T cell receptor capable of recognizing EBV antigen and use thereof

Abstract

Provided are a T cell receptor capable of recognizing an EBV antigen and the use thereof. The TCR comprises an α chain containing a variable region and / or a β chain containing a variable region, the variable region of the α chain containing a complementarity determining region 3 (CDR3) of which the amino acid sequence is as shown in SEQ ID NO: 4 or consisting of the CDR3 of which the amino acid sequence is as shown in SEQ ID NO: 4; and / or, the variable region of the β chain containing a CDR3 of which the amino acid sequence is as shown in SEQ ID NO: 7 or consisting of the CDR3 of which the amino acid sequence is as shown in SEQ ID NO: 7. The TCR can specifically bind to an EBV antigen short peptide. A T cell transcribing the TCR can specifically recognize an antigen short peptide presented by HLA-DP5 and can be specifically activated by tumor cells expressing HLA-DP5 and EBNA1, so as to generate a killing function.

Description

T cell receptors recognizing EBV antigens and their applications Technical Field

[0001] The present application relates to the field of medical technology, and in particular to a T cell receptor that recognizes EBV antigens and its application. Background Art

[0002] EBV is one of eight known human herpesviruses. It is a double-stranded DNA virus and a member of the genus Lymphotropicvirus within the subfamily Gammaherpesvirinae. It possesses a linear double-stranded DNA genome of approximately 173 kb, encompassing genes encoding approximately 100 proteins and numerous non-coding RNA and microRNA (miRNA) genes. Virus carriers and patients are the primary source of infection, primarily through saliva, but also through blood. EBV has both productive (lytic) and non-productive (latent) cycles. EBV establishes long-term latent infection in B lymphocytes and productive infection of the oral mucosal epithelium. Only the lytic cycle produces infectious virus, accompanied by cell lysis. EBV DNA is packaged as a linear genome within infectious viral particles, but persists as a closed circular chromatin genome (termed an "episome") outside the chromosomes of the nucleus of latently infected cells, representing a carrier state, establishing latent infection and selectively entering a latent phase in which only a few latent proteins are expressed. Depending on the cell type, EBV-infected cells undergo four different latency programs (0, I, II, III), with different patterns of latency protein expression. Type III latency is associated with EBV-infected primary B cells, which express all nine known latency proteins, consisting of six nuclear antigens (EBNA1 / 2 / 3A / 3B / 3C / EBNA LP) and three membrane proteins (LMP1 / 2A / 2B). EBV-infected primary B cells leave their resting state, become proliferating lymphoblasts, and transform into lymphoblastoid cell lines (LCLs). Subsequently, similar to antigen-activated primary B cells, EBV-infected lymphoblasts may enter follicles, where some cells receive survival signals and differentiate into memory B cells in germinal centers. Cells in germinal centers display a restricted pattern of EBV gene expression (EBNA1, LMP1, LMP2), which is referred to as type II latency. This is a typical form of latency observed in nasopharyngeal carcinoma, gastric cancer, and Hodgkin's lymphoma. LMP1 and LMP2 enable infected B cells in the germinal center to survive and further differentiate into memory B cells by mimicking CD40L-mediated helper T cell signals, expressing only EBNA1 (type I latency) or no latent protein at all (type 0 latency). Burkitt lymphoma is generally a latent form of latency I. As mentioned above, EBNA1 is a viral protein expressed in all latency stages except latency 0. It is also present in all EBV-positive tumors and represents the only latent protein in some tumors. EBNA1 is essential for viral genome maintenance and control of viral gene expression. Without EBNA1, the virus cannot persist and can therefore serve as a marker to distinguish virus-associated cancer cells from normal cells, providing an opportunity for targeted therapeutic intervention.

[0003] EBV infection is globally distributed and typically asymptomatic. However, EBV has a potent ability to stimulate the growth and transformation of lymphocytes, which is closely associated with malignancies such as lymphoma. EBV-associated lymphomas have an extremely poor prognosis. Traditional surgery, chemotherapy, and radiotherapy are ineffective and often associated with severe adverse reactions. Therefore, the exploration of new treatments for EBV-associated tumors is urgently needed.

[0004] Specific T cell immunotherapy utilizes T cells specific for tumor antigens to kill tumor cells. It is a highly personalized approach to tumor immunotherapy. Due to the local immunosuppressive microenvironment of the tumor, the patient's own T cells have limited ability to kill tumors. Therefore, efforts are underway to genetically modify T cells to enhance their tumor-killing capacity. Both TCR-T and CAR-T are genetically modified cell therapies. Upon binding to their target, the transferred T cell receptor (TCR) or chimeric antigen receptor (CAR) gene activates T cells, releasing granzymes, perforins, and cytokines to eliminate tumor cells. However, a challenge with CAR-T cell therapy for both solid and hematologic tumors is that the target antigens are cell surface proteins, which are not typically expressed on solid tumors. Therefore, even if CAR-T cells can effectively kill tumor cells, without the appropriate, specific cell surface target protein, CAR-T cell therapy will have no real impact. TCR-T cells, on the other hand, target short peptide-major histocompatibility complexes (pMHC). Since MHC molecules can present peptide chains obtained from protein antigens on the cell surface or inside the cell, in addition to surface antigens, TCR can also target intracellular antigens commonly expressed by solid tumors, so TCR can target more antigens than CAR.

[0005] TCRs are MHC-restricted, so it is necessary to ensure that the selected viral antigens can be presented by MHC-restricted cells. MHC has different names in different species. In humans, MHC is also called human leukocyte antigen (HLA), which can be divided into HLA class I or HLA class II. CD4 T cells mainly identify HLA class II molecules on the surface of antigen-presenting cells; CD8 T cells mainly identify HLA class I molecules on the surface of target cells. Current research on TCR-T cell therapy for the treatment of EBV-related cancers mainly focuses on CD8 T cells. However, EBNA1 contains glycine and alanine repeat (GAr) domains, which may prevent its proteasomal degradation in the HLA class I antigen processing pathway, making CD8 T cells rarely respond to EBNA1 epitopes, limiting the killing of tumor cells that only express EBNA1 by EBV-specific CD8 T cells. Therefore, attention needs to be directed toward CD4 T cells. However, research on CD4 T cell epitopes has almost entirely focused on short EBV antigen peptides presented by the HLA genotype DPB1*0401, as allele frequencies vary significantly between populations. The most frequently expressed HLA-DPB genotype in the Chinese population is DPB1*0501. Therefore, it is particularly important to identify appropriate CD4 T cell response epitopes targeting EBNA1 that are restricted to the HLA-DP5 genotype, which is more frequent in the Chinese population. Developing corresponding TCR-T drugs will also benefit more Chinese patients.

[0006] Therefore, those skilled in the art are committed to screening CD4 TCRs that can specifically recognize HLA-DP5 restricted EBNA1, so that they can play a role in T cell immunotherapy.

