TCR molecule for recognizing tumor-associated antigen and application thereof

Abstract

The invention provides a TCR capable of being specifically combined with a GLSPTVWLSV peptide fragment of HBV full-length envelope protein. The peptide fragment and HLA-A * 02 can form a compound to exist on the surface of an infected cell. The invention also provides a nucleic acid molecule for coding the TCR and a carrier containing the nucleic acid molecule. In addition, the invention also provides a cell for transducing the TCR.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to TCRs capable of recognizing short peptides derived from HBV antigens and their coding sequences, T cells transduced with the above TCRs, and their uses in the prevention and treatment of HBV-related diseases. Background Art

[0002] Hepatocellular carcinoma (HCC) is one of the most common and highly malignant cancers in the world, characterized by rapid progression, poor prognosis, and a 5-year survival rate of less than 5%. Currently, effective treatment options for liver cancer patients are limited to the early stages of the disease with a low tumor burden. The current clinical treatment methods for HCC mainly include surgical resection, chemotherapy, and radiotherapy, but the degree of patient benefit is not high, and ultimately most patients die of disease recurrence or metastasis. Some tyrosine kinase inhibitors such as sorafenib, lenvatinib, and cabozantinib have been approved for HCC, but the overall survival improvement in patients with advanced HCC has been observed to be poor in clinical practice. Immune checkpoint blockade (ICB) therapy is a new treatment method for liver cancer, and its efficacy when used alone or in combination with kinase inhibitors has different response rates and survival benefits in HCC patients (Kudo M et al. Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial. Lancet 391, 1163–1173 (2018).). Despite the progress made in existing treatment methods, effective and durable systemic treatment options for HCC are still limited.

[0003] In recent years, adoptive cell therapy (ACT) has been increasingly widely used in the treatment of HCC. ACT therapies include specific and non-specific types, mainly by administering live immune cells with or without antigen specificity to achieve the purpose of treating cancer. Non-specific adoptive cell therapy includes lymphokine-activated killer cells, autologous cytokine-induced killer cells (CIK), and natural killer cells. These immune cell-based therapies, when used in combination with conventional therapies and / or kinase inhibitors, have shown good safety in HCC patients. However, the lack of specificity limits these immunotherapies to adjuvant therapy only (Anthony T Tan & Sophia Schreiber, Adoptive T-cell therapy for HBV-associated HCC and HBV infection. Antiviral Res 176(2020)). Specific adoptive cell therapy mainly includes chimeric antigen receptor-engineered T cells and T cell receptor-engineered T cells (TCR-T) therapy, which enhance the tumor recognition and killing effects of T cells through genetic engineering to improve the treatment effect.

[0004] TCR is a receptor molecule naturally expressed on T cells for recognizing short peptide antigens, which are combined with major histocompatibility complex (MHC) molecules (also known as human leukocyte antigen (HLA)) and presented by antigen-presenting cells (APCs) (Davis et al. Ligand recognition by alpha beta T cell receptors. Annu Rev Immunol 16, 523-44(1998).). CD8+ T cells, also known as cytotoxic T cells, have TCRs that specifically recognize polypeptides bound to MHC class I molecules. CD8+ T cells are usually responsible for detecting and mediating the destruction of diseased cells, including cancerous cells and virus-infected cells.

[0005] China is a high-incidence area of HCC, and approximately 75-80% of HCC patients are induced by hepatitis B virus (HBV) infection. Virus-specific T cells in these HCC patients are often deleted or dysfunctional, resulting in a delay in the immune attack to eliminate cancer cells (Hao XS, et al. Twenty-year trends of primary liver cancer incidence rates in an urban Chinese population. Eur J Cancer Prev 12. 273–279(2003)). This problem can be overcome by TCR-T therapy. More than 90% of HBV-HCC has HBV DNA integration and may express intact or truncated forms of HBV antigens, and HBV epitopes that can be specifically recognized by T cells are processed and presented by MHC molecules on the surface of HBV-infected hepatocytes or HCC cells. Moreover, as an exogenous invader, HBV has low homology with the human endogenous genome. Therefore, it is expected to use T cells modified with HBV-specific T cell receptors (HBV-TCR T cells) to treat HBV-HCC. Currently, several cases of advanced HBV-HCC patients have been reported to be safe and have potential therapeutic effects after receiving HBsAg-specific TCR redirected T cell immunotherapy (Fanping Meng et al., Immunotherapy of HBV-related advanced hepatocellular carcinoma with short-term HBV-specific TCR expressed Tcells: results of dose escalation, phase I trial. Hepatol Int 15(6), 1402–1412(2021)).

[0006] Therefore, there is an urgent need in the art for TCRs that can recognize HBV antigens. Summary of the Invention

[0007] The object of the present invention is to provide a T cell receptor that recognizes tumor-associated antigens, especially short peptides of HBV antigens.

[0008] In a first aspect, the present invention provides a T cell receptor (TCR) that can bind to a GLSPTVWLSV polypeptide-HLA-A*02 complex.

[0009] In a preferred embodiment, the TCR comprises a variable domain of the TCR α chain and a variable domain of the TCR β chain,

[0010] The amino acid sequence of CDR1 of the variable region of the TCR α chain is selected from the following group: SEQ ID NO:8, 25, 35, 51;

[0011] The amino acid sequence of CDR2 of the variable region of the TCR α chain is selected from the following group: SEQ ID NO:9, 26, 36, 52;

[0012] The amino acid sequence of CDR3 of the variable region of the TCR α chain is selected from the following group: SEQ ID NO:10, 19, 27, 37, 43, 53;

[0013] The amino acid sequence of CDR1 of the variable region of the TCR β chain is selected from the following group: SEQ ID NO:13, 30, 46, 56;

[0014] The amino acid sequence of CDR2 of the variable region of the TCR β chain is selected from the following group: SEQ ID NO:14, 31, 47, 57; and / or

[0015] The amino acid sequence of CDR3 of the variable region of the TCR β chain is selected from the following group: SEQ ID NO:15, 22, 32, 40, 48, 58.

[0016] In a preferred embodiment, the amino acid sequences of CDR1, CDR2 and CDR3 of the variable region of the TCR α chain are selected from the following combinations: SEQ ID NO:8, 9 and 10; SEQ ID NO:8, 9 and 19; SEQ ID NO:25, 26 and 27; SEQ ID NO:35, 36 and 37; SEQ ID NO:25, 26 and 43; SEQ ID NO:51, 52 and 53;

[0017] and / or

[0018] The amino acid sequences of CDR1, CDR2 and CDR3 of the variable region of the TCR β chain are selected from the following combinations: SEQ IDNO:13, 14 and 15; SEQ ID NO:13, 14 and 22; SEQ ID NO:30, 31 and 32; SEQ ID NO:30, 31 and 40; SEQID NO:46, 47 and 48; SEQ ID NO:56, 57 and 58.

[0019] In a preferred embodiment, the amino acid sequences of CDR1, CDR2 and CDR3 of the variable region of the TCR α chain and the amino acid sequences of CDR1, CDR2 and CDR3 of the variable region of the TCR β chain are selected from the following combinations:

[0020] SEQ ID NO:8, 9 and 10 and SEQ ID NO:13, 14 and 15;

[0021] SEQ ID NO: 8, 9, and 19 and SEQ ID NO: 13, 14, and 22;

[0022] SEQ ID NO: 25, 26, and 27 and SEQ ID NO: 30, 31, and 32;

[0023] SEQ ID NO: 35, 36, and 37 and SEQ ID NO: 30, 31, and 40;

[0024] SEQ ID NO: 25, 26, and 43 and SEQ ID NO: 46, 47, and 48;

[0025] SEQ ID NO: 51, 52, and 53 and SEQ ID NO: 56, 57, and 58.

