCR combination for recognizing the hepatitis B virus antigen comprises a plurality of nucleic acid molecules, wherein, each TCR comprises a TCR a chain variable region and a TCR β chain variable region, the nucleotide sequences of the four TCR a chain variable regions are shown as SEQ ID NO: 53-SEQ ID NO: 56, and the nucleotide sequences of the four TCR β chain variable regions are shown as SEQ ID NO: 57-SEQ ID NO: 60; and / or, each TCR comprises a TCR a chain and a TCR β chain, the nucleotide sequences of the four TCR a chains are shown as SEQ ID NO: 61-SEQ ID NO: 64, and the nucleotide sequences of the four TCR β chains are shown as SEQ ID NO: 65-SEQ ID NO: 68; and / or, each TCR comprises a TCR a mutant chain and a TCR β mutant chain, the nucleotide sequences of the four TCR a mutant chains are shown as SEQ ID NO: 69-SEQ ID NO: 72, and the nucleotide sequences of the four TCR β mutant chains are shown as SEQ ID NO: 73-SEQ ID NO: 76; and / or, each TCR comprises a codon-optimized a chain and a codon-optimized β chain, the nucleotide sequences of the four codon-optimized a chains are shown as SEQ ID NO: 77-SEQ ID NO: 80, and the nucleotide sequences of the four codon-optimized β chains are shown as SEQ ID NO: 81-SEQ ID NO:

84.

5. An expression vector, characterized by, The expression vector comprises the nucleic acid molecule according to claim 4.

6. A recombinant cell, characterized in that, The recombinant cell comprises the nucleic acid molecule according to claim 4 or the expression vector according to claim 5.

7. A pharmaceutical combination comprising, The nucleic acid molecule according to claim 4 or the expression vector according to claim 5, or the recombinant cell according to claim 6.

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

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