[0007] Summary of the Invention

[0008] The purpose of the present application is to provide a T cell receptor (TCR) that recognizes EBV antigens and its application. The TCR described in the present application can bind to EBV antigen short peptides. T cells that transcribe the TCR can be specifically activated and have a strong killing effect on tumor cells expressing HLA-DP5 and EBV (EBNA1).

[0009] The specific technical solutions of this application are as follows:

[0010] 1. A T cell receptor (TCR) that recognizes an EBV antigen, wherein the TCR comprises an α chain containing a variable region and / or a β chain containing a variable region, wherein the variable region of the α chain comprises or consists of a complementarity determining region 3 (CDR3) having an amino acid sequence as shown in SEQ ID NO: 4; and / or

[0011] The variable region of the β chain comprises or consists of the complementarity determining region 3 (CDR3) having an amino acid sequence as shown in SEQ ID NO: 7.

[0012] 2. The T cell receptor (TCR) according to claim 1, wherein the variable region of the α chain comprises or consists of the complementarity determining region 1 (CDR1) having the amino acid sequence as shown in SEQ ID NO: 2; and / or

[0013] The present invention comprises a complementarity determining region 2 (CDR2) having an amino acid sequence as shown in SEQ ID NO: 3 or consists of a complementarity determining region 2 (CDR2) as shown in SEQ ID NO: 3.

[0014] 3. The T cell receptor (TCR) according to item 1 or 2, wherein the variable region of the β chain comprises or consists of the complementarity determining region 1 (CDR1) having the amino acid sequence as shown in SEQ ID NO: 5; and / or

[0015] The present invention comprises a complementarity determining region 2 (CDR2) having an amino acid sequence as shown in SEQ ID NO: 6 or consists of a complementarity determining region 2 (CDR2) as shown in SEQ ID NO: 6.

[0016] 4. The T cell receptor (TCR) according to any one of items 1 to 3, wherein

[0017] The variable region of the α chain further comprises a first leader sequence; and / or

[0018] The variable region of the beta chain also includes a second leader sequence.

[0019] 5. A T cell receptor (TCR) according to any one of items 1 to 4, wherein the amino acid sequence of the α chain variable region is as shown in SEQ ID NO: 10 or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 10, and / or the amino acid sequence of the β chain variable region is as shown in SEQ ID NO: 11 or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11.

[0020] 6. A T cell receptor (TCR) according to any one of items 1 to 5, wherein the α chain further comprises an α constant region and / or the β chain further comprises a β constant region, preferably, the constant region is a mouse constant region or a human constant region.

[0021] 7. A T cell receptor (TCR) according to any one of items 1 to 6, wherein the TCR is isolated or purified or recombinant.

[0022] 8. The T cell receptor (TCR) of any one of items 1-7, wherein the TCR is human.

[0023] 9. The T cell receptor (TCR) of any one of items 1-8, wherein the TCR is monoclonal.

[0024] 10. The T cell receptor (TCR) of any one of items 1-9, wherein the TCR is a single chain.

[0025] 11. A T cell receptor (TCR) according to any one of items 1 to 10, wherein the TCR comprises two chains.

[0026] 12. The T cell receptor (TCR) according to any one of items 1 to 11, wherein the TCR is in a cell-bound form or a soluble form, preferably a soluble form.

[0027] 13. The T cell receptor (TCR) according to any one of items 1 to 12, wherein the TCR binds to the antigen short peptide-HLA-DP5 complex, and preferably, the amino acid sequence of the antigen short peptide is as shown in SEQ ID NO: 1.

[0028] 14. A nucleic acid molecule, wherein the nucleic acid molecule comprises a protein encoding the TCR according to any one of items 1 to 13, or the α chain or β chain of the TCR.

[0029] 15. The nucleic acid molecule according to item 14, wherein the nucleotide sequence encoding the α chain comprises or consists of the nucleotide sequence shown in SEQ ID NO: 14; and / or

[0030] The nucleotide sequence encoding the β chain comprises or consists of the nucleotide sequence shown in SEQ ID NO: 15.

[0031] 16. A vector, wherein the vector comprises the nucleic acid molecule according to item 14 or 15.

[0032] 17. The vector according to item 16, wherein the vector is an expression vector.

[0033] 18. The vector according to item 16 or 17, wherein the vector is a viral vector, preferably a retroviral vector.

[0034] 19. The vector according to item 18, wherein the viral vector is a lentiviral vector.

[0035] 20. An engineered cell comprising the TCR of any one of items 1-13, the nucleic acid molecule of any one of items 14-15, or the vector of any one of items 16-19.

[0036] 21. An engineered cell according to claim 20, wherein the TCR is heterologous to the cell.

[0037] 22. The engineered cell of item 20 or 21, wherein the engineered cell is a cell line.

[0038] 23. The engineered cell according to any one of items 20-22, wherein the engineered cell is a primary cell obtained from a subject, preferably a mammalian subject, preferably a human.

[0039] 24. The engineered cell according to any one of items 20-23, wherein the engineered cell is a T cell, preferably a T cell isolated from peripheral blood.

[0040] 25. The engineered cell of item 24, wherein the T cells are CD8+ or CD4+.

[0041] 26. A method for producing the engineered cell of any one of items 20 to 25, comprising introducing the nucleic acid molecule of any one of items 14 to 15 or the vector of any one of items 16 to 19 into the cell in vitro or ex vivo.

[0042] 27. The method of claim 26, wherein the vector is a viral vector and the introduction is performed by transduction.

[0043] 28. A pharmaceutical composition comprising the T cell receptor (TCR) of any one of items 1-13, the nucleic acid molecule of any one of items 14-15, the vector of any one of items 16-19, or the engineered cell of any one of items 20-25.

[0044] 29. The pharmaceutical composition according to item 28, further comprising a pharmaceutically acceptable carrier or adjuvant.

[0045] 30. Use of the T cell receptor (TCR) described in any one of items 1-13, the nucleic acid molecule described in any one of items 14-15, the vector described in any one of items 16-19, the engineered cell described in any one of items 20-25, or the pharmaceutical composition described in any one of items 28-29 in the preparation of a drug for treating EBV-related diseases.

[0046] 31. The use according to claim 30, wherein the EBV-related disease is nasopharyngeal carcinoma, gastric cancer, Hodgkin lymphoma, Burkitt lymphoma, post-transplant lymphoproliferative disorder, nasal type extranodal natural killer / T-cell lymphoma, B-cell lymphoma or follicular dendritic cell sarcoma.