[0026] In a preferred embodiment, the TCR comprises a variable domain of the TCR α-chain and a variable domain of the TCR β-chain, wherein the variable domain of the TCR α-chain is an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID NO: 7, 17, 24, 34, 42, or 50; and / or the variable domain of the TCR β-chain is an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID NO: 12, 21, 29, 39, 45, or 55.

[0027] In a preferred embodiment, the amino acid sequence of the variable domain of the TCR α-chain of the TCR is as shown in SEQ ID NO: 7, 17, 24, 34, 42, or 50; and / or the amino acid sequence of the variable domain of the TCR β-chain is as shown in SEQ ID NO: 12, 21, 29, 39, 45, or 55.

[0028] In a preferred embodiment, the amino acid sequence of the TCR α-chain of the TCR and the amino acid sequence of the variable domain of the TCR β-chain are selected from the following combinations:

[0029] SEQ ID NO: 7 and 12; SEQ ID NO: 17 and 21; SEQ ID NO: 24 and 29; SEQ ID NO: 34 and 39; SEQ ID NO: 42 and 45; SEQ ID NO: 50 and 55.

[0030] In a preferred embodiment, the amino acid sequence of the α-chain of the TCR is selected from the group consisting of: SEQ ID NO: 6, 16, 23, 33, 41, and 49; and / or

[0031] The amino acid sequence of the β-chain of the TCR is selected from the group consisting of: SEQ ID NO: 11, 20, 28, 38, 44, and 54.

[0032] In a preferred embodiment, the amino acid sequences of the α-chain and β-chain of the TCR are selected from the following combinations: SEQ ID NO: 6 and 11; SEQ ID NO: 16 and 20; SEQ ID NO: 23 and 28; SEQ ID NO: 33 and 38; SEQ ID NO: 41 and 44; SEQ ID NO: 49 and 54.

[0033] In a preferred embodiment, the TCR is soluble.

[0034] In a preferred embodiment, the TCR is a single-chain TCR.

[0035] In a preferred embodiment, the TCR is formed by connecting the variable domain of the α-chain and the variable domain of the β-chain through a peptide linker sequence.

[0036] In a preferred embodiment, the TCR comprises (i) the variable domain of the TCR α-chain and all or part of the constant region of the TCR α-chain except for the transmembrane domain; and (ii) the variable domain of the TCR β-chain and all or part of the constant region of the TCR β-chain except for the transmembrane domain.

[0037] In a preferred embodiment, the TCR comprises the variable domain of the α-chain and the variable domain of the β-chain and all or part of the constant domain of the β-chain except for the transmembrane domain, but it does not comprise the constant domain of the α-chain, and the variable domain of the α-chain of the TCR forms a heterodimer with the β-chain.

[0038] In a preferred embodiment, a conjugate is bound to the C- or N-terminus of the α-chain and / or β-chain of the TCR.

[0039] In a preferred embodiment, the conjugate bound to the T cell receptor is a detectable label, a therapeutic agent, a PK modification moiety, or a combination of any of these substances; preferably, the therapeutic agent is an anti-CD3 antibody.

[0040] In a second aspect, the present invention provides a multivalent TCR complex comprising at least two TCR molecules, and at least one of the TCR molecules is the TCR described in the first aspect.

[0041] In a third aspect, the present invention provides an isolated nucleic acid molecule encoding the TCR described in the first aspect.

[0042] In a fourth aspect, the present invention provides a vector, which contains the nucleic acid molecule described in the third aspect; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector.

[0043] In a fifth aspect, the present invention provides an isolated host cell, which contains the vector described in the fourth aspect or the exogenous nucleic acid molecule described in the third aspect is integrated into the genome.

[0044] In a sixth aspect, the present invention provides a cell, which expresses the TCR described in the first aspect on its surface.

[0045] In a preferred embodiment, the cell is transduced with the nucleic acid molecule described in the third aspect or the vector described in the fourth aspect.

[0046] In a preferred embodiment, the cell is a T cell, NK cell, NKT cell or stem cell.

[0047] In a seventh aspect, the present invention provides a pharmaceutical composition, which contains a pharmaceutically acceptable carrier and the TCR described in the first aspect, the TCR complex described in the second aspect, the nucleic acid molecule described in the third aspect, the vector described in the fourth aspect, or the cell described in the sixth aspect.

[0048] In an eighth aspect, the present invention provides the use of the T cell receptor described in the first aspect, the TCR complex described in the second aspect, or the cell described in the sixth aspect for preparing a drug for treating tumors or infections.

[0049] In a preferred embodiment, the tumor is a tumor that specifically expresses the complete or partial HBV envelope protein; the infection is HBV infection.

[0050] In a preferred embodiment, the tumor is a tumor that specifically expresses the GLSPTVWLSV peptide segment.

[0051] In a preferred embodiment, the tumor is hepatocellular carcinoma.

[0052] In a preferred embodiment, the infection is hepatitis B.

[0053] In a ninth aspect, the present invention provides the T cell receptor described in the first aspect, the TCR complex described in the second aspect, or the cell described in the sixth aspect, which is used as a drug for treating tumors or infections.

[0054] In a preferred embodiment, the tumor is a tumor that specifically expresses the complete or partial HBV envelope protein; the infection is HBV infection.

[0055] In a preferred embodiment, the tumor is a tumor that specifically expresses the GLSPTVWLSV peptide.

[0056] In a preferred embodiment, the tumor is hepatocellular carcinoma.

[0057] In a preferred embodiment, the infection is hepatitis B.

[0058] In a tenth aspect, the present invention provides a method for treating a tumor or an infection, comprising administering a therapeutically effective amount of the T cell receptor according to the first aspect, the TCR complex according to the second aspect, the cell according to the sixth aspect, or the pharmaceutical composition according to the seventh aspect to a subject in need of treatment.

[0059] In a preferred embodiment, the tumor is a tumor that specifically expresses the complete or partial HBV envelope protein; the infection is HBV infection.

[0060] In a preferred embodiment, the tumor is a tumor that specifically expresses the GLSPTVWLSV peptide.

[0061] In a preferred embodiment, the tumor is hepatocellular carcinoma.

[0062] In a preferred embodiment, the infection is hepatitis B.

[0063] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Shows the detection results of the expression levels of different TCR molecules of the present invention on the surface of reporter cells;

[0065] Figure 2 Shows the detection results of the expression efficiency of different TCR molecules on the surface of HEK-293T cells by flow cytometry;

[0066] Figure 3 Shows the detection results of the affinity levels of different TCRs of the present invention with the target;

[0067] Figure 4 Shows the cell activation effects mediated by different TCR molecules of the present invention;

[0068] Figure 5 Shows the detection results of the specificity of different TCR molecules of the present invention for target recognition;

[0069] Figure 6Shows the expression efficiency of different TCR molecules of the present invention on primary T cells;

[0070] Figure 7 Shows the detection results of the killing effects of different TCR-T cells of the present invention on different target cells;

[0071] Figure 8 Shows the detection results of the levels of cytokines released by TCR-T cells stimulated with different concentrations of target antigen. Detailed implementation manners

[0072] After extensive and in-depth research, the inventors unexpectedly found that a TCR that can specifically bind to the GLSPTVWLSV peptide segment (SEQ ID NO: 1) of the full-length HBV envelope protein presented by MHC class I molecules, and the peptide segment GLSPTVWLSV can form a complex with HLA-A*02 and exist on the surface of infected cells. The present invention also provides a nucleic acid molecule encoding the TCR and a vector containing the nucleic acid molecule. In addition, the present invention also provides a cell transduced with the TCR of the present invention. Based on this, the present invention was completed.

