Hepatitis B virus specific TCR combination and application

Abstract

The invention relates to the field of immunology, and particularly discloses a hepatitis B virus specific TCR combination and application, the hepatitis B virus specific TCR combination is used for recognizing HBV antigen epitopes, the hepatitis B virus specific 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 in particular to a hepatitis B virus-specific TCR combination and application. Background Art

[0002] Studies have shown that the levels of viral surface antigens and viral DNA in patients with chronic HBV infection are inversely correlated with the proportion of HBV-specific CD8+ T cells, suggesting the antiviral effects of virus-specific CD8+ T cells. Therefore, the isolation and identification of virus-specific CD8+ T cells plays an important role in the prevention and treatment of chronic HBV infection and HBV-related liver cancer.

[0003] During viral infection, antigen-presenting cells process viral antigens into short antigenic peptides, which bind to HLA molecules to form stable antigenic peptide-MHC complexes expressed on the cell membrane. Specific T cells bind to these antigens through surface T cell receptors (TCRs). Under the coordination of co-stimulatory signals, a series of signal transduction and physiological functions are triggered, achieving the effect of clearing the virus. TCR-T adoptive cell therapy mainly involves in vitro transformation of the patient's autologous cells into TCRs that specifically recognize hepatitis B virus antigens, and then returning them to the patient's body, thereby achieving the purpose of efficiently clearing virus-infected cells. Transducing hepatitis B virus-specific TCRs onto the surface of healthy human T cells can give normal T cells responsiveness 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 of hepatitis B virus infection. Summary of the Invention

[0004] The main purpose of the present invention is to propose a hepatitis B virus-specific TCR combination and application, aiming to propose a hepatitis B virus-specific TCR combination, the TCR in which can specifically recognize and bind to hepatitis B virus antigen epitopes, thereby providing an important basis for TCR-T immune cell therapy of hepatitis B virus infection.

[0005] To achieve the above object, the present invention proposes a hepatitis B virus-specific TCR combination for identifying HBV antigen epitopes, wherein the hepatitis B virus-specific TCR combination includes a plurality of separated TCRs, and the plurality of separated hepatitis B virus-specific TCRs are combined to form two TCR groups, wherein the two TCR groups include a first TCR group and a second TCR group; wherein,

[0006] The first TCR group specifically recognizes a first antigen epitope, and the amino acid sequence of the first antigen epitope is shown in SEQ ID NO: 1;

[0007] The second TCR group specifically recognizes a second antigen epitope, and the amino acid sequence of the second antigen epitope is shown in SEQ ID NO:2.

[0008] In some embodiments, the first TCR group includes a first TCRα chain variable region, a first TCRβ chain variable region, a second TCRα chain variable region, a second TCRβ chain variable region, a third TCRα chain variable region, a third TCRβ chain variable region, a fourth TCRα chain variable region and a fourth TCRβ chain variable region, wherein the first TCRα chain variable region includes a first CDR1α, a first CDR2α and a first CDR3α, the first TCRβ chain variable region includes a first CDR1β, a first CDR2β and a first CDR3β, the second TCRα chain variable region includes a second CDR1α, a second CDR2α and a first CDR3β The second TCRβ chain variable region includes a second CDR1β, a second CDR2β and a second CDR3β, the third TCRα chain variable region includes a third CDR1α, a third CDR2α and a third CDR3α, the third TCRβ chain variable region includes a third CDR1β, a third CDR2β and a third CDR3β, the fourth TCRα chain variable region includes a fourth CDR1α, a fourth CDR2α and a fourth CDR3α, the fourth TCRβ chain variable region includes a fourth CDR1β, a fourth CDR2β and a fourth CDR3β, and 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 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 ID NO: 9, the amino acid sequence of the second CDR2α is shown in SEQ ID NO: 10, and 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 ID NO: 16, and the amino acid sequence of the third CDR3α is shown in SEQ ID NO: 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.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, and 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, and the amino acid sequence of the fourth CDR3β is shown in SEQ ID NO: 26; and / or,

[0009] The second TCR group includes a fifth TCRα chain variable region and a fifth TCRβ chain variable region, the fifth TCRα chain variable region includes a fifth CDR1α, a fifth CDR2α and a fifth CDR3α, the fifth TCRβ chain variable region includes a fifth CDR1β, a fifth CDR2β and a fifth CDR3β, the amino acid sequence of the fifth CDR1α is shown in SEQ ID NO: 27, the amino acid sequence of the fifth CDR2α is shown in SEQ ID NO: 28, the amino acid sequence of the fifth CDR3α is shown in SEQ ID NO: 29, the amino acid sequence of the fifth CDR1β is shown in SEQ ID NO: 30, the amino acid sequence of the fifth CDR2β is shown in SEQ ID NO: 31, and the amino acid sequence of the fifth CDR3β is shown in SEQ ID NO: 32.

[0010] In some embodiments, the isolated hepatitis B virus-specific TCRs are combined to form five TCRs, wherein:

[0011] Each TCR comprises a TCR α chain variable region and a TCR β chain variable region, the amino acid sequences of the five TCR α chain variable regions are shown in SEQ ID NO: 33 to SEQ ID NO: 37, and the amino acid sequences of the five TCR β chain variable regions are shown in SEQ ID NO: 38 to SEQ ID NO: 42; and / or,

[0012] Each TCR comprises a TCRα chain and a TCRβ chain, the amino acid sequences of the five TCRα chains are shown in SEQ ID NO:43 to SEQ ID NO:47, and the amino acid sequences of the five TCRβ chains are shown in SEQ ID NO:48 to SEQ ID NO:52; and / or,

[0013] Each TCR includes a TCRα mutant chain and a TCRβ mutant chain. The amino acid sequences of the five TCRα mutant chains are shown in SEQ ID NO:53 to SEQ ID NO:57, and the amino acid sequences of the five TCRβ mutant chains are shown in SEQ ID NO:58 to SEQ ID NO:62.