[0047] 32. A method of treating a patient suffering from an EBV-associated disease, comprising administering to a subject in need thereof the T cell receptor (TCR) of any one of items 1-13, the nucleic acid molecule of any one of items 14-15, the vector of any one of items 16-19, the engineered cell of any one of items 20-25, or the pharmaceutical composition of any one of items 28-29;

[0048] Preferably, the EBV-related disease is nasopharyngeal carcinoma, gastric cancer, Hodgkin lymphoma, Burkitt lymphoma, post-transplant lymphoproliferative disorder, nasal extranodal natural killer / T-cell lymphoma, B-cell lymphoma or follicular dendritic cell sarcoma.

[0049] 33. Use of the T cell receptor (TCR) of any one of items 1 to 13, the nucleic acid molecule of any one of items 14 to 15, the vector of any one of items 16 to 19, the engineered cell of any one of items 20 to 25, or the pharmaceutical composition of any one of items 28 to 29 for treating an EBV-related disease;

[0050] Preferably, the EBV-associated disease is nasopharyngeal carcinoma, gastric cancer, Hodgkin lymphoma, Burkitt lymphoma, post-transplant lymphoproliferative disorder, nasal-type extranodal natural killer / T-cell lymphoma, B-cell lymphoma or follicular dendritic cell sarcoma.

[0051] Effects of the Invention

[0052] The TCR described in the present application can specifically bind to EBV antigen short peptides, and the T cells that transcribe the TCR can specifically recognize the antigen short peptides presented by HLA-DP5, and can be specifically activated by tumor cells expressing HLA-DP5 and EBNA1 to produce killing function. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG1 is a schematic diagram of identifying the transfection positive rate using flow cytometry.

[0054] FIG2 is a schematic diagram showing the expression of NFAT by T cells transduced with the TCR described in the present application in response to target cells loaded with the VFLQTHIFAEVLKDAIKDLV antigen peptide.

[0055] FIG3 is a schematic diagram of the use of flow cytometry to identify TCR transfection efficiency.

[0056] FIG4 is a graph showing the ratio of cytokines released by TCR-transduced T cells.

[0057] FIG5 is a schematic diagram of the fullness of tumor cells in different groups of cells. DETAILED DESCRIPTION

[0058] The present application is described in detail below with reference to the embodiments described in the accompanying drawings, wherein like numbers in all figures represent like features. Although specific embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. Instead, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0059] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of the components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of this application shall be as defined by the attached claims.

[0060] The present application provides a T cell receptor (TCR) that recognizes EBV antigens, wherein the TCR comprises an α chain containing a variable region and / or a β chain containing a variable region, the variable region of the α chain comprises a complementarity determining region 3 (CDR3) having an amino acid sequence as shown in SEQ ID NO: 4 and consists of a complementarity determining region 3 (CDR3) as shown in SEQ ID NO: 4; and / or

[0061] The variable region of the β chain comprises or consists of the complementarity determining region 3 (CDR3) having an amino acid sequence as shown in SEQ ID NO: 7.

[0062] The amino acid sequence shown in SEQ ID NO: 4 is: AAKIYNNNDMR

[0063] The amino acid sequence shown in SEQ ID NO: 7 is: ASSLASVAGGYT

[0064] In some embodiments, the variable region of the α chain comprises or consists of the complementarity determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO: 2; and / or

[0065] comprising a complementary determining region 2 (CDR2) having an amino acid sequence as shown in SEQ ID NO: 3 or consisting of a complementary determining region 2 (CDR2) as shown in SEQ ID NO: 3. In some embodiments, the variable region of the beta chain comprises a complementary determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO: 5 or consisting of a complementary determining region 1 (CDR1) as shown in SEQ ID NO: 5; and / or

[0066] The present invention comprises a complementarity determining region 2 (CDR2) having an amino acid sequence as shown in SEQ ID NO: 6 or consists of a complementarity determining region 2 (CDR2) as shown in SEQ ID NO: 6.

[0067] The amino acid sequence shown in SEQ ID NO: 2 is: TTSDR

[0068] The amino acid sequence shown in SEQ ID NO: 3 is: LLSNGAV

[0069] The amino acid sequence shown in SEQ ID NO: 5 is: SGHTA

[0070] The amino acid sequence shown in SEQ ID NO: 6 is: FQGNSA

[0071] The T cell receptor or TCR is the only receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC). In the immune system, the binding of the antigen-specific TCR to the pMHC complex triggers direct physical contact between T cells and antigen-presenting cells (APCs). Then, other cell surface molecules of both T cells and APCs interact, which triggers a series of subsequent cell signaling and other physiological reactions, thereby allowing T cells with different antigen specificities to exert immune effects on target cells.

[0072] The TCR is a molecule containing variable α and β chains or variable γ and δ chains, and the molecule is capable of binding to the peptide-specific binding of MHC molecules, and in some embodiments, TCR is in αβ form. Generally, the TCRs in the form of αβ and γδ are generally similar in structure, but the T cells expressing them can have different anatomical locations or functions, and TCR can be found on the cell surface or found in soluble form. Generally, TCR is found on the surface of T cells (T lymphocytes), where it is generally responsible for identifying antigens that are bound to major histocompatibility complex (MHC) molecules.

[0073] The variable domain of a TCR contains complementarity determining regions (CDRs), which are typically the primary contributors to antigen recognition, binding capacity, and specificity for peptides, MHC, and / or MHC-peptide complexes. The CDRs of a TCR, or a combination thereof, form all or substantially all of the antigen-binding site of a given TCR molecule. Individual CDRs within the variable region of a TCR are typically separated by framework regions (FRs). CDR3 is the primary CDR responsible for antigen binding or specificity, or the most important of the three CDRs on a given TCR variable region for antigen recognition and / or for interaction with the processed peptide portion of a peptide-MHC complex. In some cases, CDR1 of the α chain can interact with the N-terminal portion of certain antigenic peptides; in some cases, CDR1 of the β chain can interact with the C-terminal portion of certain antigenic peptides; in some cases, CDR2 has the strongest effect or is the primary responsible CDR for interaction or recognition of the MHC portion of an MHC-peptide complex; in some cases, the variable region antigen of the β chain contains additional hypervariable regions (CDR4 or HVR4), which are typically involved in superantigen binding rather than antigen recognition.

[0074] In some embodiments, the variable region of the α chain further comprises a first leader sequence; and / or

[0075] The variable region of the beta chain also includes a second leader sequence.

[0076] The first leader sequence of the variable region of the α chain and the second leader sequence of the variable region of the β chain are well known to those skilled in the art. For example, the first leader sequence of the variable region of the α chain can use an amino acid sequence such as the leader sequence shown in SEQ ID NO: 8, and the second leader sequence of the variable region of the β chain can use an amino acid sequence such as the leader sequence shown in SEQ ID NO: 9.

