A tcr capable of simultaneously recognizing hbv core antigen epitope and envelope protein epitope and a coding sequence thereof

Abstract

The application relates to a TCR capable of simultaneously recognizing an HBV core antigen epitope and an envelope protein epitope and a coding sequence thereof, the TCR can simultaneously combine LLDYQGMLPV-HLA-A0201 and FLPSDFFPSV-HLA-A0201 complexes, the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, the full-length amino acid sequence of CDR3 of the TCR alpha chain variable domain is CARNTGNQFYF (SEQ ID NO:1), and the full-length amino acid sequence of CDR3 of the TCR beta chain variable domain is CASSPSTGTSYGYTF (SEQ ID NO:2). By adopting the technical scheme, the application provides a TCR capable of simultaneously and specifically recognizing an antigen epitope FLPSDFFPSV (SEQ ID NO:3) from an HBV core antigen and an antigen epitope LLDYQGMLPV (SEQ ID NO:4) from an HBV envelope protein, the short peptides can form complexes with HLA-A0201 and are presented to the cell surface together.

Description

Technical Field

[0001] This invention relates to a TCR capable of simultaneously recognizing two HBV antigenic epitopes and their coding sequences, and also to the use of the TCR in the prevention and treatment of HBV-related diseases. Background Technology

[0002] HBV infection is a leading cause of liver cancer and chronic hepatitis worldwide, accounting for more than 50% of liver cancer cases. HBV core antigen and envelope protein are important structural proteins in the hepatitis B virus (HBV) genome, encoding the core protein and surface antigen, respectively, and participating in viral particle assembly and immune responses. The HBV core antigen-derived epitope FLPSDFFPSV (SEQ ID NO:3) and the HBV envelope protein-derived epitope LLDYQGMLPV (SEQ ID NO:4) are important therapeutic targets for HBV-related diseases. Currently, there is some research on the immune recognition of HBV antigens and T cell responses, but the TCR capable of simultaneously recognizing multiple HBV antigen epitopes remains unclear.

[0003] TCR-T (T-cell receptor engineered T-cell) adoptive immunotherapy is an immunotherapy strategy that uses genetically engineered T cells from a patient to precisely recognize and kill specific viral antigens. Its core involves introducing a specific TCR gene into T cells, enabling them to recognize antigens presented on the cell surface via the master histocompatibility complex (MHC), triggering an immune response. Therefore, one of the current research focuses is screening for TCRs that can specifically recognize HBV-related antigenic peptides and transducing them into T cells for cellular immunotherapy. However, it remains unclear which antigenic peptides can effectively induce specific T cell clones, and whether TCRs that simultaneously recognize multiple HBV antigenic epitopes exist. Therefore, there is an urgent need to develop TCRs that specifically recognize HBV antigenic peptides and related technologies. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art. This invention provides a TCR for recognizing multiple antigenic epitopes of HBV and its coding sequence. This TCR can specifically recognize the complex formed by the core antigen short peptide FLPSDFFPSV (SEQ ID NO:3) and the envelope protein short peptide LLDYQGMLPV (SEQ ID NO:4) with HLA-A0201.

[0005] The technical solution of the present invention is a TCR that recognizes multiple antigenic epitopes of HBV. This TCR can simultaneously bind to the LLDYQGMLPV-HLA-A0201 and FLPSDFFPSV-HLA-A0201 complexes. The TCR contains a TCRα chain variable domain and a TCRβ chain variable domain, wherein the full-length amino acid sequence of the TCRα chain variable domain CDR3 is CARNTGNQFYF (SEQ ID NO:1), and the full-length amino acid sequence of the TCRβ chain variable domain CDR3 is CASSPSTGTSYGYTF (SEQ ID NO:2).

[0006] The core amino acid sequences of the variable domains CDR1, CDR2, and CDR3 of the TCRα chain are as follows:

[0007] αCDR1-SSNFYA (SEQ ID NO:5)

[0008] αCDR2-MTLNGDE (SEQ ID NO:6)

[0009] αCDR3-ARNTGNQFY (SEQ ID NO:7)

[0010] The core amino acid sequences of the variable domains CDR1, CDR2, and CDR3 of the TCRβ chain are as follows:

[0011] βCDR1-MNHEY (SEQ ID NO:8)

[0012] βCDR2-SVGAGI (SEQ ID NO:9)

[0013] βCDR3-ASSPSGTSYGYT (SEQ ID NO: 10).