[0014] The present invention provides a nucleic acid molecule combination, comprising a plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules encode and form the hepatitis B virus-specific TCR combination as described above, wherein the hepatitis B virus-specific TCR combination comprises five TCRs, wherein:

[0015] Each TCR comprises a TCR α chain variable region and a TCR β chain variable region, the nucleotide sequences of the five TCR α chain variable regions are shown in SEQ ID NO: 63 to SEQ ID NO: 67, and the nucleotide sequences of the five TCR β chain variable regions are shown in SEQ ID NO: 68 to SEQ ID NO: 72; and / or,

[0016] Each TCR comprises a TCRα chain and a TCRβ chain, the nucleotide sequences of the five TCRα chains are shown in SEQ ID NO:73 to SEQ ID NO:77, and the nucleotide sequences of the five TCRβ chains are shown in SEQ ID NO:78 to SEQ ID NO:82; and / or,

[0017] Each TCR comprises a TCRα mutant chain and a TCRβ mutant chain, the nucleotide sequences of the five TCRα mutant chains are shown in SEQ ID NO:83 to SEQ ID NO:87, and the nucleotide sequences of the five TCRβ mutant chains are shown in SEQ ID NO:88 to SEQ ID NO:92; and / or,

[0018] Each TCR includes α chain codon-optimized nucleotides and β chain codon-optimized nucleotides, the five α chain codon-optimized nucleotide sequences are shown in SEQ ID NO:93 to SEQ ID NO:97, and the five β chain codon-optimized nucleotide sequences are shown in SEQ ID NO:98 to SEQ ID NO:102.

[0019] The present invention also provides an expression vector comprising the above-mentioned nucleic acid molecule combination.

[0020] The present invention also provides a recombinant cell, which comprises the above-mentioned nucleic acid molecule combination or the above-mentioned expression vector.

[0021] The present invention also provides a drug combination comprising any one of the above-described hepatitis B virus-specific TCR combinations or the above-described isolated TCR or the above-described nucleic acid molecule combination, the above-described expression vector, and the above-described recombinant cell.

[0022] The present invention also proposes a use of the hepatitis B virus-specific TCR combination as described above, the isolated TCR as described above, the nucleic acid molecule combination 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, which is used to prevent and / or treat hepatitis B virus and / or related diseases caused by hepatitis B virus.

[0023] The multiple TCR groups in the hepatitis B virus-specific TCR combination provided by the present invention can specifically recognize multiple antigen epitopes respectively, can endow normal cells with responsiveness to hepatitis B virus antigens, and provide a new solution for the prevention and treatment of hepatitis B virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 This is a diagram showing the results of identifying the reactivity of hepatitis B virus-specific TCRs (C341-8, C342-3) to hepatitis B virus proteins in Example 1;

[0026] Figure 2 This is a diagram showing the results of identifying the reactivity of hepatitis B virus-specific TCRs (C346-6, C346-7, and C346-10) to hepatitis B virus proteins in Example 1;

[0027] Figure 3 Schematic diagram of the detection results of the peptide epitope identified and recognized by TCR (C341-8) under the condition of epitope presentation by homologous LCL cells in Example 1;

[0028] Figure 4 Schematic diagram of the detection results of the peptide epitope identified by TCR (C342-3) under the condition of epitope presentation by homologous LCL cells in Example 1;

[0029] Figure 5 Schematic diagram of the detection results of the peptide epitope identified and recognized by TCR (C346-6) under the condition of epitope presentation by homologous LCL cells in Example 1;

[0030] Figure 6 Schematic diagram of the detection results of the peptide epitope identified by TCR (C346-7) under the condition of epitope presentation by homologous LCL cells in Example 1;

[0031] Figure 7Schematic diagram of the detection results of the peptide epitope identified by TCR (C346-10) under the conditions of epitope presentation by homologous LCL cells in Example 1;

[0032] Figure 8 This is a diagram illustrating the HLA molecule recognition by the TCR (C341-8) in Example 1;

[0033] Figure 9 This is a diagram illustrating the HLA molecule recognition by the TCR (C342-3) in Example 1;

[0034] Figure 10 This is a diagram illustrating the HLA molecules recognized by the TCRs (C346-6 (A), C346-7 (B), and C346-10 (C)) in Example 1;

[0035] Figure 11 This is a functional verification diagram of TCR (C341-8) in Example 1;

[0036] Figure 12 This is a functional verification diagram of TCR (C342-3) in Example 1;

[0037] Figure 13 This is a functional verification diagram of TCR (C346-6) in Example 1;

[0038] Figure 14 This is a functional verification diagram of TCR (C346-7) in Example 1;

[0039] Figure 15 This is a functional verification diagram of TCR (C346-10) in Example 1.

[0040] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0041] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0042] It should be noted that, in the embodiments, those without specifying specific conditions, are carried out according to conventional conditions or the conditions recommended by the manufacturer. Those for reagents or instruments used that do not specify the manufacturer are conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes. Taking "A and / or B" as an example, it includes schemes A, B, or A and B that meet the same time. In addition, the technical solutions between the various embodiments can be combined with each other, but must be based on those of ordinary skill in the art. When the combination of the technical solutions is contradictory or cannot be realized, it should be considered that the combination of such technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work belong to the scope of protection of the present invention.

[0043] During viral infection, antigen-presenting cells process viral antigens into short antigenic peptides, which bind to HLA molecules to form stable antigenic peptide-MHC complexes expressed on the cell membrane. Specific T cells bind to these antigens through surface T cell receptors (TCRs). Under the coordination of co-stimulatory signals, a series of signal transduction and physiological functions are triggered, achieving the effect of clearing the virus. TCR-T adoptive cell therapy mainly involves in vitro transformation of the patient's autologous cells into TCRs that specifically recognize hepatitis B virus antigens, and then returning them to the patient's body, thereby achieving the purpose of efficiently clearing virus-infected cells. Transducing hepatitis B virus-specific TCRs onto the surface of healthy human T cells can give normal T cells responsiveness 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 of hepatitis B virus infection.

[0044] In view of this, the present invention proposes a hepatitis B virus-specific TCR combination and application for identifying HBV antigen epitopes, wherein the hepatitis B virus-specific TCR combination includes multiple separated TCRs, and the multiple separated hepatitis B virus-specific TCRs are combined to form two TCR groups, and the two TCR groups include a first TCR group and a second TCR group; wherein,

[0045] The first TCR group specifically recognizes a first antigen epitope, and the amino acid sequence of the first antigen epitope is shown in SEQ ID NO: 1;

[0046] The second TCR group specifically recognizes a second antigen epitope, and the amino acid sequence of the second antigen epitope is shown in SEQ ID NO:2.

[0047] The present invention targets the above two antigenic epitopes and obtains a hepatitis B virus-specific TCR combination that can specifically recognize HBV virus. The TCR in this combination can specifically recognize and target hepatitis B virus antigenic epitopes, thereby providing an important basis for TCR-T immune cell therapy of hepatitis B virus infection.

[0048] Specifically, the amino acid sequence of the first antigen epitope is shown in SEQ ID NO: 1, specifically expressed as: YRPPNAPIL-HLA-C*07:02; the amino acid sequence of the second antigen epitope is shown in SEQ ID NO: 2, specifically expressed as: YRPPNAPIL-HLA-C*06:02.