[0077] The amino acid sequence shown in SEQ ID NO: 8 is: MKKLLAMILWLQLDRLSG

[0078] The amino acid sequence shown in SEQ ID NO: 9 is: MGTRLLFWVAFCLLGADHT

[0079] In the present application, the leader sequence refers to a signal peptide, which is a short (5-30 amino acids in length) peptide chain that directs the transfer of newly synthesized proteins to the secretory pathway.

[0080] In some embodiments, the amino acid sequence of the α chain variable region is as shown in SEQ ID NO: 10, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 10, and / or the amino acid sequence of the β chain variable region is as shown in SEQ ID NO: 11, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11.

[0081] The amino acid sequence shown in SEQ ID NO: 10 is:

[0082] The amino acid sequence shown in SEQ ID NO: 11 is:

[0083] The amino acid sequence of the α chain variable region having at least 90% sequence identity with SEQ ID NO: 10 can be, for example, an amino acid sequence having at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with SEQ ID NO: 10;

[0084] The amino acid sequence of the variable region of the beta chain having at least 90% sequence identity with SEQ ID NO: 11 can be, for example, an amino acid sequence having at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with SEQ ID NO: 11.

[0085] In this application, "identity" in the context of amino acid sequences refers to the number of residues that are identical in two sequences when aligned for maximum correspondence. There are many different algorithms known in the art that can be used to measure percent amino acid identity (i.e., Basic Local Alignment Tool or Unless otherwise indicated, the default parameters for specific programs or algorithms were used.

[0086] In some embodiments, the α chain further comprises an α constant region and / or the β chain further comprises a β constant region, preferably, the constant region is a mouse constant region or a human constant region,

[0087] For example, the amino acid sequence of the mouse α constant region is shown in SEQ ID NO: 12; and / or

[0088] The amino acid sequence of the mouse β constant region is shown in SEQ ID NO:13.

[0089] The amino acid sequence shown in SEQ ID NO: 12 is:

[0090] The amino acid sequence shown in SEQ ID NO: 13 is:

[0091] The constant region of the TCR can contain a short linker sequence in which cysteine ​​residues form a disulfide bond, thereby connecting the two chains of the TCR. The TCR can have additional cysteine ​​residues in each of the α and β chains, such that the TCR contains two disulfide bonds in the constant region.

[0092] In some embodiments, artificial disulfide bonds are introduced between the residues of the constant regions of the α chain and the β chain of the TCR, and the positions of the disulfide bonds that can be introduced are well known to those skilled in the art.

[0093] In some embodiments, the TCR is isolated or purified or recombinant.

[0094] In some embodiments, the TCR is human.

[0095] In some embodiments, the TCR is monoclonal.

[0096] In some embodiments, the TCR is single chain.

[0097] In some embodiments, the TCR comprises two chains.

[0098] TCRs can be obtained from biological sources, such as from cells (e.g., from T cells (e.g., cytotoxic T cells)), T cell hybridomas, or other publicly available resources, for example, TCRs can be derived from one of a variety of animal species, such as human, mouse, rat, or other mammals, such as typically from human.

[0099] In some embodiments, the TCR may be in a cell-bound form or in a soluble form, preferably a soluble form.

[0100] The soluble form of the TCR refers to a TCR in which mutations occur in its hydrophobic core region. These mutations in the hydrophobic core region are preferably mutations that can improve the stability of the soluble TCR of the present application.

[0101] In one embodiment, the TCR is capable of binding to the antigen short peptide-HLA-DP5 complex. Preferably, the amino acid sequence of the antigen short peptide is as shown in SEQ ID NO: 1.

[0102] The antigen peptide-HLA-DP5 complex refers to a complex formed by the binding of HLA-DP5 and an antigen peptide. Proteins are degraded by the proteasome in cells into peptides of varying lengths, some of which bind to HLA to form complexes that are presented on the cell surface. The antigen peptide-HLA-DP5 complex recognized by the TCR can be expressed on the cell membrane or present as a soluble protein in solution.

[0103] In the present application, DP5 is composed of an A chain and a B chain, the A chain is DPA1*0202, and the B chain is DPB1*0501, and the TCR can specifically recognize the HLA-DPA1*0202 / HLA-DPB1*0501 complex.

[0104] Among them, for the A chain of DP5, the amino acid sequence of HLA-DPA1*0202 is shown in SEQ ID NO: 16, and its amino acid sequence is:

[0105] For the B chain of DP5, the amino acid sequence of HLA-DPB1*0501 is shown in SEQ ID NO: 17, and its amino acid sequence is:

[0106] The amino acid sequence shown in SEQ ID NO: 1 is:

[0107] VFLQTHIFAEVLKDAIKDLV

[0108] In some embodiments, the TCR comprises an α chain comprising a variable region and / or a β chain comprising a variable region, the variable region of the α chain comprising or consisting of a complementarity determining region 3 (CDR3) having an amino acid sequence as shown in SEQ ID NO: 4; and / or

[0109] The variable region of the β chain comprises or consists of the complementary determining region 3 (CDR3) having an amino acid sequence as shown in SEQ ID NO: 7. In some embodiments, the variable region of the α chain comprises or consists of the complementary determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO: 2; and / or

[0110] comprising or consisting of the complementarity determining region 2 (CDR2) as shown in SEQ ID NO: 3;

[0111] Preferably, the variable region of the β chain comprises or consists of the complementarity determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO: 5; and / or

[0112] comprising or consisting of the complementarity determining region 2 (CDR2) as shown in SEQ ID NO: 6;

[0113] Preferably, the variable region of the α chain further comprises a first leader sequence; and / or

[0114] The variable region of the beta chain further includes a second leader sequence;

[0115] Preferably, the amino acid sequence of the α chain variable region is as shown in SEQ ID NO: 10 or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 10, and / or the amino acid sequence of the β chain variable region is as shown in SEQ ID NO: 11 or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11. In some embodiments, the α chain further comprises an α constant region and / or the β chain further comprises a β constant region, preferably, the constant region is a mouse constant region or a human constant region;

[0116] Preferably, the TCR is isolated or purified or recombinant;

[0117] Preferably, the TCR is human;

[0118] Preferably, the TCR is monoclonal;

[0119] Preferably, the TCR is single chain;

[0120] Preferably, the TCR comprises two chains;

[0121] Preferably, the TCR is in a cell-bound or soluble form, preferably a soluble form. In some embodiments, the TCR binds to an antigen short peptide-HLA-DP5 complex, and preferably, the amino acid sequence of the antigen short peptide is as shown in SEQ ID NO: 1.

[0122] The present application provides a nucleic acid molecule comprising a nucleic acid encoding the TCR described above or the α chain or β chain of the TCR.

[0123] In some embodiments, the nucleotide sequence encoding the α chain comprises or consists of the nucleotide sequence shown in SEQ ID NO: 14; and / or

[0124] The nucleotide sequence encoding the β chain comprises or consists of the nucleotide sequence shown in SEQ ID NO: 15.