[0014] In another preferred embodiment, the TCR includes a TCRα chain variable domain that is an amino acid sequence with at least 90% sequence similarity to SEQ ID NO:18, and a TCRβ chain variable domain that is an amino acid sequence with at least 90% sequence similarity to SEQ ID NO:20.

[0015] In another preferred embodiment, the TCR is in a soluble form.

[0016] In another preferred embodiment, the TCR is in single-chain form.

[0017] In another preferred embodiment, the TCR is a multivalent TCR complex, characterized in that it contains at least two TCR molecules, and each TCR is linked by a flexible linker peptide (such as G4S), and the complex is capable of recognizing multiple antigenic epitopes of HBV.

[0018] In another preferred embodiment, the N-terminus or C-terminus of the α-chain and β-chain of the TCR is bound to a coupling molecule, which is selected from fluorescent markers, biotin, Fc fragments or PEG, etc., and the binding does not affect the ability of the TCR to recognize the LLDYQGMLPV-HLA-A0201 and FLPSDFFPSV-HLA-A0201 complexes.

[0019] A second aspect of the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule described in the first aspect of the present invention, or a complementary strand thereof.

[0020] In another preferred embodiment, the nucleic acid molecule encodes the nucleotide sequence SEQ ID NO:17 of the TCRα chain variable domain.

[0021] In another preferred embodiment, the nucleic acid molecule encodes the nucleotide sequence SEQ ID NO:19 of the TCRβ chain variable domain.

[0022] A third aspect of the present invention provides a vector comprising the nucleic acid molecule described in the second aspect of the present invention; preferably, the vector is a lentiviral vector.

[0023] A fourth aspect of the present invention provides a host cell comprising the vector described in the third aspect of the present invention or having the nucleic acid molecule described in the second aspect of the present invention integrated into its genome.

[0024] A fifth aspect of the present invention provides a cell that transduces the nucleic acid molecule described in the second aspect of the present invention or the vector described in the third aspect of the present invention; preferably, the cell is a T cell or a stem cell.

[0025] A sixth aspect of the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR as described in the first aspect of the present invention, a nucleic acid molecule as described in the second aspect of the present invention, a carrier as described in the third aspect of the present invention, or a cell as described in the fifth aspect of the present invention.

[0026] The seventh aspect of the present invention provides the use of the TCR described in the first aspect, the nucleic acid molecule described in the second aspect, the vector described in the third aspect, or the cell described in the fifth aspect in the preparation of a medicament for treating diseases related to HBV infection, such as liver cancer and hepatitis. Attached Figure Description

[0027] Figure 1 To analyze the overlap between the TCR patterns of CD8+ T cells identifying LLDYQGMLPV-HLA-A0201 tetramer and those identifying FLPSDFFPSV-HLA-A0201 tetramer CD8+ T cells;

[0028] Figure 2 This is a molecular docking diagram of the TCR, which identifies the HBV multiantigen epitope, binding to the LLDYQGMLPV-HLA-A0201 and FLPSDFFPSV-HLA-A0201 complexes as identified in this invention. The pink peptide chain represents the TCR gene α chain (TRA), and the yellow peptide chain represents the TCR gene β chain (TRB).

[0029] Figure 3 The results of the LDH killing function assay of effector T cells transduced with the HBV multiantigen epitope TCR identified in this invention were verified; the blue bars represent the control group blocked with MHC antibody, and the orange bars represent the experimental group.

[0030] Figure 4 To transduce the activation markers of the TCR of this invention after effector T cells kill target cells, the formula detection results are as follows: 4-1BB is a T cell activation marker, and CD8 is a CD8+ T cell marker. The two markers can determine the proportion of activated CD8+ T cells in the total number of T cells. Detailed Implementation

[0031] The technical solutions in this embodiment will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] It should be noted that in the description of this invention, all directional indications (such as up, down, forward, backward, etc.) are only used to explain the relative positional relationship and movement of the components in a certain specific posture (as shown in the figure). If the specific posture changes, the directional indication will also change accordingly.