[0073] Term definitions

[0074] The terms used herein have the meanings commonly understood by those skilled in the art. For a clear understanding of the present invention, some terms are defined as follows.

[0075] MHC molecule

[0076] MHC molecules are proteins of the immunoglobulin superfamily and can be class I or class II MHC molecules. Therefore, they are specific for antigen presentation, and different individuals have different MHCs, which can present different short peptides in a protein antigen to the surface of their respective APC cells. The MHC in humans is usually referred to as the HLA gene or HLA complex.

[0077] GLSPTVWLSV peptide segment

[0078] The GLSPTVWLSV peptide is located at amino acids 348 - 357 of the full-length HBV envelope protein and forms a complex with HLA-A*02 on the surface of infected cells. T cells that recognize this peptide-HLA complex have been reported (Webster et al., Longitudinal analysis of CD8+T cells specific for structural and nonstructural hepatitis B virus proteins in patients with chronic hepatitis B:implications for immunotherapy. J Virol 78(11),5707–5719(2004).). The GLSPTVWLSV peptide shows a high degree of sequence conservation among all HBV genotypes. Therefore, the GLSPTVWLSV polypeptide-HLA-A*02 complex provides an ideal target for TCR-T immunotherapy of HBV-HCC or chronic HBV infections.

[0079] T cell receptor

[0080] The T cell receptor used herein has the meaning commonly understood by those skilled in the art. It is the only receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC). In the immune system, the binding of antigen-specific TCR to the pMHC complex triggers direct physical contact between T cells and antigen-presenting cells (APCs). Then, other cell membrane surface molecules on both T cells and APCs interact, which leads to a series of subsequent cell signaling and other physiological responses, enabling T cells with different antigen specificities to exert immune effects on their target cells.

[0081] The TCR is a cell membrane surface glycoprotein existing in the form of a heterodimer of an α chain / β chain or a γ chain / δ chain. In 95% of T cells, the TCR heterodimer is composed of an α and a β chain, while 5% of T cells have a TCR composed of a γ and a δ chain. The natural αβ heterodimeric TCR has an α chain and a β chain, and the α chain and the β chain constitute the subunits of the αβ heterodimeric TCR. Generally speaking, each of the α and β chains contains a variable region, a joining region, and a constant region. The β chain usually also contains a short diversity region between the variable region and the joining region, but this diversity region is often regarded as part of the joining region. Each variable region contains 3 CDRs (complementary determining regions), CDR1, CDR2, and CDR3, embedded in the framework regions. The CDR regions determine the binding of the TCR to the pMHC complex, among which CDR3 is recombined from the variable region and the joining region and is called the hypervariable region. The α and β chains of the TCR are generally regarded as having two "domains" each, namely the variable domain and the constant domain. The variable domain is composed of the joined variable region and the joining region. The sequences of the TCR constant domains can be found in the public database of the International Immunogenetics Information System (IMGT). For example, the constant domain sequence of the α chain of the TCR molecule is "TRAC*01", and the constant domain sequences of the β chain of the TCR molecule are "TRBC1*01" or "TRBC2*01". In addition, the α and β chains of the TCR also contain a transmembrane region and a cytoplasmic region, and the cytoplasmic region is very short.

[0082] In the present invention, the terms "the polypeptide of the present invention", "the TCR of the present invention", and "the T cell receptor of the present invention" can be used interchangeably.

[0083] During antigen processing, the antigen is degraded inside the cell and then carried to the cell surface by MHC molecules. The T cell receptor can recognize the peptide-MHC complex on the surface of antigen-presenting cells. Therefore, the present invention provides a TCR molecule capable of binding to the GLSPTVWLSV polypeptide-HLA-A*02 complex. Preferably, the TCR molecule is isolated or purified. Each of the α and β chains of the TCR has 3 complementary determining regions (CDRs).

[0084] In a preferred embodiment of the present invention, the amino acid sequence of CDR1 of the variable domain of the α-chain of the TCR is selected from the group consisting of: SEQ ID NO:8, 25, 35, 51; the amino acid sequence of CDR2 of the variable domain of the α-chain of the TCR is selected from the group consisting of: SEQ ID NO:9, 26, 36, 52; the amino acid sequence of CDR3 of the variable domain of the α-chain of the TCR is selected from the group consisting of: SEQ ID NO:10, 19, 27, 37, 43, 53; the amino acid sequence of CDR1 of the variable domain of the β-chain of the TCR is selected from the group consisting of: SEQ ID NO:13, 30, 46, 56; the amino acid sequence of CDR2 of the variable domain of the β-chain of the TCR is selected from the group consisting of: SEQ ID NO:14, 31, 47, 57; and / or, the amino acid sequence of CDR3 of the variable domain of the β-chain of the TCR is selected from the group consisting of: SEQ ID NO:15, 22, 32, 40, 48, 58.

[0085] The amino acid sequences of the CDR regions of the present invention described above can be embedded into any suitable framework structure to prepare chimeric TCRs. As long as the framework structure is compatible with the CDR regions 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 regions disclosed in the present invention. Therefore, the TCR molecules of the present invention refer to TCR molecules comprising the above-mentioned CDR region sequences of the α- and / or β-chains and any suitable framework structure. The variable domain of the TCR α-chain of the present invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity with SEQ ID NO:7, 17, 24, 34, 42 or 50; and / or the variable domain of the TCR β-chain of the present invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity with SEQ ID NO:12, 21, 29, 39, 45 or 55.

[0086] In a preferred example of the present invention, the TCR molecule of the present invention is a heterodimer composed of an α-chain and a β-chain. Specifically, on the one hand, the α-chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the amino acid sequence of the variable domain of the α-chain comprises CDR1, CDR2 and CDR3 of the above-mentioned α-chain. Preferably, the TCR molecule comprises the amino acid sequence SEQ ID NO:7, 17, 24, 34, 42 or 50 of the variable domain of the α-chain. On the other hand, the β-chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the amino acid sequence of the variable domain of the β-chain comprises CDR1, CDR2 and CDR3 of the above-mentioned β-chain. Preferably, the TCR molecule comprises the amino acid sequence SEQ ID NO:12, 21, 29, 39, 45 or 55 of the variable domain of the β-chain.

[0087] In a preferred embodiment of the present invention, the TCR molecule of the present invention is a single-chain TCR molecule composed of part or all of the α-chain and / or part or all of the β-chain. Descriptions of single-chain TCR molecules can be found in the reference Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-12658. According to the description in the literature, those skilled in the art can easily construct single-chain TCR molecules containing the CDRs region of the present invention. Specifically, the single-chain TCR molecule contains Vα, Vβ, and Cβ, preferably connected in the order from the N-terminus to the C-terminus.

[0088] The amino acid sequence of the variable domain of the α-chain of the single-chain TCR molecule contains CDR1, CDR2, and CDR3 of the above-mentioned α-chain. Preferably, the single-chain TCR molecule contains the amino acid sequence of the variable domain of the α-chain SEQ ID NO: 7, 17, 24, 34, 42, or 50. The amino acid sequence of the variable domain of the β-chain of the single-chain TCR molecule contains CDR1, CDR2, and CDR3 of the above-mentioned β-chain. Preferably, the single-chain TCR molecule contains the amino acid sequence of the variable domain of the β-chain SEQ ID NO: 12, 21, 29, 39, 45, or 55.