[0125] Wherein, the nucleotide sequence shown in SEQ ID NO: 14 is:

[0126] The nucleotide sequence shown in SEQ ID NO: 15 is:

[0127] The nucleic acid molecule may comprise naturally and / or non-naturally occurring nucleotides and bases, including, for example, those with backbone modifications, and the nucleic acid molecule refers to a polymer of nucleotides, which may contain naturally and / or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. A nucleotide sequence refers to the linear sequence that makes up a nucleic acid molecule.

[0128] In some cases, the nucleic acid molecule contains cDNA, and in some cases, the nucleic acid molecule can be modified for use in the constructs described herein, such as for codon optimization. In some cases, for the purpose of cloning into a vector, the sequence can be designed to contain a terminal restriction site sequence.

[0129] In some cases, nucleic acid molecules encoding a TCR can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of encoding nucleic acids within or isolated from one or more given cells.

[0130] In one embodiment, the nucleotide sequence encoding the alpha chain and / or the nucleotide sequence encoding the beta chain are codon optimized. Generally, codon optimization involves balancing the percentage of the selected codons with the abundance of disclosed human transfer RNAs so that none of them is overloaded or restricted. In some cases, this may be necessary because most amino acids are encoded by more than one codon, and codon usage varies from organism to organism. Codon usage differences between transfected genes and host cells may affect protein expression and immunogenicity of nucleic acid constructs. Generally, for codon optimization, codons are selected to select those codons that are balanced with human usage frequency. Generally, the redundancy of amino acid codons makes different codons encode a kind of amino acid. In some embodiments, when selecting a codon for replacement, it may be necessary that the resulting mutation is a silent mutation so that the codon change does not affect the amino acid sequence. Generally, the last nucleotide of the codon can remain unchanged and will not affect the amino acid sequence.

[0131] The present application provides a vector comprising the nucleic acid molecule described above.

[0132] For example, one or more nucleic acids encoding one or both chains of the TCR described above are cloned into one or more suitable expression vectors, which can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification or for expression or for both, such as plasmids and viruses.

[0133] The vector may contain regulatory sequences (such as transcription and translation initiation and termination codons) that are specific to the type of host into which the vector is to be introduced (e.g., bacteria, fungi, plants, or animals), taking into account whether the vector is DNA-based or RNA-based. The vector may also contain a non-natural promoter operably linked to the nucleotide sequence encoding the TCR. The promoter may be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and the promoter found in the long terminal repeat sequence of the mouse stem cell virus, and other promoters known to those skilled in the art are also contemplated.

[0134] In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector, preferably a retroviral vector. In some embodiments, the viral vector is a lentiviral vector.

[0135] The present application provides an engineered cell comprising the TCR described above, the nucleic acid molecule described above, or the vector described above. In some embodiments, the TCR is heterologous to the cell. In some embodiments, the engineered cell is a cell line. In some embodiments, the engineered cell is a primary cell obtained from a subject, preferably a mammalian subject, preferably a human. In some embodiments, the engineered cell is a T cell, preferably a T cell isolated from peripheral blood. In some embodiments, the T cell is CD8+ or CD4+.

[0136] In some embodiments, the engineered cell is a cell that is derived from blood, bone marrow, lymph or lymphoid organs, and is a cell of the immune system, such as a cell of innate immunity or adaptive immunity, such as bone marrow or lymphoid cells (including lymphocytes, typically T cells and / or NK cells). Other exemplary cells include stem cells, such as pluripotent stem cells and multipotent stem cells, including induced pluripotent stem cells (iPSCs). Cell is typically primary cells, such as directly separating from experimenter and / or separating and freezing from experimenter those. In some embodiments, cell includes one or more subsets of T cells or other cell types, such as whole T cell populations, CD+ cells, CD8+ cells and subgroups thereof.

[0137] Subtypes and subsets of T cells and / or CD+ and / or CD8+ T cells include naive T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes (such as stem cell memory T (T SCM ), central memory T(T CM ), effect memory T(T EM ) or terminally differentiated effector memory T cells), tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, etc.

[0138] In some embodiments, the cell is a natural killer (NK) cell. In some embodiments, the cell is a monocyte or granulocyte, such as a myeloid cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil, and / or a basophil.

[0139] The present application provides a method for producing the engineered cells described above, which comprises introducing the nucleic acid molecules described above or the vector described above into cells in vitro or ex vivo.

[0140] In some embodiments, the vector is a viral vector and the introducing is by transduction.

[0141] The present application provides a pharmaceutical composition comprising the aforementioned T cell receptor (TCR), the aforementioned nucleic acid molecule, the aforementioned vector or the aforementioned engineered cell.

[0142] In some embodiments, it further comprises a pharmaceutically acceptable carrier or adjuvant.

[0143] The pharmaceutically acceptable carrier or adjuvant refers to a component in a pharmaceutical composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers or adjuvants include but are not limited to buffers, excipients, stabilizers or preservatives.

[0144] The pharmaceutical composition can utilize timed release, delayed release, and sustained release delivery systems so that delivery of the composition occurs before sensitization of the treated area and has sufficient time to cause sensitization. Many types of release delivery systems are available and known. Such systems can be used to avoid repeated administration of the composition, thereby increasing the convenience of the subject and the physician.

[0145] The present application provides the use of the aforementioned T cell receptor (TCR), the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned engineered cell or the aforementioned pharmaceutical composition in the preparation of a drug for treating EBV-related diseases.

[0146] In one embodiment, the EBV-associated disease is nasopharyngeal carcinoma, gastric cancer, Hodgkin lymphoma, Burkitt lymphoma, post-transplant lymphoproliferative disorder, extranodal natural killer / T-cell lymphoma, nasal type, B-cell lymphoma, or follicular dendritic cell sarcoma.

[0147] The present application provides a method for treating patients suffering from EBV-related diseases, comprising administering the above-mentioned T cell receptor (TCR), the above-mentioned nucleic acid molecule, the above-mentioned vector, the above-mentioned engineered cell or the above-mentioned pharmaceutical composition to a subject in need thereof. Preferably, the EBV-related disease is nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, post-transplantation lymphoproliferative disorder, nasal extranodal natural killer / T-cell lymphoma, B-cell lymphoma or follicular dendritic cell sarcoma.

[0148] The present application provides the use of the aforementioned T cell receptor (TCR), the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned engineered cell, or the aforementioned pharmaceutical composition in treating EBV-related diseases;

[0149] Preferably, the EBV-associated disease is nasopharyngeal carcinoma, gastric cancer, Hodgkin lymphoma, Burkitt lymphoma, post-transplant lymphoproliferative disorder, nasal-type extranodal natural killer / T-cell lymphoma, B-cell lymphoma or follicular dendritic cell sarcoma.