[0049] It should be noted that, taking YRPPNAPIL-HLA-C*07:02 as an example, YRPPNAPIL is the peptide segment of the antigen epitope, HLA-C*07:02 is the subtype of the HLA molecule, and HLA-A belongs to a category of the HLA family.

[0050] It should be further explained that the T cell receptor (TCR) is a membrane protein on the surface of T cells, which can recognize short antigenic peptides on the surface of corresponding target cells, and the human leukocyte antigen HLA, commonly known as the MHC molecule, is a type of gene on the short arm of chromosome 6. Each person's HLA is 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 short peptide-major histocompatibility antigen (pMHC) complex triggers direct physical contact between T cells and antigen-presenting cells (APCs). Then, other cell membrane surface molecules of both T cells and APCs interact, causing a series of subsequent cell signaling and other physiological reactions, thereby enabling T cells with different antigen specificities to exert immune effects on their target cells.

[0051] pMHC (Peptide-Major Histocompatibility Complex) and TCR (T-cell Receptor) are important components of the immune system.

[0052] pMHC is a complex consisting of an antigenic peptide and a major histocompatibility complex (MHC) molecule. MHC molecules are a class of membrane molecules whose main function is to display antigenic peptides to T cells.

[0053] It should be noted that each TCR includes a TCRα chain and a TCRβ chain, and the TCRα chain or TCRβ chain contains at least a variable region. The variable region has the main function of specifically binding 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 the antibody, namely CDR1, CDR3 and CDR3, which together constitute the site that binds to the antigen, determine the specificity, and are responsible for identifying and binding to antigens, thereby exerting an immune effect.

[0054] In some embodiments, the first TCR group includes a first TCRα chain variable region, a first TCRβ chain variable region, a second TCRα chain variable region, a second TCRβ chain variable region, a third TCRα chain variable region, a third TCRβ chain variable region, a fourth TCRα chain variable region and a fourth TCRβ chain variable region, wherein the first TCRα chain variable region includes a first CDR1α, a first CDR2α and a first CDR3α, the first TCRβ chain variable region includes a first CDR1β, a first CDR2β and a first CDR3β, the second TCRα chain variable region includes a second CDR1α, a second CDR2α and a first CDR3β The second TCRβ chain variable region includes a second CDR1β, a second CDR2β and a second CDR3β, the third TCRα chain variable region includes a third CDR1α, a third CDR2α and a third CDR3α, the third TCRβ chain variable region includes a third CDR1β, a third CDR2β and a third CDR3β, the fourth TCRα chain variable region includes a fourth CDR1α, a fourth CDR2α and a fourth CDR3α, the fourth TCRβ chain variable region includes a fourth CDR1β, a fourth CDR2β and a fourth CDR3β, and 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 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 ID NO: 9, the amino acid sequence of the second CDR2α is shown in SEQ ID NO: 10, and 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 ID NO: 16, and the amino acid sequence of the third CDR3α is shown in SEQ ID NO: 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.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, and 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, and the amino acid sequence of the fourth CDR3β is shown in SEQ ID NO: 26; and / or,

[0055] The second TCR group includes a fifth TCRα chain variable region and a fifth TCRβ chain variable region, the fifth TCRα chain variable region includes a fifth CDR1α, a fifth CDR2α and a fifth CDR3α, the fifth TCRβ chain variable region includes a fifth CDR1β, a fifth CDR2β and a fifth CDR3β, the amino acid sequence of the fifth CDR1α is shown in SEQ ID NO: 27, the amino acid sequence of the fifth CDR2α is shown in SEQ ID NO: 28, the amino acid sequence of the fifth CDR3α is shown in SEQ ID NO: 29, the amino acid sequence of the fifth CDR1β is shown in SEQ ID NO: 30, the amino acid sequence of the fifth CDR2β is shown in SEQ ID NO: 31, and the amino acid sequence of the fifth CDR3β is shown in SEQ ID NO: 32.

[0056] Specifically, the hepatitis B virus-specific TCRs identified by the present invention include five groups of TCRs, namely C341-8, C346-6, C346-7, C346-10 and C342-3. Among them, C341-8, C346-6, C346-7 and C346-10 can bind to the YRPPNAPIL-HLA-C*07:02 complex (first antigen epitope), and C342-3 can bind to the YRPPNAPIL-HLA-C*06:02 complex (second antigen epitope). Moreover, these TCRs can confer specific responses to hepatitis B virus antigens on normal cells after transduction into primary T cells, providing new methods for the prevention and treatment of hepatitis B virus infection. Strategy, the specific sequences of the CDRs of the α chain variable region and the β chain variable region of the above-mentioned TCR are shown in Table 1, wherein the first TCR group includes a first TCR, a second TCR, a third TCR and a fourth TCR, the first TCR includes a first TCR α chain variable region and a first TCR β chain variable region, the second TCR includes a second TCR α chain variable region and a second TCR β chain variable region, the third TCR includes a third TCR α chain variable region and a third TCR β chain variable region, the fourth TCR includes a fourth TCR α chain variable region and a fourth TCR β chain variable region, and the second TCR group includes a fifth TCR, and the fifth TCR includes a fifth TCR α chain variable region and a fifth TCR β chain variable region. The present invention analyzes single-cell sequencing data of liver puncture samples from patients with chronic hepatitis B virus infection, selects several TCR clones for in vitro synthesis and insertion into lentiviral vectors, constructs T cell lines stably expressing TCRs through lentiviral infection, stimulates with hepatitis B virus polypeptides, and identifies hepatitis B virus-specific TCRs by detecting the activation level of the cells, and clarifies the antigenic peptides and HLA molecules they recognize, namely the two antigenic epitopes described herein.

[0057] In some specific embodiments, the five TCRs are αβ heterodimers, which contain the TCR α chain constant region TRAC*01 and the β chain constant region TRBC*02. The sequences of the TCR constant domains can be found in the public database of the International Immunogenetics Information System (IMGT), such as the constant domain sequence of the TCR molecule α chain is "TRAC*01", and the constant domain sequence of the TCR molecule β chain is "TRBC1*01" or "TRBC2*01", wherein an artificial disulfide bond is introduced between the α chain and β chain constant regions of the TCR of the present invention, and 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.