[0089] In a preferred embodiment of the present invention, the constant domain of the TCR molecule of the present invention is a human constant domain. Those skilled in the art know or can obtain the amino acid sequence of the human constant domain by referring to relevant books or the public database of IMGT (International Immunogenetics Information System). For example, the constant domain sequence of the α-chain of the TCR molecule of the present invention can be "TRAC*01", and the constant domain sequence of the β-chain of the TCR molecule can be "TRBC1*01" or "TRBC2*01". The 53rd position of the amino acid sequence given in IMGT's TRAC*01 is Arg, which is represented here as: Arg53 of exon 1 of TRAC*01, and so on for the others. Preferably, the amino acid sequence of the α-chain of the TCR molecule of the present invention is SEQ ID NO: 6, 16, 23, 33, 41, and 49, and / or the amino acid sequence of the β-chain is SEQ ID NO: 11, 20, 28, 38, 44, and 54.

[0090] Naturally occurring TCR is a membrane protein stabilized by its transmembrane region. Just as immunoglobulins (antibodies) serve as antigen recognition molecules, TCR can also be developed for diagnostic and therapeutic applications, in which case soluble TCR molecules are required. Soluble TCR molecules do not include their transmembrane regions. Soluble TCR has a wide range of uses. It can not only be used to study the interaction between TCR and pMHC, but also serve as a diagnostic tool for detecting infections or as a biomarker for autoimmune diseases. Similarly, soluble TCR can be used to deliver therapeutic agents (such as cytotoxic compounds or immunostimulatory compounds) to cells presenting specific antigens. Additionally, soluble TCR can bind to other molecules (such as anti-CD3 antibodies) to redirect T cells, thereby targeting cells presenting specific antigens. The TCR molecules of the present invention are all soluble, thus providing more options for the development of TCR-T cell therapy or TCR-like protein drugs.

[0091] To obtain soluble TCR, the transmembrane region of TCR can be removed. Preferably, such TCR can comprise (i) all or part of the TCRα chain except its transmembrane domain, and (ii) all or part of the TCRβ chain except its transmembrane domain, wherein both (i) and (ii) comprise the variable domain and at least a part of the constant domain of the TCR chain, and the α chain and the β chain form a heterodimer. More preferably, such TCR can comprise the variable domains of the α chain and the β chain and all or part of the constant domain of the β chain except the transmembrane domain, but it does not comprise the constant domain of the α chain, and the variable domain of the α chain of the TCR forms a heterodimer with the β chain.

[0092] The TCR of the present invention can be a stable soluble single-chain TCR formed by connecting the variable domains of the α and β chains of TCR with a flexible peptide chain. It should be noted that the flexible peptide chain in the present invention can be any peptide chain suitable for connecting the variable domains of the TCRα and β chains.

[0093] The TCR of the present invention can also be provided in the form of a multivalent complex. The multivalent TCR complex of the present invention comprises a polymer formed by combining two, three, four or more TCRs of the present invention. For example, a tetramer can be generated using the tetrameric domain of p53, or a complex formed by combining multiple TCRs of the present invention with another molecule. The TCR complex of the present invention can be used for tracking or targeting cells presenting specific antigens in vitro or in vivo, and can also be used as an intermediate for generating other multivalent TCR complexes with such applications.

[0094] The TCR of the present invention can be used alone or combined with a conjugate in a covalent or other manner, preferably in a covalent manner. The conjugate includes a detectable label (for diagnostic purposes, where the TCR is used to detect the presence of cells presenting the GLSPTVWLSV polypeptide-HLA-A*02 complex), a therapeutic agent, a PK (protein kinase) modification moiety, or a combination or conjugate of any of the above substances.

[0095] Detectable labels for diagnostic purposes include, but are not limited to: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0096] Therapeutic agents that can be combined or conjugated with the TCR of the present invention include, but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539); 2. Biological toxins (Chaudhary et al., 1989, Nature 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy 51, 565); 3. Cytokines, such as IL-2, etc. (Gillies et al., 1992, PNAS 89, 1428; Card et al., 2004, Cancer Immunology and Immunotherapy 53, 345; Halin et al., 2003, Cancer Research 63, 3202); 4. Antibody Fc fragments (Mosquera et al., 2005, The Journal Of Immunology 174, 4381); 5. Antibody scFv fragments (Zhu et al., 1995, International Journal of Cancer 62, 319); 6. Gold nanoparticles / nanorods (Lapotko et al., 2005, Cancer letters 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115); 7. Virus particles (Peng et al., 2004, Gene therapy 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research 65, 11631); 9. Nanomagnetic particles; 10. Prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. Chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0097] In addition, the TCR of the present invention may also be a chimeric TCR comprising sequences derived from more than one species. For example, studies have shown that murine TCRs can be expressed more efficiently in human T cells than human TCRs. Thus, the TCRs of the present invention may comprise human variable domains and murine constant domains. A drawback of this approach is that it may trigger an immune response. Therefore, when used in adoptive T cell therapy, there should be a regulatory regimen for immunosuppression to allow the engraftment of murine-expressing T cells.

[0098] It should be understood that amino acid names are represented by the internationally accepted single-letter or three-letter English notations in this text, and the correspondence between the single-letter and three-letter notations of amino acid names is as follows: Ala(A), Arg(R), Asn(N), Asp(D), Cys(C), Gln(Q), Glu(E), Gly(G), His(H), Ile(I), Leu(L), Lys(K), Met(M), Phe(F), Pro(P), Ser(S), Thr(T), Trp(W), Tyr(Y), Val(V).

[0099] Nucleic acid molecule

[0100] The present invention provides nucleic acid molecules encoding the TCR of the present invention or portions thereof, which portions may be one or more CDRs, variable domains of the α and / or β chains, and the α chain and / or β chain.

[0101] The nucleotide sequence of the nucleic acid molecule of the present invention may be single-stranded or double-stranded, the nucleic acid molecule may be RNA or DNA, and may contain or not contain introns. Preferably, the nucleotide sequence of the nucleic acid molecule of the present invention does not contain introns but is capable of encoding the polypeptide of the present invention.

[0102] It should be understood that due to the degeneracy of the genetic code, different nucleotide sequences may encode the same polypeptide. Thus, the nucleic acid sequences encoding the TCR of the present invention may be the same as or degenerate variants of the nucleic acid sequences shown in the present invention.

[0103] The nucleotide sequence may be codon-optimized. Different cells have different codon usages, and according to the cell type, the codons in the sequence can be changed to increase the expression level. Codon usage tables for mammalian cells and many other organisms are well known to those skilled in the art.

[0104] The full-length sequence or fragments of the nucleic acid molecule of the present invention can generally be obtained by, but not limited to, PCR amplification, recombination, or artificial synthesis methods. Currently, it is possible to completely obtain the DNA sequence encoding the TCR of the present invention (or its fragments, or its derivatives) by chemical synthesis. Then this DNA sequence can be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. The DNA can be the coding strand or the non-coding strand.

[0105] vector

[0106] The present invention also relates to vectors comprising the nucleic acid molecules of the present invention, including expression vectors, i.e., constructs capable of expressing in vivo or in vitro. Commonly used vectors include bacterial plasmids, bacteriophages, and animal and plant viruses.

[0107] Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors, retroviral vectors, lentiviral vectors, baculoviral vectors.

[0108] Preferably, the vector can transfer the nucleotide of the present invention into cells, such as T cells, such that the cells express a TCR specific for HBV antigen. Ideally, the vector should be able to express continuously at a high level in T cells.