[0150] The TCR described in the present application can specifically bind to the antigenic short peptide of EBV-positive tumor cells. The T cells transduced with the TCR can be specifically activated and have a strong killing effect on target cells. The TCR can be used for immunotherapy of EBV-positive tumors such as nasopharyngeal carcinoma.

[0151] Example

[0152] This application provides general and / or specific descriptions of the materials and experimental methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., percentage by weight. All reagents or instruments used without manufacturer indication are commercially available conventional reagents.

[0153] Example 1: Cloning of VFLQTHIFAEVLKDAIKDLV antigen short peptide-specific T cells and TCR gene acquisition

[0154] Peripheral blood lymphocytes from healthy volunteers with the HLA-DP5 genotype were stimulated using a synthetic short peptide, VFLQTHIFAEVLKDAIKDLV (SEQ ID NO: 1, Jiangsu GenScript Biotechnology Co., Ltd.). The VFLQTHIFAEVLKDAIKDLV short peptide was loaded onto a biotin-labeled HLA-DP5 protein in a phosphate buffer solution, and pHLA monomers were prepared according to conventional methods in the art. These monomers were combined with APC-labeled streptavidin (BD Company) to form APC-labeled tetramers according to conventional methods in the art. The tetramers and anti-CD4-FITC double-positive cells were enriched according to conventional methods in the art to obtain specific T cells. The obtained double-positive cells were flow-sorted to obtain single cells, and the single cells obtained by sorting were amplified using a one-step RT-PCR kit (QIAGEN, catalog number 210212) for TCR α and β chains, respectively, and the PCR products were sequenced. The sequencing results were compared with the sequences in the public database of IMGT (International Immunogenetics Information System) to obtain the nucleotide sequences of the TCR α chain variable region sequence and the β chain variable region and their CDR1, CDR2, and CDR3 information, as shown in SEQ ID NOs: 14-15 and 2-7, respectively.

[0155] Example 2: Construction of VFLQTHIFAEVLKDAIKDLV antigen short peptide-specific TCR lentiviral vector and lentiviral packaging

[0156] (1) Construction of TCR lentiviral vector

[0157] The VFLQTHIFAEVLKDAIKDLV TCRα and β chain variable region sequences were cloned into a pLKO-based expression plasmid (Addgene). The α or β chain variable domains were cloned into a pLKO-based expression plasmid containing the mouse α or β constant region using the Multi-Fragment Recombination Cloning Kit (Novozyme Biotech, catalog number C113) using standard methods. The ligated plasmids were transformed into competent E. coli strain Stbl3 cells (Beijing Quanshijin Biotechnology Co., Ltd.) and plated on LB / agar plates containing 100 μg / ml ampicillin. After overnight incubation at 37°C, single colonies were picked and grown overnight at 37°C in 10 ml of LB containing 100 μg / ml ampicillin with shaking. The cloned plasmid was purified using a miniprep kit (TIANGEN catalog # DP118-02) and sequenced to obtain VFLQTHIFAEVLKDAIKDLV TCR (ie, TCR-VFL).

[0158] (2) Lentiviral packaging

[0159] Assay medium: 10% FBS (ExCell, catalog number FSP500), DMEM (ThermoFisher, catalog number C11995500BT)

[0160] Prepare 293T cells (American Type Culture Collection, catalog number CRL-1573) in a 10cm dish and begin plasmid transfection when no more than 80% confluency is reached. Use a 1:1 ratio of viral packaging plasmid to VFLQTHIFAEVLKDAIKDLV TCR plasmid, for a total of 10 μg. Mix the plasmids in serum-free DMEM medium with polyethylenimine (PEI). Add this mixture to the 293T cells and incubate at 37°C. After 72 hours, concentrate the supernatant using a 100 kDa ultrafiltration tube to collect the viral vector.

[0161] Example 3: Construction and functional identification of a Jurkat cell line expressing a VFLQTHIFAEVLKDAIKDLV antigen peptide-specific TCR

[0162] Nuclear Factor of Activated T Cells (NFAT) Reporter Gene Expression Method

[0163] The following experiment was performed to demonstrate the specific activation response of TCR-transduced T cells to target cells. NFAT expression was measured using flow cytometry as a readout of T cell activation.

[0164] (1) Reagents

[0165] Assay medium: 10% FBS (ExCell, catalog number FSP500), RPMI1640 (ThermoFisher, catalog number C11875500BT)

[0166] (2) Method

[0167] Target cell preparation

[0168] The target cells used in this experiment were 293T-CIITA-DP5 cells (293T cells were deposited with ATCC under the catalog number CRL-3216). Target cells were prepared in experimental culture medium at a concentration of 1.0 × 106 cells / ml, and 50 μl was plated per well to obtain 50,000 cells / well.

[0169] Effector cell preparation

[0170] The effector cells in this experiment were J.RT3-T3.5-CD4-NFAT-ZsGreen (JK4NF) cells transduced with the TCR of the present application, and JK4NF cells not transfected with the TCR of the present application were used as a control group.

[0171] JK4NF cells (J.RT3-T3.5 cells were deposited in ATCC with a deposit catalog number of TIB-152) were added with the lentivirus carrying the TCR gene of the present application obtained in Example 2 at an MOI (multiplicity of infection) of 1. After 72 hours, the transfection positive rate was determined by flow cytometry. The results are shown in FIG1 . As can be seen from FIG1 , the transfection positive rate was about 100%. The concentration of the effector cells after expansion culture was adjusted to 1.0×10 6 50 μl was taken from each well to obtain 50,000 cells / well.

[0172] Preparation of short peptide solution

[0173] The original concentration of 5 mg / ml short peptide (VFLQTHIFAEVLKDAIKDLV) was diluted to 200 μg / ml, and then diluted down in 10-fold ratio to 20 μg / ml, 2 μg / ml, 0.2 μg / ml, 0.02 μg / ml, 0.002 μg / ml, 0.0002 μg / ml, and 0.00002 μg / ml.

[0174] 100 μl was taken from each well to make the final concentrations of the short peptides in the 96-well plate 10 μg / ml, 1 μg / ml, 0.1 μg / ml, 0.01 μg / ml, 0.001 μg / ml, 0.0001 μg / ml, and 0.00001 μg / ml, respectively.

[0175] Finally, 50 μl of target cells, 50 μl of effector cells, and 100 μl of short peptide dilutions of corresponding concentrations were added to each well of a 96-well flat-bottom plate and incubated in a cell culture incubator at 37 degrees for 12 h.

[0176] (3) Results

[0177] The expression of NFAT in the TCR-transduced T cells of the present application in response to target cells loaded with the VFLQTHIFAEVLKDAIKDLV antigen peptide was tested by the above method. The expression level curve of NFAT was plotted using Graphpad Prism 8, and the results are shown in Figure 2.

[0178] As can be seen from FIG2 , T cells transduced with the TCR of the present application have a good activation response to target cells loaded with their specific short peptides.