[0058] In some embodiments, the plurality of separated TCRs form five TCRs, wherein,

[0059] Each TCR comprises a TCR α chain variable region and a TCR β chain variable region, wherein the five TCR α chain variable region amino acids specifically comprise C341-8 α chain variable region amino acids, C346-6 α chain variable region amino acids, C346-7 α chain variable region amino acids, C346-10 α chain variable region amino acids, and C342-3 α chain variable region amino acids, and the specific sequences of the five TCR α chain variable region amino acids are shown in SEQ ID NO:33 to SEQ ID NO:37. Similarly, the specific sequences of the five TCR β chain variable region amino acids are shown in SEQ ID NO:38 to SEQ ID NO:42; and / or,

[0060] Each TCR comprises a TCR α chain and a TCR β chain, wherein the amino acids of the five TCR α chains specifically comprise C341-8 α chain amino acids, C346-6 α chain amino acids, C346-7 α chain amino acids, C346-10 α chain amino acids, and C342-3 α chain amino acids, and their specific sequences are shown in SEQ ID NO:43 to SEQ ID NO:47. Similarly, the specific amino acid sequences of the five TCR β chains are shown in SEQ ID NO:48 to SEQ ID NO:52; and / or,

[0061] Each TCR includes a TCRα mutant chain and a TCRβ mutant chain, and the amino acids of the five TCRα mutant chains specifically include the amino acids of the C341-8α mutant chain, the amino acids of the C346-6α mutant chain, the amino acids of the C346-7α mutant chain, the amino acids of the C346-10α mutant chain and the amino acids of the C342-3α mutant chain, and their specific sequences are shown in SEQ ID NO:53 to SEQ ID NO:57. Similarly, the amino acid sequences of the five TCRβ mutant chains are shown in SEQ ID NO:58 to SEQ ID NO:62.

[0062] The present invention also provides a nucleic acid molecule combination, comprising a plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules encode and form the hepatitis B virus-specific TCR combination as described above, wherein the hepatitis B virus-specific TCR combination comprises five TCRs, wherein:

[0063] Each TCR comprises a TCR α chain variable region and a TCR β chain variable region, the nucleotides of the five TCR α chain variable regions specifically comprise nucleotides of the C341-8 α chain variable region, nucleotides of the C346-6 α chain variable region, nucleotides of the C346-7 α chain variable region, nucleotides of the C346-10 α chain variable region, and nucleotides of the C342-3 α chain variable region, and their specific sequences are shown in SEQ ID NO:63 to SEQ ID NO:67. Similarly, the nucleotides of the five TCR β chain variable regions specifically comprise nucleotides of the C341-8 β chain variable region, nucleotides of the C346-6 β chain variable region, nucleotides of the C346-7 β chain variable region, nucleotides of the C346-10 β chain variable region, and nucleotides of the C342-3 β chain variable region, and their specific sequences are shown in SEQ ID NO:68 to SEQ ID NO:72; and / or,

[0064] Each TCR comprises a TCR α chain and a TCR β chain, and the nucleotides of the five TCR α chains specifically include nucleotides of the C341-8 α chain, nucleotides of the C346-6 α chain, nucleotides of the C346-7 α chain, nucleotides of the C346-10 α chain, and nucleotides of the C342-3 α chain, and their specific sequences are shown in SEQ ID NO:73 to SEQ ID NO:77. Similarly, the nucleotide sequences of the five TCR β chains are shown in SEQ ID NO:78 to SEQ ID NO:82; and / or,

[0065] Each TCR comprises a TCRα mutant chain and a TCRβ mutant chain, and the nucleotides of the five TCRα mutant chains specifically include nucleotides of the C341-8α mutant chain, nucleotides of the C346-6α mutant chain, nucleotides of the C346-7α mutant chain, nucleotides of the C346-10α mutant chain, and nucleotides of the C342-3α mutant chain, and their specific sequences are shown in SEQ ID NO:83 to SEQ ID NO:87. Similarly, the nucleotide sequences of the five TCRβ mutant chains are shown in SEQ ID NO:88 to SEQ ID NO:92; and / or,

[0066] Each TCR includes α chain codon-optimized nucleotides and β chain codon-optimized nucleotides, and the five α chain codon-optimized nucleotides specifically include C341-8 α chain codon-optimized nucleotides, C346-6 α chain codon-optimized nucleotides, C346-7 α chain codon-optimized nucleotides, C346-10 α chain codon-optimized nucleotides and C342-3 α chain codon-optimized nucleotides, whose specific sequences are shown in SEQ ID NO:93 to SEQ ID NO:97. Similarly, the five β chain codon-optimized nucleotide sequences are shown in SEQ ID NO:98 to SEQ ID NO:102.

[0067] Specifically, TCRs can be modified with various mutations that do not alter the affinity or off-rate of the TCR for the target antigen, but are designed to prevent mispairing of the introduced TCR with endogenous autologous TCRs. These mutations are made in codon-optimized nucleotide sequences.

[0068] Furthermore, each TCR includes a TCRα mutant chain and a TCRβ mutant chain. The five TCRs are C341-8, C346-6, C346-7, C346-10, and C342-3. Among them, the 183rd amino acid in the α chain amino acid sequence of C341-8 is mutated from T to C, and the corresponding α chain nucleotides are mutated from ACT to TGC at positions 547, 548, and 549; the 492nd amino acid in the β chain amino acid sequence of C341-8 is mutated from S to C, and the 510th amino acid is mutated from C to A, and the corresponding β chain nucleotides are mutated from TCT to TGC at positions 1474, 1475, and 1476, and from TGC to GCA at positions 1528, 1529, and 1530;

[0069] The 179th amino acid in the α-chain amino acid sequence of C346-6 mutated from T to C, and the corresponding nucleotides in the α-chain mutated from ACC to TGC at positions 535, 536, and 537. The 485th amino acid in the β-chain amino acid sequence of C346-6 mutated from S to C, and the 503rd amino acid mutated from C to A. The corresponding nucleotides in the β-chain mutated from TCC to TGC at positions 1453, 1454, and 1455, and from TGC to GCA at positions 1507, 1508, and 1509.

[0070] The 172nd amino acid in the α-chain amino acid sequence of C346-7 mutated from T to C, and the corresponding nucleotides in the α-chain mutated from ACC to TGC at positions 514, 515, and 516; the 480th amino acid in the β-chain amino acid sequence of C346-7 mutated from S to C, and the 498th amino acid mutated from C to A, and the corresponding nucleotides in the β-chain mutated from TCG to TGC at positions 1438, 1439, and 1440, and from TGC to GCA at positions 1492, 1493, and 1494;

[0071] The 176th amino acid in the α-chain amino acid sequence of C346-10 mutated from T to C, and the corresponding nucleotides in the α-chain mutated from ACC to TGC at positions 526, 527, and 528. The 480th amino acid in the β-chain amino acid sequence of C346-10 mutated from S to C, and the 498th amino acid mutated from C to A. The corresponding nucleotides in the β-chain mutated from TCC to TGC at positions 1438, 1439, and 1440, and from TGC to GCA at positions 1492, 1493, and 1494.