[0109] cell

[0110] The present invention also relates to host cells genetically engineered with the vectors or coding sequences of the present invention. The host cells contain the vectors of the present invention or the nucleic acid molecules of the present invention are integrated into the chromosomes. The host cells are selected from: prokaryotic cells and eukaryotic cells, such as Escherichia coli, yeast cells, CHO cells, etc.

[0111] In addition, the present invention also includes isolated cells expressing the TCR of the present invention, which can be, but are not limited to, T cells, NK cells, NKT cells, stem cells, especially T cells. The T cells can be derived from T cells isolated from a subject, or can be a mixed cell population isolated from a subject, such as a part of a peripheral blood lymphocyte (PBL) population. For example, the cells can be isolated from peripheral blood mononuclear cells (PBMC), and can be CD4+ helper T cells or CD8+ cytotoxic T cells. The cells can be in a mixed population of CD4+ helper T cells / CD8+ cytotoxic T cells. Generally, the cells can be activated with antibodies (such as anti-CD3 or anti-CD28 antibodies) so that they can be more easily transfected, for example, transfected with a vector containing a nucleotide sequence encoding the TCR molecule of the present invention.

[0112] Alternatively, the cells of the present invention can also be or be derived from stem cells, such as hematopoietic stem cells (HSC). Transferring genes into HSC does not result in the expression of TCR on the cell surface because stem cells do not express CD3 molecules on their surface. However, when the stem cells differentiate into lymphoid precursors that migrate to the thymus, the expression of CD3 molecules will initiate the expression of the introduced TCR molecules on the surface of thymocytes.

[0113] There are many methods suitable for T cell transfection with DNA or RNA encoding the TCR of the present invention (e.g., Robbins et al., (2008) J. Immunol. 180:6116-6131). T cells expressing the TCR of the present invention can be used for adoptive immunotherapy. Those skilled in the art are aware of many suitable methods for adoptive therapy (e.g., Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).

[0114] Therapeutic method

[0115] Treatment can be carried out by isolating T cells from patients or volunteers infected with HBV virus-induced HCC or chronic hepatitis, introducing the TCR of the present invention into the above T cells, and then transfusing these genetically engineered modified cells back into the patient's body. Therefore, the present invention provides a method for treating HBV-related diseases, including transfusing isolated T cells expressing the TCR of the present invention, preferably, the T cells are derived from the patient himself, into the patient's body. Generally, it includes (1) isolating the patient's T cells, (2) in vitro transducing the T cells with the nucleic acid molecule of the present invention or a nucleic acid molecule capable of encoding the TCR molecule of the present invention, and (3) transfusing the genetically engineered modified T cells into the patient's body. The number of isolated, transfected and transfused cells can be determined by the physician.

[0116] Advantages of the present invention:

[0117] (1) The TCR of the present invention can specifically bind to the complex composed of the GLSPTVWLSV peptide segment and HLA-A*02, and the effector cells transduced with the TCR of the present invention can be specifically activated;

[0118] (2) The effector cells transduced with the TCR of the present invention can specifically kill GLSPTVWLSV peptide segment-positive target cells;

[0119] (3) After being stimulated by target cells, the effector cells transduced with the TCR of the present invention can secrete a large amount of cytokines; and

[0120] (4) The present invention provides TCR molecules with different levels of affinity activity for the target pMHC complex, and all are soluble, which can provide more options for the development of TCR-T cell therapy or TCR-like protein drugs.

[0121] The following specific examples further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions noted in the following examples, they are generally carried out under conventional conditions, such as the conditions described in (Sambrook and Russell et al., Molecular Cloning - A Laboratory Manual (Third Edition) (2001) CSHL Press), or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources without special instructions.

[0122] Example:

[0123] Example 1

[0124] 1.1 Antigen - specific T - cell activation

[0125] Peripheral blood T cells were activated by co - culturing healthy donor PBMCs (Peripheral blood mononuclear cells, Miaoshun (Shanghai) Biotechnology Co., Ltd.) with peptide - loaded DCs (Dendritic cells), or by using the method of peptide immunization, or by using the method of immunization with HLA - molecule - and - peptide - loaded DCs. Then, T cells were stained with fluorescent - tagged multimers (such as Tetramer), and T cells that could be stained by the multimer were sorted using a flow cytometer. This group of T cells might be antigen - specifically activated T cells. The peptide sequence used in this study was: GLSPTVWLSV (SEQ ID NO:1). The peptide sequence used in this example was common to HBV proteins.

[0126] 1.2 TCR sequence acquisition

[0127] Screening and cloning of TCR were carried out with reference to the literature (Paria BC, Levin N, Lowery FJ, et al. Rapid Identification and Evaluation of Neoantigen-reactive T-Cell Receptors From Single Cells. Journal of Immunotherapy (Hagerstown, Md.: 1997). 2021 Jan; 44(1): 1-8. Pai, J.A., Satpathy, A.T. High-throughput and single-cell T cell receptor sequencing technologies. Nat Methods 18, 881-892 (2021). De Simone M, Rossetti G and Pagani M (2018) Single Cell T Cell Receptor Sequencing: Techniques and Future Challenges. Front. Immunol. 9: 1638. doi: 10.3389 / fimmu.2018.01638). The screened TCR clones were named TCR-01, TCR-02, TCR-03, TCR-04, TCR-05, and TCR-06 respectively. The full-length amino acid sequences of the TCR variable regions are shown in Table 1. The variable region subtypes and CDR region sequences of TCR were analyzed through the IMGT website to obtain the sequences of the CDR1 region, CDR2 region, and CDR3 region of different TCRs (the amino acid sequences are shown in Table 1).

[0128] TCR Sequence Listing

[0129] Table 1. Amino Acid Sequence Listing

[0130]

[0131]

[0132]

[0133] Example 2

[0134] 2.1 Construction of TCR Gene Vector

[0135] The variable region sequences of the TCR alpha and TCR beta genes cloned in Example 1 were respectively ligated to the constant regions of murine TCR alpha and TCR beta to construct the full-length sequences of TCR alpha and TCR beta. Then, the paired full-length sequences of TCR alpha and TCR beta were ligated through the 2A fragment and constructed into a lentiviral vector for lentiviral packaging. The lentivirus was concentrated by centrifugation and stored in a -80 °C refrigerator. The basic experimental steps are as follows:

[0136] 1) On the first day, HEK-293T cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were inoculated: The cells were inoculated at approximately 1.0×10^7 cells / T175 flask (cultured in 30 ml of medium) so that the cell density reached 80 - 90% on the next day for transfection.

[0137] 2) On the second day, plasmid transfection: Before transfection, the medium needed to be changed to DMEM medium with 10% FBS but without antibiotics. First, plasmid complexes were prepared separately: The following plasmids were added to 1.5 ml of Opti-MEM (Thermo Fisher Scientific; 31985-070) and mixed well: 18 μg of the psPAX2 plasmid (Addgene; catalog number: 12260), 9 μg of the pMD2.G plasmid (Addgene; catalog number: 12259), and 18 μg of the viral vector plasmid. The lentiviral vector plasmids carrying the TCR-01, TCR-02, TCR-03, TCR-04, TCR-05, and TCR-06 genes were respectively named: lenti-TCR-01, lenti-TCR-02, lenti-TCR-03, lenti-TCR-04, lenti-TCR-05, and lenti-TCR-06. Then, transfection reagent complexes were prepared: According to the mass ratio of plasmid to PEI of 1:3, 45 μL (3 mg / mL) of PEI (polysciences: 24765) was added to 1.5 mL of Opti-MEM and mixed well, and left at room temperature for 5 min; then the transfection reagent complex was added to the plasmid complex, mixed well, and left for 20 min. Finally, the transfection complex was slowly dropped into the 293T cell culture dish, gently mixed, and cultured in a cell culture incubator at 37 °C with 5% CO2.