[0033] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0034] like Figure 1 As shown, through the study of HBV-infected liver tissue infiltrating lymphocytes, a TCR that can simultaneously recognize the HBV antigen short peptides LLDYQGMLPV (SEQ ID NO:4) and FLPSDFFPSV (SEQ ID NO:3) was found. The antigen short peptides can form a complex with HLA-A0201 and be presented together to the cell surface.

[0035] The present invention also provides a nucleic acid molecule encoding the TCR.

[0036] In a preferred embodiment of the present invention, the core amino acid sequences of the α-chain variable domains CDR1, CDR2, and CDR3 of the TCR are as follows:

[0037] αCDR1-SSNFYA (SEQ ID NO:5)

[0038] αCDR2-MTLNGDE (SEQ ID NO:6)

[0039] αCDR3-ARNTGNQFY (SEQ ID NO:7)

[0040] The core amino acid sequences of the variable domains CDR1, CDR2, and CDR3 of the TCRβ chain are as follows:

[0041] βCDR1-MNHEY (SEQ ID NO:8)

[0042] βCDR2-SVGAGI (SEQ ID NO:9)

[0043] βCDR3-ASSSPSTGTSYGYT (SEQ ID NO:10)

[0044] The CDR region amino acid sequence of the present invention can be inserted into any suitable framework structure to prepare a chimeric TCR. As long as the framework structure is compatible with the CDR region of the TCR of the present invention, those skilled in the art can design or synthesize TCR molecules with corresponding functions based on the CDR region disclosed in the present invention.

[0045] Therefore, the TCR molecule of the present invention refers to a TCR molecule containing the above-mentioned α and β chain CDR region sequences and any suitable framework structure.

[0046] The variable domain of the TCRα chain of the present invention is an amino acid sequence having at least 90%, more preferably 95% sequence identity with SEQ ID NO:18; the variable domain of the TCRβ chain of the present invention is an amino acid sequence having at least 90%, more preferably 95% sequence identity with SEQ ID NO:20.

[0047] In a preferred embodiment of the present invention, the TCR molecule is a heterodimer composed of an α chain and a β chain. Specifically, the α chain of the heterodimer TCR includes a variable domain and a constant region, and the amino acid sequence of the variable domain of the α chain includes CDR1 (SEQ ID NO:5), CDR2 (SEQ ID NO:6), and CDR3 (SEQ ID NO:7) of the aforementioned α chain.

[0048] Preferably, the TCR molecule contains the amino acid sequence SEQ ID NO:18 as a variable domain of the α chain. On the other hand, the β chain of the heterodimeric TCR also contains a variable domain and a constant region, wherein the amino acid sequence of the variable domain of the β chain includes CDR1 (SEQ ID NO:8), CDR2 (SEQ ID NO:9), and CDR3 (SEQ ID NO:10) of the β chain.

[0049] Preferably, the TCR molecule contains the amino acid sequence SEQ ID NO:20 as a variable domain of the β chain.

[0050] The TCR of the present invention can be used independently or combined with coupling molecules via covalent bonds or other means, preferably via covalent bonding.

[0051] The conjugated molecules are selected from fluorescent markers, biotin, Fc fragments, or PEG, and their binding does not affect the ability of TCR to recognize the LLDYQGMLPV-HLA-A0201 and FLPSDFFPSV-HLA-A0201 complexes.

[0052] Nucleic acid molecules

[0053] A second aspect of the present invention discloses a nucleic acid molecule that encodes the TCR molecule or a fragment thereof described in the first aspect of the present invention; the fragment may include one or more CDRs, variable domains of the α strand and β strand, or the entire α strand and β strand.