[0072] The 179th amino acid in the α-chain amino acid sequence of C342-3 mutated from T to C, and the corresponding α-chain nucleotides mutated from ACC to TGC at positions 535, 536, and 537; the 483rd amino acid in the β-chain amino acid sequence of C342-3 mutated from S to C, and the 501st amino acid mutated from C to A, and the corresponding β-chain nucleotides mutated from TCT to TGC at positions 1447, 1448, and 1449, and from TGT to GCA at positions 1501, 1502, and 1503.

[0073] The present invention also provides an expression vector, which 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, where synthesis of the encoded protein can occur. The vector includes a plasmid, a phagemid or a particle for gene therapy.

[0074] The present invention also provides a recombinant cell, comprising the nucleic acid molecule or the expression vector as described above, wherein the recombinant cell comprises a nucleic acid molecule encoding the TCR as described above or a vector comprising the nucleic acid molecule.

[0075] The present invention also proposes a drug combination comprising any one of the above-described hepatitis B virus-specific TCR combinations or the above-described isolated TCR or the above-described nucleic acid molecule, the above-described expression vector, and the above-described recombinant cell. In addition to the active ingredients, the drug combination of the present invention may also include additional ingredients, which are mixed with an acceptable carrier to form a pharmaceutical composition for treating or alleviating a disease.

[0076] The present invention also proposes a use of the above-mentioned hepatitis B virus-specific TCR combination, the above-mentioned isolated TCR or the above-mentioned nucleic acid molecule, the above-mentioned expression vector, the above-mentioned recombinant cell or the above-mentioned pharmaceutical composition in the preparation of a drug, which is used to prevent and / or treat hepatitis B virus and / or related diseases caused by hepatitis B virus.

[0077] The multiple TCR groups in the hepatitis B virus-specific TCR combination provided by the present invention can specifically recognize multiple antigen epitopes respectively, can endow normal cells with responsiveness to hepatitis B virus antigens, and provide a new solution for the prevention and treatment of hepatitis B virus.

[0078] The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0079] This study was approved by the Ethics Committee of Shenzhen Third People's Hospital (approval number: 2021-210), and participants provided written informed consent for sample collection and subsequent analysis.

[0080] Experimental Materials:

[0081] pHR-SFFV-IRES-EGFP: Addgene, 46911;

[0082] pMD2.G;Addgene,12259;

[0083] pSPAX2: Addgene, 12260.

[0084] Example 1: Acquisition of Hepatitis B Virus-Specific TCR and Lentiviral Packaging

[0085] The present invention collects liver biopsy tissue samples from patients with chronic hepatitis B virus, treats the surgically removed tissue samples 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. Single cells are prepared using a 10X genomics instrument, and PCR and library construction procedures are performed according to the company's instructions. The constructed single-cell library is subjected to second-generation sequencing. The single-cell sequencing results are analyzed to obtain TCR nucleotide and amino acid sequence information. The analysis results show that the amino acid sequences of CDR1, CDR2, and CDR3 of the α and β chains of the TCR are shown in Table 1, in order:

[0086] Table 1

[0087]

[0088] The full-length sequences of the α chain and β chain of TCR (C341-8, C346-6, C346-7, C346-10 and C342-3) were spliced ​​based on the sequencing information. The full-length nucleotide sequence of the α chain is shown in SEQ ID NOs. 63-67, and the full-length nucleotide sequence of the β chain is shown in SEQ ID NOs. 68-72. The spliced ​​TCR α chain full-length nucleic acid sequence and the full-length nucleic acid sequence of the β chain were connected by T2A to form a TCRα-T2A-TCRβ nucleic acid fragment. The spliced ​​TCRα-T2A-TCRβ nucleic acid fragment was then codon-optimized and cloned into the lentiviral expression vector pHR-SFFV-IRES-EGFP by Nanjing GenScript Biotechnology Co., Ltd. to obtain the lentiviral vector pHR-SFFV-TCR-IRES-EGFP containing the TCR sequence of the present invention. The codon-optimized full-length nucleotide sequence of the TCR α chain is shown in SEQ ID NOs. 83-87, and the codon-optimized full-length nucleotide sequence of the TCR β chain is shown in SEQ ID NOs. 88-92. Using a second-generation lentiviral packaging system, 293T cells were co-transfected with three plasmids (pHR-SFFV-TCR-IRES-EGFP, two plasmids containing other components necessary for the construction of infectious but non-replicating lentiviral particles, pMD2.G and pSPAX2) to prepare lentivirus and concentrate the virus. The specific steps are described using the 175cm2 culture flask packaging lentivirus test as an example:

[0089] Take 15×10 6 293T cells were cultured in 175 cm 2 Culture flasks were incubated in a 37°C, 5% CO2 incubator overnight. Transfection was performed when the cells grew to about 60% density in the culture flasks. Cell culture medium was changed 1 hour before transfection by adding 30 mL of fresh DMEM complete medium. Each 175 cm 2The plasmid dosage for the culture flask is: 10 μg pMD2.G, 30 μg pSPAX2, 40 μg pHR-SFFV-C152-1-IRES-EGFP, the ratio of transfection reagent PEI to plasmid is 4:1, i.e. 320 μg; prepare solution A by adding the above three plasmids to 750 μL serum-free DEME and vortex mixing; solution B is 750 μL serum-free DEME with corresponding PEI added and vortex mixing; solution A and solution B are mixed and vortexed thoroughly, let stand at room temperature for 20 minutes, added dropwise to the prepared 293T cells, and the culture flask is gently shaken to mix the culture medium, and cultured at 37°C and 5% CO2; 72 hours after transfection, the culture supernatant is collected and centrifuged (500g, 4°C, 5 minutes), the supernatant after centrifugation is filtered through a 0.45 μm filter, and finally 5×PEG8000 is added to concentrate the virus, mixed at 4°C for 2 hours, and let stand at 4°C overnight; centrifuged overnight (4000g, 4°C, 20 minutes), the supernatant is completely discarded, and 200 μL The precipitate was dissolved in PBS and the dissolved lentiviral sample was frozen at -80°C.

[0090] As a control, empty pHR-SFFV-IRES-EGFP lentivirus packaging was performed simultaneously, and the packaging steps were the same as the above pHR-SFFV-TCR-IRES-EGFP lentivirus packaging steps.