[0179] Example 4: In vitro functional characterization of primary T cells expressing VFLQTHIFAEVLKDAIKDLV antigen short peptide-specific TCR

[0180] Intracellular staining method

[0181] The following experiment was performed to demonstrate that the VFLQTHIFAEVLKDAIKDLV antigen short peptide-specific TCR produces a specific activation response to HLA-DP5-positive EBNA1-positive target cells.

[0182] (1) Reagents

[0183] Assay medium: 10% FBS (ThermoFisher, catalog number 10099-044), RPMI1640 (ThermoFisher, catalog number C11875500BT), Cytofix / Cytoperm TM Fixation / Permeabilization Kit (Becton, Dickinson and Company, USA, catalog number 554714)

[0184] (2) Method

[0185] Effector T cell preparation

[0186] The effector cells (T cells) of this experiment were T cells transduced with the TCR of the present application, and T cells of the same volunteer that were not transfected with the TCR of the present application were used as a control group.

[0187] Peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation from healthy volunteers with HLA-DP5 genotype. TM Primary CD4 T cells were obtained by sorting with Human CD4 T Cell Isolation Kit (STEMCELL, catalog number 17952) and plated in 24-well plates at a rate of 5.0 × 10 5 / 500μl was placed in the well, and a total of 1x10 6 T cells were stimulated with T Cell TransAct magnetic beads and then cultured in a 37°C, 5% CO2 incubator. After 24 hours, the cell clumping was observed and the TCR lentivirus obtained in Example 2 was added at an MOI (multiplicity of infection) of 1 for transduction. The cells were then expanded in 1640 medium containing 10% FBS and 200 IU / ml IL-2 until 3-4 days after transduction. The TCR transfection efficiency was determined by flow cytometry (the results are shown in Figure 3). The concentration of the effector cells after expansion was adjusted to 5.0×10 5 Positive cells / ml, 100 μl was taken from each well to obtain 50,000 positive cells / well.

[0188] Target cell preparation

[0189] The target cells used in this experiment were C666-1 (nasopharyngeal carcinoma cells deposited with ATCC, naturally expressing HLA-DP molecules) cells constructed by overexpressing the EBNA1 gene via a lentiviral vector. 50,000 cells were plated per well. An HLA-DP neutralizing antibody (Anti-HLA-DP antibody [B7 / 21], purchased from abcam, catalog number ab20897) was also added as a negative control.

[0190] Cell co-culture

[0191] Add 100 μl of target cells and 100 μl of effector cells to each well of a 96-well flat-bottom plate. Add BD GolgiStop™ (0.66 μl / ml culture medium) and BD GolgiPlug (1 μl / ml culture medium) to each well, mix thoroughly, and incubate at 37°C for 12 hours. For the negative control, add Anti-DP antibody.

[0192] Flow cytometry

[0193] The co-incubated cells were collected and transferred from the 96-well flat-bottom plate to a 96-well V-bottom plate, and the cell surface markers were stained with antibodies PE anti-human CD4 Antibody (purchased from Biolegend, catalog number 357404) and APC anti-mouse-TCRβchain Antibody (purchased from Biolegend, catalog number 109212), incubated at 4°C for 20 minutes, and washed once with 1× PBS.

[0194] The intracellular staining step was performed according to the manufacturer's instructions (BD, catalog number 554714). 100 μl of Fixation / Permeabilization solution was added to each well of the above 96-well V-bottom plate and incubated at 4°C for 20 minutes. Then 250 μl of 1× BD Perm / Wash was added to each well. TM Wash the cells twice with 1×BD Perm / Wash TM Dilute the Pacific Blue antibody in the buffer at a ratio of 1:100 TM anti-human IFNγAntibody, PE / Cyanine7 anti-human IL-2Antibody, Alexa 700 anti-human TNF-α Antibody was used to stain the intracellular substances of the cells. The cells were incubated at 4°C for 30 minutes and washed with 1× BD Perm / Wash TM The cells were washed once with PBS. Finally, the cells were resuspended in 1× PBS and analyzed by flow cytometry. The function of the TCR-transduced T cells of the present invention was tested by intracellular staining (as described above). The ratio of cytokine release by the T cells in each well was plotted using Graphpad Prism 8. The results are shown in Figure 4.

[0195] (3) Results

[0196] As shown in Figure 4, TCR-VFL expressing a TCR specific for the VFLQTHIFAEVLKDAIKDLV antigen peptide strongly activated the C666-1-EBNA1 cell line, while control T cells showed no activation response. Addition of an HLA-DP neutralizing antibody inhibited TCR-VFL recognition of the C666-1-EBNA1 cell line.

[0197] Example 5: In vitro functional characterization of primary T cells expressing VFLQTHIFAEVLKDAIKDLV antigen short peptide-specific TCR

[0198] A. Incucyte real-time live cell analysis

[0199] method

[0200] The following experiment was performed to demonstrate the killing effect of VFLQTHIFAEVLKDAIKDLV antigen short peptide-specific TCR on HLA-DP5-positive EBNA1-positive target cells.

[0201] Detection of tumor cell growth curve using the Incucyte real-time live cell analysis system

[0202] (1) Reagents

[0203] Assay medium: 10% FBS (ThermoFisher, catalog number 10099-044), RPMI1640 (ThermoFisher, catalog number C11875500BT)

[0204] (2) Method

[0205] Target cell preparation

[0206] The target cells used in this experiment were C666-1-EBNA1 cells, which were constructed by overexpressing the EBNA1 gene using a lentiviral vector using C666-1 (purchased from Zhejiang Meisen Cell Technology Co., Ltd., catalog number CTCC-400-0109). Target cells were prepared in experimental culture medium and the target cell concentration was adjusted to 2.0×10 5 100 μl was taken per well to obtain 20,000 cells / well.

[0207] Effector T cell preparation

[0208] The effector cells (T cells) of this experiment were T cells transduced with the TCR of the present application, and T cells of the same volunteer that were not transfected with the TCR of the present application were used as a control group.

[0209] Peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation from healthy volunteers with HLA-DP5 genotype. PBMCs were plated at 1.0×10 per well in a 24-well plate. 6 / 500μl was placed in the well, and a total of 2x10 6T cells were stimulated with T Cell TransAct magnetic beads and then cultured in a 37°C, 5% CO2 incubator. After 24 hours, the cell clumping was observed and the TCR lentivirus obtained in Example 2 was added at an MOI (multiplicity of infection) of 1 for transduction. The cells were then expanded in 1640 medium containing 10% FBS and 200 IU / ml IL-2 until 3-4 days after transduction. The TCR transfection efficiency was determined by flow cytometry, as shown in Figure 3. The concentration of the effector cells after expansion was adjusted to 6×10 5 positive cells / ml.