[0054] The core nucleotide sequences encoding the CDR1, CDR2, and CDR3 variable domains of the α chain of the TCR molecule in the second aspect of this invention are as follows:

[0055] αCDR1-TCCAGCAATTTTTATGCC (SEQ ID NO:11)

[0056] αCDR2-ATGACTTTAAATGGGGATGAA (SEQ ID NO:12)

[0057] αCDR3- GCCCGGAACACCGGTAACCAGTTCTAT (SEQIDNO:13)

[0058] The core nucleotide sequences encoding the CDR1, CDR2, and CDR3 variable domains of the TCR molecule in the second aspect of this invention are as follows:

[0059] βCDR1-ATGAACCATGAATAC (SEQ ID NO:14)

[0060] βCDR2-TCAGTTGGTGCTGGTATC (SEQ ID NO:15)

[0061] βCDR3- GCCAGCAGTCCTTCTACCGGGACATCGTATGGCTACACC (SEQ ID NO:16)

[0062] Therefore, the nucleotide sequences encoding the TCRα chain provided by the present invention include, but are not limited to, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13; meanwhile, the nucleotide sequences encoding the TCRβ chain may include, but are not limited to, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16.

[0063] The nucleotide sequence of the nucleic acid molecule can be single-stranded or double-stranded, and can be either RNA or DNA, and can selectively contain or exclude introns.

[0064] Preferably, the nucleic acid molecule of the present invention has an intron-free structure and can efficiently express the polypeptide of the present invention. For example, the nucleic acid sequence used to express the variable region of the TCR α chain of the present invention is preferably a nucleic acid molecule capable of encoding the amino acid sequence shown in SEQ ID NO:1; the nucleic acid sequence used to express the variable region of the TCR β chain is preferably a nucleic acid molecule capable of encoding the amino acid sequence shown in SEQ ID NO:2. Alternatively, the amino acid sequence of the variable region of the TCR α chain may be as shown in SEQ ID NO:11 to SEQ ID NO:13, and the amino acid sequence of the variable region of the TCR β chain may be as shown in SEQ ID NO:14 to SEQ ID NO:16, and the corresponding nucleic acid sequence may be its respective coding sequence.

[0065] It should be noted that, due to the degeneracy of the genetic code, different nucleotide sequences may encode the same polypeptide. Therefore, the nucleic acid sequence encoding the TCR of this invention can be either completely identical to the sequence listed in the accompanying drawings or a degenerate variant thereof. For example, a "degenerate variant" refers to a nucleic acid molecule that, despite differences in nucleotide sequence from SEQ ID NO:13, is still able to encode the protein sequence shown in SEQ ID NO:1.

[0066] The following specific embodiments provide a more detailed description of the present invention. Experimental conditions not specifically described in the embodiments were performed using conventional experimental methods. Unless otherwise specified, all experimental materials and reagents involved in the embodiments are commercially available.

[0067] Short peptides LLDYQG and FLPSD (Jiangsu Genscript Biotech Co., Ltd.) were synthesized. These peptides were then renatured with biotin-labeled HLA-A0201 to prepare pMHC haploids. These haploids were then combined with PE-labeled streptavidin (BD Biosciences) to form PE-labeled tetramers.

[0068] Freshly excised HBV-positive liver tissue was cut into 2-4 mm pieces using sterile surgical scissors and forceps, and placed in 24-well plates containing 2 mL of complete T-cell culture medium per well. The plates were then cultured in a 37°C, 5% CO2 incubator to isolate tumor-infiltrating lymphocytes (TILs).

[0069] TILs were stained with PE-labeled tetramers, and CD8+ and tetramer double-positive cells were screened for single-cell TCR sequencing. LLDYQG and FLPSD tetramer TCR immunochromatograms were analyzed, and TCR genes appearing simultaneously in both immunochromatograms were screened and cloned.

[0070] A lentiviral vector containing a candidate TCR gene was synthesized, and the specificity of the candidate TCR gene was verified by LDH cell killing and T cell activation assays. Target cells were COS7-HLA-A0201 cells loaded with LLDYQG or FLPSD, while the control group was COS7-HLA-A0201 cells without the TCR gene. The results showed that the candidate TCR gene could specifically recognize both LLDYQG and FLPSD-loaded COS7-HLA-A0201 cells simultaneously.