[0091] Example 2: TCR specificity identification and hepatitis B virus antigen peptide screening

[0092] The present invention transforms the TCR-knocked T cell line Jurkat 76 (J76) by transducing CD8 molecules and inserting a luciferase reporter gene downstream of NFAT, so that after the T cell line is specifically activated, the specific activation level of the cell can be characterized by detecting the fluorescent signal, which is conducive to high-throughput identification of the specificity of TCR. The cell line is referred to as J76.8 cells. The TCR (C341-8, C346-6, C346-7, C346-10 and C342-3) of the present invention is expressed on the surface of the J76.8 cell line by a lentiviral infection method. The cell is referred to as J76.8-TCR cell. The specific method is as follows: 1 mL of the lentiviral supernatant of the TCR packaged in Example 1 is taken before adding PEG8000 for concentration and used to infect the cell line; J76.8 cells are counted and 1×10 5 Place 10 J76.8 cells into one well of a 24-well plate; add 1 mL of TCR lentiviral supernatant and infect by centrifugation (700 g, 32°C, 1.5 hours); after centrifugation, supplement with 1 mL of 1640 complete medium and culture at 37°C, 5% CO2 for 3 days; flow cytometry detection of GFP positivity and CD3+TCRαβ+ double-positivity rates of more than 90% indicates successful construction of J76.8-TCR cells.

[0093] The present invention also constructs a lymphoblastoid cell line (LCL) homologous to TCR (C341-8, C346-6, C346-7, C346-10 and C342-3) as an antigen-presenting cell for specific identification and antigen peptide screening. The specific method is as follows: B95-8 cells are cultured in 1640 medium containing 2% FBS low-concentration serum for two weeks to produce a supernatant containing EBV virus; the supernatant containing EBV virus is collected and centrifuged (500g, 4°C, 5 minutes) to remove cell debris, and then filtered through a 0.45μm sterile filter; peripheral mononuclear cells (PBMC) of patients derived from TCR C152-1 are revived, and 1×10 6 The standard of 10 cells / mL virus supernatant was used. The corresponding volume of EBV supernatant was taken to resuspend the cells and infect at 37℃ for 2 hours. The infected cells were centrifuged (350g, 4℃, 5 minutes), the supernatant was removed, and the cells were washed with 2×10 5 The cells were resuspended in 1640 complete medium (containing 0.4 μg / mL cyclosporine A) at a density of 10 cells / mL; the resuspended cells were cultured in a 48-well plate at a standard rate of 1 mL / well at 37°C, 5% CO2 for three weeks, with half the medium replaced weekly with half of fresh 1640 complete medium (containing 0.4 μg / mL cyclosporine A); after three weeks, the cells began to aggregate and lymphoblastoid cell lines (LCLs) were formed.

[0094] The constructed J76.8-TCR (C341-8, C346-6, C346-7, C346-10 and C342-3) cells were co-incubated with LCL cells. Under the conditions of HBV protein peptide stimulation, the fluorescence signal of J76.8-TCR cells was detected to identify the specificity of TCR and the antigen peptide recognized. The specific method was as follows: 1×10 5 J76.8-TCR cells and 1×10 5 Homologous LCL cells were added with 3.5 μg / mL hepatitis B virus peptide library (15 amino acids per polypeptide, each polypeptide overlaps with the previous polypeptide by 4 amino acids, the peptide library used covers the entire hepatitis B virus genome, synthesized by Nanjing GenScript Biotechnology Co., Ltd.), with a total volume of 200 μL per well. An unstimulated control group was also set up, and the cells were stimulated in a 37°C, 5% CO2 incubator for 6 hours; 600g, centrifuged for 3 minutes, the supernatant discarded, and each well was resuspended with 50 μL PBS, transferred to a 96-well white plate, 50 μL luciferase detection substrate (product of Novizan) was added, mixed and reacted for 3 minutes, and chemiluminescence was detected by microplate reader.

[0095] The specific screening process is to first screen the various protein peptide libraries of hepatitis B virus, then subdivide the large protein peptide library into a small peptide library containing 10 single peptides for further screening, and then further divide the small peptide library that can activate TCR into single peptides for screening. Finally, the positive 15-amino acid single peptide is split into nine-amino acid single peptides in sequence for screening, and finally the specific antigen epitope of TCR is confirmed as shown in SEQ ID NO.1-2.

[0096] The results are as follows Figure 1 and 2 As shown in the figure, after co-incubation of J76.8-TCR with LCL, the upregulated expression of CD69 was used as a marker to confirm the HBV proteins recognized by these TCRs. The horizontal axis represents the synthetic peptide library that constitutes the HBV antigen (polymerase protein PLM, surface protein LS, core protein Core, and X protein). Among them, NS is a nonspecific 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 vertical axis represents the upregulated expression of CD69 molecules by TCR-receiving signal cells. The expression ratio of this molecule is used as an indicator of the activation strength of T cells under different stimuli. Among them, DMSO treatment is used as a negative control (NS), PMA / ionomycin and anti-CD3 antibodies are used as positive controls. The reactivity of different TCRs under the same antigen stimulation can be compared to screen for TCRs with higher reactivity to HBV proteins. The above five TCRs are all core protein-specific TCRs.

[0097] Luciferase Assay was then used to demonstrate that J76.8-TCR cells were significantly activated under stimulation with a hepatitis B virus protein peptide library. Further experiments demonstrated that the epitope that caused these J76.8-TCR cell activation was located on the hepatitis B virus core protein (Core40-2). Specifically, taking C341-8 as an example, that is, Figure 3As shown, the horizontal axis indicates different experimental treatment conditions, which are related to the presented peptide or stimulation method. NS (Negative Control): Negative control group, without any peptide or stimulatory factors, serves as a baseline control. Core: represents the whole protein of hepatitis B virus core antigen (HBcAg). Anti-CD3 is an anti-CD3 antibody used for nonspecific activation of T cells and is often used as a positive control. PMA is a strong cell activator, similar to anti-CD3, used to assess the overall activity of T cells and is often used as another positive control. The vertical axis represents the fold change of expression or response. To synthesize the peptide library of Core protein, we first screened a small mixed peptide library consisting of ten peptides to confirm that the target peptide existed in Core31-40 and Core41-50. Then, we stimulated the peptides separately to confirm that the peptides Core40 and Core41 had signals, indicating that the epitope existed in these peptides. Finally, based on the peptides Core40 and Core41 (15 amino acids in length), we designed peptides with a length of 9 or 10 amino acids covering these peptides. We selected the peptide with the strongest stimulation signal to confirm the final epitope Core40-2 (YRPPNAPIL). Figure 3 As shown, and so on, as Figure 3-7 As shown, C341-8, C346-6, C346-7, C346-10 and C342-3 recognize YRPPNAPIL on the core protein (Core) of hepatitis B virus.