[0138] 3) On the fifth day, harvest the virus: Collect the supernatant of the culture medium 72 h after transfection, and centrifuge at 2000 rpm for 10 min to remove cell debris. Filter the supernatant using a 0.45 μM filter membrane, and transfer the filtrate to a dedicated centrifuge tube for balancing. Ultracentrifuge at 25,000 rpm for 2 h using an ultracentrifuge. After pouring off the supernatant, resuspend the lentivirus with X-VIVO-15 medium, aliquot the lentivirus, and store it in an ultra-low temperature freezer at -80 °C. Prepare lentiviruses containing TCR-01, TCR-02, TCR-03, TCR-04, TCR-05, and TCR-06 genes respectively according to this procedure.

[0139] 2.2 Preparation of TCR-reporter gene cell lines

[0140] The reporter gene cell line used in this example is Jurkat cells that have knocked out endogenous TCRα and β genes, carry the NFAT promoter and luciferase gene, and express CD8a and CD8b genes, named Jurkat-TCRKO-031 (Smith-Garvin J.E. et al., 2009. T Cell Activation. Ann. Rev. Immunol. 27:591-619). Add the lentiviruses carrying candidate TCR genes to the reporter gene cells respectively. After 2-3 days, detect the expression of TCR by multimer staining and flow cytometry.

[0141] The basic steps are as follows:

[0142] 1) Synthesize HLA-A02-restricted HPV target polypeptide (purchased from GenScript, SEQ ID No.: 1), and first prepare a PE-fluorescein-conjugated MHCⅠ-peptide complex multimer Dextramer according to the instructions of the kit (Immudex, product number: U-LX02) for detecting TCRs that specifically recognize the target polypeptide;

[0143] 2) Take Jurkat-TCRKO-031 cells in the logarithmic growth phase and inoculate them into a 6-well plate at a density of 5×10 5 / well, and make up the volume of the culture medium in each well to 2 mL;

[0144] 3) Take 100 μL of the packaged lentivirus solution and add it drop by drop to the well plate, and gently shake to fully mix the virus solution with the culture medium;

[0145] 4) Place the orifice plate in an incubator at 37°C with 5% CO2 for 48 h. Name the engineered cells transfected with TCR-01, TCR-02, TCR-03, TCR-04, TCR-05, and TCR-06 genes as TCR-P1, TCR-P2, TCR-P3, TCR-P4, TCR-P5, and TCR-P6, respectively. The NTD group is untransfected cells, serving as negative control cells.

[0146] 5) After 48 h, take out 200 μL of the cell suspension, centrifuge to collect the cells, and wash the cells once with FACS buffer (PBS containing 2% FBS).

[0147] 6) Resuspend the cells with 100 μL of FACS buffer respectively. Add 1 μL of APC anti-human CD3 and 2 μL of the above-prepared Dextramer to each tube. After mixing evenly, incubate at 4°C for 30 minutes.

[0148] 7) After incubation, wash the cells twice with FACS buffer and centrifuge to collect the cells.

[0149] 8) Resuspend the cells with 100 μL of FACS buffer and detect the proportion of CD3 and Dextramer positive cell populations by flow cytometry. The flow cytometry results are as Figure 1 shown.

[0150] The results show that the 6 TCR sequences screened in the present invention can be integrated into the cell genome by lentiviral transfection and expressed on the cell surface. The transfection efficiency of the 6 TCR molecules all reaches more than 80%. The Dextramer staining results show that the 6 TCR molecules can all bind to Dextramer. Among them, the binding activities of TCR-P4, TCR-P2, and TCR-P1 are relatively high (>60%), followed by TCR-P3 and TCR-P6 (about 30%), and TCR-P5 is the weakest.

[0151] 2.3 Affinity evaluation

[0152] To further evaluate the affinity levels of the 6 TCR molecules of the present invention for the target, in this example, the pCDNA3.4 plasmid expressing human CD3 and the TCR molecules are co-transfected into HEK-293T cells, and the binding levels of TCR are detected with different concentrations of peptide-MHC I tetramer. The specific steps are as follows:

[0153] 1) Biotinylate the expressed peptide-MHC fusion protein (Bio Ying) according to the instructions (Thermo, catalog number: 21338), and then add streptavidin labeled with PE to prepare the Tetramer tetramer (Immatics, catalog number: MP S-866).

[0154] 2) Take HEK-293T cells in good growth state and seed the cells into a 6-well plate at a density of 5×10 5 / well. Place the plate in a cell culture incubator and incubate overnight;

[0155] 3) Dilute 1.5 μg of CD3 plasmid and 4.5 μg of PEI with 100 μL of Opti-MEM medium respectively. Slowly add the PEI dilution to the CD3 dilution and incubate at room temperature for 20 minutes;

[0156] 4) Slowly add the above 200 μL of plasmid-PEI mixture to one well of the 6-well plate incubated overnight. Place the plate in a cell culture incubator and incubate for 4 - 6 hours;

[0157] 5) After incubation, change to fresh medium. Add 100 μL of lentivirus solution carrying the candidate TCR gene to each well respectively. Place the plate back into the incubator and incubate for 48 hours;

[0158] 6) Take out the plate, digest and collect the cells with 0.25% trypsin, wash the cells once with FACS buffer, and centrifuge to collect the cells;

[0159] 7) Resuspend the cells with FACS buffer at a density of 2×10 6 / mL. Take 25 μL of the cell suspension and add it to a 96-well U-bottom plate, and inoculate 10 wells for each sample;

[0160] 8) Dilute the APC anti-CD3 antibody (Biolegend, catalog number: 300412) with FACS buffer at a ratio of 1:50; Dilute the Tetramer prepared in step 1 with FACS buffer in a gradient manner, with the highest concentration being 17.28 μg / mL, and a total of 10 concentration points are diluted at a 3-fold gradient;

[0161] 9) Add 25 μL of the CD3 antibody dilution and 50 μL of the corresponding Tetramer dilution to each well respectively, and incubate at 4°C in the dark for 30 minutes;

[0162] 10) Wash the cells twice with FACS buffer, centrifuge to collect the cells, resuspend the cells with 100 μL of FACS buffer, and detect the TCR transfection efficiency and target affinity level by flow cytometry.

[0163] The detection results show that the 6 TCR molecules of the present invention can be effectively co-transfected with the CD3 plasmid and expressed in a mammalian cell system, and the transfection efficiency is equivalent, which is 30 - 40% ( Figure 2)。Taking the average fluorescence intensity value (MFI) of PE bound to the surface of CD3⁺ cell population as the vertical axis and the working concentration of Tetramer as the horizontal axis to fit a non-linear curve, the results show that the EC 50 concentration values of TCR-P1, TCR-P2, TCR-P3, TCR-P4, TCR-P5 and TCR-P6 binding to Tetramer are 0.332, 0.722, 0.389, 1.423, 8.857 and 1.102 μg / mL respectively. Generally speaking, among the six molecules of the present invention, the EC 50 values and dose curves of TCR-P1 and TCR-P3 molecules binding to the target polypeptide are relatively consistent, and the affinity is at the highest level; the binding activities of TCR-P2, TCR-P4 and TCR-P6 are second, and TCR-P5 is the lowest( Figure 3 ).