[0071] The full-length nucleotide sequence of the TCR α-chain variable region, SEQ ID NO:17, is ATGGAGAAGAATCCTTTGGCAGCCCCATTACTAATCCTCTGGTTTCATCTTGACTGCGTGAGCAGCATACTGAACGTGGAACAAAGTCCTCAGTCACTGCATGTTCAGGAGGGAGACAGCACCAATTTCACCTGCAGCTTCCCTTCCAGCAATTTTTATGCCTTACACTGGTACAGATGGGAAACTGCAAAAAGCCCCGAGGCCTTGTTTGTAATGACTTTAAATGGGGATGAAAAGAAGAAAGGACGAATAAGTGCCACTCTTAATACCAAGGAGGGTTACAGCTATTTGTACATCAAAGGATCCCAGCCTGAAGACTCAGCCACATACCTCTGTGCCCGGAACACCGGTAACCAGTTCTATTTTGGGACAGGGACAAGTTTGACGGTCATTCCA (SEQ ID NO:17).

[0072] The full-length amino acid sequence of the TCR α-chain variable region, SEQ ID NO:18, is MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIP (SEQ ID NO:18).

[0073] The full-length nucleotide sequence of the TCRβ chain variable region, SEQ ID NO:19, is ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTCCTTCTACCGGGACATCGTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTA (SEQ ID NO:19).

[0074] The full-length amino acid sequence of the TCRβ chain variable region, SEQ ID NO:20, is MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPSTGTSYGYTFGSGTRLTVV (SEQ ID NO:20).

Claims

1. A TCR that simultaneously recognizes HBV core antigen epitopes and envelope protein epitopes, characterized in that, This TCR can simultaneously bind to the LLDYQGMLPV-HLA-A0201 and FLPSDFFPSV-HLA-A0201 complexes. This TCR contains a TCR α-chain variable domain and a TCR β-chain variable domain, wherein: The core amino acid sequences of the variable domains CDR1, CDR2, and CDR3 of the TCRα chain are as follows: αCDR1-SSNFYA (SEQ ID NO:5) αCDR2-MTLNGDE (SEQ ID NO:6) αCDR3-ARNTGNQFY (SEQ ID NO:7) The core amino acid sequences of CDR1, CDR2, and CDR3 in the TCRβ chain are as follows: βCDR1-MNHEY (SEQ ID NO:8) βCDR2-SVGAGI (SEQ ID NO:9) βCDR3-ASSPSGTSYGYT (SEQ ID NO: 10).

2. The TCR that simultaneously recognizes HBV core antigen epitopes and envelope protein epitopes according to claim 1, characterized in that, The TCR contains an α-chain variable domain that has at least 90% sequence similarity to an amino acid sequence of SEQ ID NO:18, and a β-chain variable domain that has at least 90% sequence similarity to an amino acid sequence of SEQ ID NO:

20.

3. The TCR that simultaneously recognizes HBV core antigen epitopes and envelope protein epitopes according to claim 1 or 2, characterized in that, The N- or C-terminus of the α- and β-chains of its TCR are bound to a coupling molecule selected from fluorescent markers, biotin, Fc fragments, or PEG. The binding does not affect the ability of the TCR to recognize the LLDYQGMLPV-HLA-A0201 and FLPSDFFPSV-HLA-A0201 complexes.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a nucleic acid sequence or its complementary sequence encoding the TCR molecule of any one of claims 1-3 above.

5. A carrier, characterized in that, The vector contains the nucleic acid molecule as described in claim 4; the vector is a lentiviral vector.

6. An isolated host cell, characterized in that, The host cell contains the vector of claim 5, or the host cell's chromosome contains an exogenous nucleic acid molecule of claim 4.

7. A cell, characterized in that, The cell is transduced with the nucleic acid molecule of claim 4 or the vector of claim 5; the cell is a T cell or a stem cell.

8. A pharmaceutical composition, characterized in that, It includes a pharmaceutically acceptable carrier and the TCR as described in any one of claims 1-3, the nucleic acid molecule as described in claim 4, or the cell as described in claim 7.

9. The use of the TCR as described in claim 1, characterized in that, Used to prepare drugs for the treatment of liver cancer, hepatitis, or diseases related to HBV infection.

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