[0210] Cell co-culture and live cell real-time analysis

[0211] 20,000 target cells, 10,000 effector cells (1:2 group) or 4,000 effector cells (1:5 group) were added to each well of a 96-well plate. The 96-well plate was placed in an Incucyte machine and photographed every hour for 72 hours.

[0212] (3) Results

[0213] T cells expressing a TCR specific for the VFLQTHIFAEVLKDAIKDLV antigen peptide, TCR-VFL, significantly inhibited the growth of C666-1-EBNA1 tumor cells. The tumor cell confluence (i.e., the ratio of tumor cell area to total well area) of each well was analyzed using Incucyte and derived, as shown in Figure 5.

[0214] As can be seen from Figure 5, for the TCR-VFL group, the tumor cell density was significantly reduced, indicating that T cell TCR-VFL expressing VFLQTHIFAEVLKDAIKDLV antigen short peptide specific TCR can significantly inhibit the growth of C666-1-EBNA1 tumor cells.

[0215] In summary, the T cells transduced with the TCR in the present application can specifically recognize the VFLQTHIFAEVLKDAIKDLV short peptide presented by HLA-DP5, and can be specifically activated by tumor cells expressing HLA-DP5 and EBNA1 to produce killing function.

[0216] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application in any other manner. Any person skilled in the art may utilize the above disclosed technical content to modify or modify the present application into equivalent embodiments with equivalent variations. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present application and are based on the technical essence of the present application shall still fall within the scope of protection of the present application.

Claims

1. A T cell receptor (TCR) that recognizes EBV antigen, wherein: The TCR comprises an α chain comprising a variable region and / or a β chain comprising a variable region, wherein the variable region of the α chain comprises or consists of a complementary determining region 3 (CDR3) having an amino acid sequence as shown in SEQ ID NO: 4; and / or The variable region of the β chain comprises or consists of the complementarity determining region 3 (CDR3) as shown in SEQ ID NO:

7.

2. The T cell receptor (TCR) according to claim 1, wherein The variable region of the α chain comprises or consists of the complementary determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO: 2; and / or comprising or consisting of the complementarity determining region 2 (CDR2) as shown in SEQ ID NO: 3; Preferably, the variable region of the β chain comprises or consists of the complementarity determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO: 5; and / or comprising or consisting of the complementarity determining region 2 (CDR2) as shown in SEQ ID NO:6; Preferably, the variable region of the α chain further comprises a first leader sequence; and / or The variable region of the beta chain also includes a second leader sequence; Preferably, the amino acid sequence of the α chain variable region is as shown in SEQ ID NO: 10 or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 10, and / or the amino acid sequence of the β chain variable region is as shown in SEQ ID NO: 11 or an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

11.

3. The T cell receptor (TCR) according to any one of claims 1 to 2, wherein The α chain further comprises an α constant region and / or the β chain further comprises a β constant region, preferably, the constant region is a mouse constant region or a human constant region; Preferably, the TCR is isolated or purified or recombinant; Preferably, the TCR is human; Preferably, the TCR is monoclonal; Preferably, the TCR is a single chain; Preferably, the TCR comprises two chains; Preferably, the TCR is in a cell-bound form or in a soluble form, preferably a soluble form.

4. The T cell receptor (TCR) according to any one of claims 1 to 3, wherein The TCR binds to the antigen short peptide-HLA-DP5 complex. Preferably, the amino acid sequence of the antigen short peptide is as shown in SEQ ID NO:

1.

5. A nucleic acid molecule, wherein The nucleic acid molecule comprises a nucleic acid encoding the TCR of any one of claims 1 to 13 or the α chain or β chain of the TCR; Preferably, the nucleotide sequence encoding the α chain comprises or consists of the nucleotide sequence shown in SEQ ID NO: 14; and / or The nucleotide sequence encoding the β chain comprises the nucleotide sequence shown in SEQ ID NO:15 or consists of the nucleotide sequence shown in SEQ ID NO:

15.

6. A vector, wherein The vector comprises the nucleic acid molecule of claim 5.

7. The carrier according to claim 6, wherein The vector is an expression vector; Preferably, the vector is a viral vector, preferably a retroviral vector; Preferably, the viral vector is a lentiviral vector.

8. An engineered cell comprising the TCR of any one of claims 1-4, the nucleic acid molecule of claim 5, or the vector of any one of claims 6-7.

9. The engineered cell according to claim 8, wherein The TCR is heterologous to the cell; Preferably, the engineered cell is a cell line; Preferably, the engineered cells are primary cells obtained from a subject, preferably, the subject is a mammalian subject, preferably a human; Preferably, the engineered cells are T cells, preferably T cells isolated from peripheral blood; Preferably, the T cells are CD8+ or CD4+.

10. A method for producing the engineered cell according to any one of claims 8 to 9, comprising introducing the nucleic acid molecule according to claim 5 or the vector according to any one of claims 6 to 7 into the cell in vitro or ex vivo; Preferably, the vector is a viral vector and the introducing is performed by transduction.

11. A pharmaceutical composition comprising the T cell receptor (TCR) according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the vector according to any one of claims 6 to 7, or the engineered cell according to any one of claims 8 to 9; Preferably, it further comprises a pharmaceutically acceptable carrier or adjuvant.

12. Use of the T cell receptor (TCR) according to any one of claims 1 to 4, the nucleic acid molecule according to any one of claims 5, the vector according to any one of claims 6 to 7, the engineered cell according to any one of claims 8 to 9, or the pharmaceutical composition according to claim 11 in the preparation of a drug for treating a disease associated with EBV; Preferably, the EBV-associated disease is nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, post-transplant lymphoproliferative disorder, extranodal natural killer / T-cell lymphoma of the nasal type, B-cell lymphoma or follicular dendritic cell sarcoma.

13. A method for treating a patient suffering from an EBV-related disease, comprising administering to a subject in need thereof the T cell receptor (TCR) of any one of claims 1-4, the nucleic acid molecule of any one of claims 5, the vector of any one of claims 6-7, the engineered cell of any one of claims 8-9, or the pharmaceutical composition of claim 11; Preferably, the EBV-related disease is nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, post-transplantation lymphoproliferative disorder, nasal extranodal natural killer / T-cell lymphoma, B-cell lymphoma or follicular dendritic cell sarcoma.

14. Use of the T cell receptor (TCR) according to any one of claims 1 to 4, the nucleic acid molecule according to any one of claims 5, the vector according to any one of claims 6 to 7, the engineered cell according to any one of claims 8 to 9, or the pharmaceutical composition according to claim 11 in treating EBV-related diseases; Preferably, the EBV-associated disease is nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, post-transplant lymphoproliferative disorder, extranodal natural killer / T-cell lymphoma of the nasal type, B-cell lymphoma or follicular dendritic cell sarcoma.

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