[0098] Example 3: Identification of HLA molecules recognized by TCR

[0099] In Example 2, the TCRs of the present invention (C341-8, C346-6, C346-7, C346-10, and C342-3) were identified as recognizing hepatitis B virus antigen peptides. However, LCLs homologous to the TCRs include six HLA class I molecule types, necessitating further clarification of the HLA molecule types presenting the antigen peptides. To achieve this goal, in Example 3, homologous LCL cells were co-incubated with J76.8-TCR cells and stimulated with the aforementioned antigen peptides. Similarly, the fluorescent signal indicating the cell activation level was detected to identify the HLA molecule type bound to the antigen peptide.

[0100] The results are as follows Figures 8-10 As shown, J76.8-TCR cells were activated under antigenic peptide stimulation only when co-incubated with homologous LCL (Lymphoblastoid cell lines, immortalized cell lines prepared from patient-derived cells that act as antigen-presenting cells) and K562 cells expressing HLA-C*07:02 or HLA-C*06:02. Figures 8-10In the figure, the horizontal axis represents the antigen epitopes (HBV antigen peptides) identified by the above different TCRs; the vertical axis represents the increase in luciferase expression after J76.8-TCR cell activation. After subtracting the control treatment, the signal value increased by antigen peptide treatment is obtained, indicating specific activation. The HLA molecule identification diagram recognized by these TCRs is shown in Figure 2. Figures 8-10 As shown, they are C*07:02 (C341-8, C346-7, C346-6, C346-10) and C*06:02 (C342-3). Figure 8 For example, this chart shows the fold change (Fold Change) of expression or response of different cell lines (K562, K562-C*07:02, and LCL19) under the two experimental conditions of Core1 and Core41. The chart shows that under the irrelevant control peptide Core1 condition, the response or expression of the three cell lines did not change much; however, under the target peptide Core41 condition, the response of the LCL19 cell line and K562-C*07:02 was significantly higher than that of K562, showing extremely high Fold Change values. This indicates that under the Core41 condition, C*07:02 is responsible for presenting the Core40-2 target peptide and activating TCR signaling. The remaining figures are similar.

[0101] Example 4: Mutation of the TCR constant region

[0102] To verify whether exogenously transducing T cells with TCRs (C341-8, C346-6, C346-7, C346-10, and C342-3) can confer the function of secreting inflammatory and anti-cytokines to normal cells, the present invention requires further testing by transducing TCRs into primary T cells. To reduce the mismatch rate between the exogenous TCR and the cell's own TCR molecules during transduction of primary T cells and to improve the stability of the TCR molecules, this example introduces an artificial disulfide bond between the α-chain constant region and the β-chain constant region of the TCR molecule. Specifically, the threonine at position 48 of the TCR α chain constant region TRAC*01 was mutated to cysteine, and the serine at position 57 of the TCR β chain constant region TRBC*02 was mutated to cysteine; in addition, the cysteine ​​at position 75 of the TCR β chain constant region TRBC*02 was mutated to alanine to prevent the cysteine ​​at this site from mispairing with the cysteine ​​at position 48 of the mutated α chain constant region TRAC*01 to form a disulfide bond, thereby ensuring the formation of an artificial disulfide bond between the cysteine ​​at position 48 of the mutated α chain constant region TRAC*01 and the cysteine ​​at position 57 of the β chain constant region TRBC*02, thereby reducing the mispairing rate of TCR transduction of primary T cells.

[0103] Example 5: TCR transduction of primary T cells

[0104] To prove that TCR has functional activity after recognizing antigen epitopes, this example uses lentiviral infection to transduce TCR onto the surface of primary T cells. The transduction efficiency of TCR is determined by staining and detecting the expression of GFP carried by the lentiviral vector. The specific infection method is as follows: PBMCs are recovered, centrifuged (400g, 4°C, 5 minutes), and counted; according to 1×10 6 The cells were cultured in appropriate well plates using the standard of 10 cells / mL culture medium. The corresponding volume of human serum culture medium was added. T cells were activated by adding 10 μL / mL anti-CD3 + anti-CD28 activation mixture (Miltenyi Biotec) and 50 U / mL IL-2 (Peprotech). Primary T cells were collected 24 hours after activation, centrifuged (400 g, 4°C, 5 minutes), resuspended in human serum culture medium, and counted. 2.5 × 10 cells were added to each well. 5 The cells were divided into 48-well plates according to the standard of 10 cells, and the packaged point-mutated TCR (C341-8, C346-6, C346-7, C346-10 and C342-3) lentivirus was added to each well (the lentivirus packaging method was the same as in Example 1). Correspondingly, the packaged blank vector lentivirus was used as a control group. Each well was supplemented with human serum medium to a volume of 500 μL and infected by centrifugation (1000g, 32°C, centrifugation for 1 hour); after centrifugation, each well was supplemented with 500 μL of human serum medium, 50 U / mL IL-2 was added, and cultured at 37°C, 5% CO2 for 3 days.

[0105] Example 6: Functional detection of TCR

[0106] To verify whether the five TCRs of the present invention can be transduced into primary T cells and activated by hepatitis B antigen peptides to produce corresponding inflammatory cytokines and cytotoxic cytokines, this example transduced TCRs into primary T cells using the method of Example 5, and then co-incubated with homologous LCLs. Under the stimulation condition of hepatitis B virus antigen peptides, the cytokine levels were detected by flow cytometry.

[0107] The specific test method is: add 5×10 5 TCR-transduced primary T cells and 5×10 5Homologous LCL cells were added with 0.01 μg / mL novel coronavirus antigen peptide, 1 μg / mL anti-CD28 antibody (Biolegend product), and 3 μL / well CD107a-PE / Cy7 flow cytometry antibody (Biolegend product), with a total volume of 200 μL per well. An irrelevant peptide stimulation control group and an equal amount of DMSO control group were also set up; after stimulation for 2 hours in a 37°C, 5% CO2 incubator, a protein transport blocker (Biolegend product) was added to block the secretion of cytokines after stimulation, and the cells were cultured for 18 hours; 600g, centrifuged for 3 minutes, the supernatant was discarded, and surface antibody staining was performed, the cells were stained in the dark at 4°C for 30 minutes, and washed twice with PBS; the supernatant was discarded, and 100 μL of membrane permeabilization solution (BD product) was added to each well, the membrane was permeated for 20 minutes, and the membrane was washed twice with the matching membrane wash solution; 50 μL of intracellular flow cytometry antibody prepared with membrane wash solution was added to each well, the cells were stained in the dark at 4°C for 30 minutes, and the membrane was washed twice with the membrane wash solution, and 200 μL Cells were resuspended in 2% paraformaldehyde and analyzed by flow cytometry. Antibodies used for flow cytometry include: CD3-BUV805, CD8-BUV737, CD4-BB700, Viability-eFluor506, Perforin-BV421, CD107a-PE / Cy7, TNF-PE-CF574, IFN-γ-APC, Granzyme-PE, and IL-2-APC / Cy7.