[0164] 2.4 Biological activity evaluation

[0165] Using T2 cells as target cells, loading polypeptides at different concentrations, activating reporter cells, and calculating the EC 50 value. The basic steps are as follows:

[0166] 1) Take T2 cells in the logarithmic growth phase, resuspend them in complete medium to 2×10 6 / mL, and inoculate the cell suspension into a 96-well U-bottom deep-well plate at 100 μL / well;

[0167] 2) Dilute the target polypeptide with complete medium, with the initial concentration of 5 μM, and perform 3-fold gradient dilution to 10 concentration points. Corresponding to each well, add 100 μL of the diluted polypeptide solution to the cells in step 1, mix well, and incubate in a 37 °C incubator for 1 - 2 hours to load the polypeptide onto the surface of T2 cells;

[0168] 3) After incubation, wash off the excess polypeptide with medium, centrifuge to collect the cells, resuspend the cells with 0.9 mL of medium per well, and inoculate the T2 cells loaded with different concentrations of polypeptide into a 96-well white opaque cell culture plate at 50 μL / well;

[0169] 4) Take the TCR-P1, TCR-P2, TCR-P3, TCR-P4, TCR-P5 and TCR-P6 cells expressing the TCR molecules of the present invention prepared in Example 2, adjust the cell density to 1×10 6 / mL with complete medium, add them to the 96-well plate at 50 μL / well (using untransfected Jurkat-TCRKO-031 cells as negative control), mix well on a microplate shaker, and then transfer the cells to a 37 °C incubator for 4 - 6 hours;

[0170] 5) After the incubation, add the D-luciferin reaction solution (purchased from Thermo Fisher Scientific, catalog number L2912) to the culture plate at 100 μL / well, react for 10 minutes at room temperature in the dark, and then use a microplate reader (Thermo Fisher Scientific, Varioskan.LUX) to detect the fluorescence signal value (RLU);

[0171] 6) Use Graphpad Prism software for data analysis, fit the dose-dependent curve, and calculate the EC 50 value. The results are as Figure 4 shown. The EC 50 values corresponding to TCR-P1, TCR-P2, TCR-P3, TCR-P4, TCR-P5, and TCR-P6 are 0.0392, 0.111, 0.115, 0.504, 0.172, and 0.0157 μM, respectively.

[0172] The results show that Jurkat-TCRKO-031 cells expressing the 6 TCR molecules shown in the present invention can all be activated by T2 cells loaded with target peptides and express luciferase signals. Among them, TCR-P1 and TCR-P3 have the highest activation effect and relatively low EC50 values, showing good biological activity. Although the EC50 values of TCR-P5 and TCR-P6 activated by T2 cells loaded with target peptides are also relatively low, their activation signals are weak.

[0173] Example 3: Analysis of off-target effects of TCR molecules

[0174] Referring to the experimental steps in Example 2.4, use polypeptides with similar sequences (purchased from GenScript, SEQ ID No.: 2-5) as potential off-target polypeptides, use T2 cells loaded with different off-target polypeptides as target cells, and Jurkat-TCRKO-031 expressing the TCR molecules described in this patent as effector cells. After co-culture, detect the luciferase activity. The basic steps are as follows:

[0175] 1) Dissolve the off-target polypeptide in DMSO to a stock solution of 10 mM. Take 3 μL of the polypeptide stock solution and add it to 1 mL of complete medium (RPMI 1640 medium containing 10% FBS) to make its working concentration 30 μM;

[0176] 2) Take T2 cells in good growth state, centrifuge to collect the cells, and resuspend the cells in the above medium containing different polypeptides to a density of 1×10 6 / mL. Place the cell suspension in the cell culture incubator and incubate for 1 hour;

[0177] 3) Take the Jurkat-TCRKO-031 cells expressing the candidate TCR prepared in Example 1, resuspend them in the medium to 1×106 / mL, add the cell suspension into a 96-well white opaque cell culture plate (purchased from Thermo, product number 136101) at 50 μL per well;

[0178] 4) Wash the T2 cells loaded with the polypeptide twice with the culture medium, resuspend the cells at a density of 2×10 5 / mL, add the T2 cells loaded with different polypeptides into the corresponding wells at 50 μL per well, use the T2 cells without loaded polypeptide as the negative control, and the T2 cells loaded with the HBV target polypeptide as the positive control;

[0179] 5) Place the well plate on a microplate shaker and shake for 10 minutes to mix the cells evenly, then remove the well plate and incubate it in an incubator for 4 - 6 hours;

[0180] 6) After the incubation, add the D-luciferin reaction solution (purchased from Thermo Fisher Scientific, product number L2912) into the culture plate at 100 μL per well, react for 10 minutes at room temperature in the dark, and then use a microplate reader (Thermo Fisher Scientific, Varioskan.LUX) to detect the fluorescence signal value (RLU);

[0181] 7) Use Graphpad Prism software for data analysis, and the results are as Figure 5 shown.

[0182] The results show that all 6 TCR molecules of the present invention can specifically bind to the target polypeptide and transmit T cell activation signals downstream, without recognizing off-target polypeptides, showing good specificity and safety, and having the value for further development.

[0183] The sequences of the off-target analysis polypeptides used in this example are shown in the following table:

[0184] Serial number Polypeptide name Polypeptide sequence 1 HBV GLSPTVWLSV(SEQ ID NO:1) 2 OTP1 AQPTVWLTI(SEQ ID NO:2) 3 OTP2 SLSPTVWFL(SEQ ID NO:3) 4 OTP3 LVSGLSPTV(SEQ ID NO:4) 5 OTP4 GLSPTVAVL(SEQ ID NO:5)

[0185] Example 4: Evaluation of TCR-T cell function

[0186] 4.1 Preparation of TCR-T cells

[0187] Transfect the primary human T cells with the lentivirus solution carrying 6 different TCR genes prepared in Example 2 to prepare TCR-T cells carrying different TCR genes. Name the TCR-T cells carrying 6 TCR clone type T genes as: TCR-T-01, TCR-T-02, TCR-T-03, TCR-T-04, TCR-T-05, and TCR-T-06, and use the antibody against the mouse constant region to detect the expression of the TCR gene. The specific steps are as follows:

[0188] 1) Resuscitate CD3+ T cells derived from healthy human peripheral blood (purchased from Miaoshun), resuspend the cells with T cell medium containing 300 IU / mL IL-2 to a density of 1×10 6 / mL, add the T cell activator CD3 / CD28 magnetic beads (purchased from ACRO, product number: MBS-C001) at a ratio of 1:1 of cells to magnetic beads, mix well, and then inoculate the cells into a 6-well plate for culture;

[0189] 2) After culturing for 48 hours, aspirate the cells, remove the magnetic beads by magnetic adsorption, and centrifuge to collect the cells;

[0190] 3) Resuspend the cells with fresh medium and inoculate them into a new 6-well plate, inoculating 5×10 5 cells per well, add 100 μL of lentivirus solution carrying different TCR genes, and use T cells without adding virus solution as the negative control CT. Place the cells in an incubator and continue culturing for 5 days;

[0191] 4) Take 100 μL of the cell suspension, centrifuge to collect the cells, resuspend the cells with 100 μL of FACS buffer, add 1 μL of APC anti-mouse TCRβ (Biolegend, product number: 109212) antibody to each tube of cells, mix well, and incubate at 4°C for 30 minutes;

[0192] 5) Wash the cells twice, resuspend the cells with 100 μL of FACS buffer, and detect the expression efficiency of TCR on T cells by flow cytometry.

[0193] The results show that all 6 TCR molecules of the present invention can be successfully expressed on T cells, and the expression efficiencies are relatively consistent, at 30 - 50% ( Figure 6 ), and the prepared TCR-T cells can be used for subsequent biological function evaluation.

[0194] 4.2 Cell killing detection

[0195] The TCR-T cells prepared by the present invention can specifically recognize and kill target cells. In this experiment, 2 types of cells were used as target cells, namely T2 cells loaded with target polypeptides, and the hepatoblastoma cell line HepG2.2.15 that can express HBV antigens and secrete intact HBV particles. The TCR-T cells prepared in the above examples were co-cultured with the target cells respectively to detect the killing effect and evaluate the biological function of TCR-T. The specific steps are as follows:

[0196] 1) Prepare a lentivirus solution expressing GFP luciferase according to the method of Example 2.1, and transfect different target cells with the lentivirus solution to obtain a cell line labeled with luciferase, labeled as: T2-GFP and HepG2.2.15-GFP;

[0197] 2) Prepare a 30 μM polypeptide dilution with complete medium. Take T2-GFP cells in good growth state, resuspend the cells with the medium containing the polypeptide to a density of 1×10 6 / mL, place the cell suspension in a cell culture incubator for 1 hour, and after incubation, wash the cells with the medium to remove the excess polypeptide;

[0198] 3) Resuspend the T2-GFP and HepG2.2.15-GFP cells loaded with the polypeptide with the medium to 2×10 5 / mL, and seed the target cells into a 96-well flat-bottom opaque cell culture plate at 50 μL / well respectively, and temporarily place it in a 37 °C incubator;

[0199] 4) Adjust the positive ratio of 6 kinds of TCR-T cells to 30% with CT, and resuspend the 6 kinds of TCR-T with the medium to 1×10 7 / mL, 3.3×10 6 / mL, 6.7×10 5 / mL, 1.3×10 5 / mL and 4.4×10 4 / mL at five different cell densities. Use only the untransfected CT cells activated as a negative control. Seed the above-mentioned effector cells at different densities into a 96-well culture plate at 50 μL / well respectively. After mixing well, place it in a 37 °C incubator for 24 hours;

[0200] 5) Take out the co-cultured opaque 96-well flat-bottom plate, add 100 μL of an equal volume of D-luciferin substrate (Thermo Fisher Scientific: 88293) to the wells, mix well and react in the dark for 10 minutes, and detect the fluorescence intensity in a microplate reader using the chemiluminescence mode. Since luciferase is only expressed in the target cells, the remaining luciferase activity in the wells is directly related to the number of live target cells. Use the medium added to the target cells to obtain the maximum luciferase activity as a control, and calculate the apoptosis ratio of the target cells by subtracting the live cell fluorescence signal value, which is the killing effect of TCR-T cells on the target cells. The results are as Figure 7 shown.

[0201] The results show that the 6 TCR molecules of the present invention can all mediate T cell killing of 2 kinds of target cells, and the killing effect on T2 cells loaded with the target polypeptide is significantly stronger than that of HepG2.2.15, suggesting that its cytotoxic effect is related to the antigen expression abundance of the target cells; the killing levels of the 6 TCR molecules on the target cells are not very different, and among them, TCR-T-01, TCR-T-03 and TCR-T-04 have strong killing effects on both 2 kinds of antigen-positive target cells ( Figure 7 ).

[0202] 4.3 Detection of cytokine secretion

[0203] To further evaluate the level of specifically activating the TCR-T cells of the present invention by target cells and releasing cytokines, referring to the operation steps of Example 2.4, after co-culturing T2 cells loaded with target polypeptides at different concentrations and the 6 types of TCR-T cells prepared in Example 3.1 for 24 hours at an effector-to-target ratio of 5:1, the level of IFN-γ cytokine secreted by T cells in the culture supernatant was detected. The specific steps are as follows:

[0204] 1) Take T2 cells in the logarithmic growth phase, resuspend them in complete medium to 2×10 6 / mL, and inoculate the cell suspension into a 96-well U-bottom deep well plate at 100 μL / well;

[0205] 2) Dilute the target polypeptide with complete medium, with an initial concentration of 200 μM, and 5-fold gradient to 10 concentration points. Add the diluted polypeptide solution to the cells in step 1 at 100 μL / well correspondingly, mix well, and place it in an incubator at 37 °C for 1-2 hours to allow the polypeptide to be loaded onto the surface of T2 cells;

[0206] 3) After incubation, wash off the excess polypeptide with medium, centrifuge to collect the cells, resuspend the cells by adding 0.9 mL of medium per well, and inoculate the T2 cells loaded with different concentrations of polypeptide into a 96-well white opaque cell culture plate at 50 μL / well;

[0207] 4) Take the 6 types of TCR-T cells prepared in Example 3.1, adjust the cell density to 1×10 6 / mL with complete medium, add it to the 96-well plate at 50 μL / well (using untransfected CT cells as a negative control), mix well on a microplate oscillator, and then transfer the cells to an incubator at 37 °C for 24 hours;

[0208] 5) After incubation, collect 50 μL of the culture supernatant, transfer it to a new U-bottom 96-well plate, and use an ELISA kit (Thermo Fisher Scientific; catalog number 88-7316) to detect the secretion of IFN-γ cytokine in T cells. The preparation of the plate and the detection of the supernatant cytokine are carried out according to the instructions provided by the kit;

[0209] 6) Use Graphpad Prism software for data analysis, fit the dose-dependent curve, and calculate the EC 50 value. The results are as shown in Figure 4As shown, the EC50 values corresponding to TCR-T-01, TCR-T-02, TCR-T-03, TCR-T-04, TCR-T-05, and TCR-T-06 are 0.396, 0.0635, 0.188, 0.353, 0.260, and 0.402 μM, respectively.

[0210] The results show that all six TCR-T cells can effectively recognize T2 loaded with target polypeptides, activate and transmit immune signals, and secrete IFN-γ cytokines. Among them, the levels of IFN-γ factor released by TCR-T-02, TCR-T-03, and TCR-T-05 molecules are relatively high, and their E 50 values are lower.

[0211] Based on the above results, the TCR sequences of the present invention all have good affinity activity and biological functions, indicating their value for further development and application.

[0212] All the documents mentioned in the present invention are cited in this application as references, just as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A T cell receptor (TCR) that is capable of binding to a GLSPTVWLSV polypeptide-HLA-A*02 complex.

2. A multivalent TCR complex comprising at least two TCR molecules, and at least one of the TCR molecules is the TCR of claim 1.

3. An isolated nucleic acid molecule encoding the TCR of claim 1.

4. A vector containing the nucleic acid molecule of claim 3; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector.

5. An isolated host cell containing the vector of claim 4 or having the exogenous nucleic acid molecule of claim 3 integrated into its genome.

6. A cell that expresses the TCR of claim 1 on its surface.

7. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the TCR of claim 1, the TCR complex of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, or the cell of claim 6.

8. Use of the T cell receptor of claim 1, the TCR complex of claim 2, or the cell of claim 6 for the preparation of a medicament for treating tumors or infections.

9. The T cell receptor of claim 1, the TCR complex of claim 2, or the cell of claim 6, used as a medicament for treating tumors or infections.

10. A method for treating tumors or infections, comprising administering to a subject in need of treatment a therapeutically effective amount of the T cell receptor of claim 1, the TCR complex of claim 2, the cell of claim 6, or the pharmaceutical composition of claim 7.

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