[0108] The test results are as follows Figure 11-15 As shown in the figure, NS represents no stimulation, Unrelated peptide represents irrelevant peptide stimulation, and specific peptide stimulation, wherein the specific peptides are the peptide segments screened above, such as Core32-10-1 and LS96-10-2 peptide segments. Compared with the unstimulated control group and the irrelevant antigen peptide control group, after stimulation with the corresponding antigen peptides, the production of inflammatory cytokines IFN-γ and TNFα of T cells transduced with the TCR of the present invention is enhanced, as well as the expression of the cytokine CD107a is increased, proving that these TCRs are functionally active and can confer responsiveness to hepatitis B virus antigens on normal T cells, and are expected to become a new immune cell therapy strategy for hepatitis B virus infection.

[0109] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of the present invention.

Claims

1. A hepatitis B virus-specific TCR combination for recognizing HBV antigen epitopes, characterized in that: The hepatitis B virus-specific TCR combination includes a plurality of separated TCRs, and the plurality of separated hepatitis B virus-specific TCR combinations 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 antigen epitope, and the amino acid sequence of the first antigen epitope is 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 shown in SEQ ID NO:

2.

2. The hepatitis B virus-specific TCR combination according to claim 1, wherein The first TCR group includes a first TCRα chain variable region, a first TCRβ chain variable region, a second TCRα chain variable region, a second TCRβ chain variable region, a third TCRα chain variable region, a third TCRβ chain variable region, a fourth TCRα chain variable region and a fourth TCRβ chain variable region, wherein the first TCRα chain variable region includes a first CDR1α, a first CDR2α and a first CDR3α, the first TCRβ chain variable region includes a first CDR1β, a first CDR2β and a first CDR3β, the second TCRα chain variable region includes a second CDR1α, a second CDR2α and a second CDR 3α, the second TCRβ chain variable region includes a second CDR1β, a second CDR2β and a second CDR3β, the third TCRα chain variable region includes a third CDR1α, a third CDR2α and a third CDR3α, the third TCRβ chain variable region includes a third CDR1β, a third CDR2β and a third CDR3β, the fourth TCRα chain variable region includes a fourth CDR1α, a fourth CDR2α and a fourth CDR3α, the fourth TCRβ chain variable region includes a fourth CDR1β, a fourth CDR2β and a fourth CDR3β, and the amino acid sequence of the first CDR1α is as shown in SEQ The amino acid sequence of the first CDR2α 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 ID NO: 9, the amino acid sequence of the second CDR2α is shown in SEQ ID NO: 10, and 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 ID NO: 16, and the amino acid sequence of the third CDR3α is shown in SEQ ID NO: The amino acid sequence of the third CDR1β 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.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, and 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, and the amino acid sequence of the fourth CDR3β is shown in SEQ ID NO: 26; and / or, The second TCR group includes a fifth TCRα chain variable region and a fifth TCRβ chain variable region, the fifth TCRα chain variable region includes a fifth CDR1α, a fifth CDR2α and a fifth CDR3α, the fifth TCRβ chain variable region includes a fifth CDR1β, a fifth CDR2β and a fifth CDR3β, the amino acid sequence of the fifth CDR1α is shown in SEQ ID NO: 27, the amino acid sequence of the fifth CDR2α is shown in SEQ ID NO: 28, the amino acid sequence of the fifth CDR3α is shown in SEQ ID NO: 29, the amino acid sequence of the fifth CDR1β is shown in SEQ ID NO: 30, the amino acid sequence of the fifth CDR2β is shown in SEQ ID NO: 31, and the amino acid sequence of the fifth CDR3β is shown in SEQ ID NO:

32.

3. The hepatitis B virus-specific TCR combination according to claim 1, wherein The plurality of separated TCRs form five TCRs, wherein, Each TCR comprises a TCR α chain variable region and a TCR β chain variable region, the amino acid sequences of the five TCR α chain variable regions are shown in SEQ ID NO: 33 to SEQ ID NO: 37, and the amino acid sequences of the five TCR β chain variable regions are shown in SEQ ID NO: 38 to SEQ ID NO: 42; and / or, Each TCR comprises a TCRα chain and a TCRβ chain, the amino acid sequences of the five TCRα chains are shown in SEQ ID NO:43 to SEQ ID NO:47, and the amino acid sequences of the five TCRβ chains are shown in SEQ ID NO:48 to SEQ ID NO:52; and / or, Each TCR includes a TCRα mutant chain and a TCRβ mutant chain. The amino acid sequences of the five TCRα mutant chains are shown in SEQ ID NO: 53 to SEQ ID NO: 57, and the amino acid sequences of the five TCRβ mutant chains are shown in SEQ ID NO: 58 to SEQ ID NO:

62.

4. A nucleic acid molecule combination, characterized in that: The nucleic acid molecule combination includes a plurality of nucleic acid molecules, and the plurality of nucleic acid molecules encode and form the hepatitis B virus-specific TCR combination according to any one of claims 1 to 3, wherein the hepatitis B virus-specific TCR combination includes five TCRs, wherein: Each TCR comprises a TCR α chain variable region and a TCR β chain variable region, the nucleotide sequences of the five TCR α chain variable regions are shown in SEQ ID NO: 63 to SEQ ID NO: 67, and the nucleotide sequences of the five TCR β chain variable regions are shown in SEQ ID NO: 68 to SEQ ID NO: 72; and / or, Each TCR comprises a TCRα chain and a TCRβ chain, the nucleotide sequences of the five TCRα chains are shown in SEQ ID NO:73 to SEQ ID NO:77, and the nucleotide sequences of the five TCRβ chains are shown in SEQ ID NO:78 to SEQ ID NO:82; and / or, Each TCR comprises a TCRα mutant chain and a TCRβ mutant chain, the nucleotide sequences of the five TCRα mutant chains are shown in SEQ ID NO: 83 to SEQ ID NO: 87, and the nucleotide sequences of the five TCRβ mutant chains are shown in SEQ ID NO: 88 to SEQ ID NO: 92; and / or, Each TCR includes α chain codon-optimized nucleotides and β chain codon-optimized nucleotides, the five α chain codon-optimized nucleotide sequences are shown in SEQ ID NO:93 to SEQ ID NO:97, and the five β chain codon-optimized nucleotide sequences are shown in SEQ ID NO:98 to SEQ ID NO:

102.

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

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

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

8. Use of the nucleic acid molecule combination according to claim 4, the expression vector according to claim 5, 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 hepatitis B virus-related diseases.

Images