CN112940108A

CN112940108A - T cell receptor for recognizing EBV antigen and application of T cell receptor

Info

Publication number: CN112940108A
Application number: CN202110295939.5A
Authority: CN
Inventors: 王子兵; 陈琳; 张建功; 李红乐; 寇贺丹
Original assignee: Henan Cancer Hospital; Guangzhou Medical University
Current assignee: Henan Cancer Hospital; Guangzhou Medical University
Priority date: 2021-03-19
Filing date: 2021-03-19
Publication date: 2021-06-11
Anticipated expiration: 2041-03-19
Also published as: CN112940108B

Abstract

The invention discloses a T cell receptor for identifying an EBV antigen and application of the T cell receptor. The T Cell Receptor (TCR) of the invention comprises an alpha chain comprising a variable region comprising complementarity determining region 3(CDR3) having amino acid sequence AVVNNNDMR (SEQ ID NO: 3); and/or the variable region of the beta strand comprises complementarity determining region 3(CDR3) comprising ASSPGRWYEQY (SEQ ID NO: 6). The TCR disclosed by the invention can be specifically combined with the EBV antigen short peptide, and the T cell transduced with the TCR can be specifically activated and has a strong killing effect on a target cell.

Description

T cell receptor for recognizing EBV antigen and application of T cell receptor

Technical Field

The invention relates to the technical field of medicines, in particular to a T cell receptor for identifying an EBV antigen and application of the T cell receptor.

Background

EBV (Epstein-Barr virus), also known as human herpesvirus 4 (human herpesvirus 4), is a double stranded DNA virus. Humans are the only host of EBV, carrying the virus in approximately > 90% of the adult population worldwide.

The infection pathway of EBV is via contact transmission with saliva. Most primary infections occur in childhood and are usually without or with only non-specific symptoms, whereas infections in late adolescents or in adulthood lead to infectious mononucleosis. Once infection occurs, EBV is often latent in peripheral lymphocytes, making infected individuals a lifelong carrier of EBV. Carrying EBV has no serious consequences in most cases, as long as the virus is constantly in a latent inactive state. However, in rare cases, latent virus is activated, which leads to chronic active infection (chronic active infection) and the development of malignant tumors of the epithelial, mesenchymal and lymphatic systems, such as nasopharyngeal carcinoma, gastric carcinoma, Hodgkin lymphoma (Hodgkin lymphoma), Burkitt lymphoma (Burkitt lymphoma) and the like. Epidemiological studies have shown that 90% of patients with nasopharyngeal carcinoma, 10% of patients with gastric carcinoma, 40% of patients with hodgkin's lymphoma, and 95% of patients with epidemic burkitt's lymphoma present EBV positivity. It is worth mentioning that the incidence of nasopharyngeal carcinoma is high in the southern china and southeast asia (especially for the Guangdong male), 30 cases occur in 10 ten thousand, while only <1 case occurs in 10 ten thousand worldwide, and 100% of undifferentiated nasopharyngeal carcinomas (the most common type) and most differentiated nasopharyngeal carcinomas are EBV positive.

EBV is usually latent in malignant cells, meaning a state in which there is a persistent viral infection but no active viral replication release in the body. EBV can be latent in memory B cells as well as epithelial cells. It is currently believed that one EBV genome is carried in each million B cells in individuals following recovery from acute EBV infection. EBV expresses only limited proteins and RNA during latency, which is also one of its mechanisms to escape immune surveillance. Latent-expressed EBV proteins include: EBV nuclear antigen (EBNA) -1,2,3, EBNA leader protein (EBNA-LP) and Latent Membrane Protein (LMP) -1, 2. EBV has three forms of latency (I, II, III), and in different cells (or tumors), it usually exists in different forms of latency. LMP2A protein is expressed in latency stage II and latency stage III, and LMP2A expression can be detected in gastric cancer, nasopharyngeal cancer and Hodgkin lymphoma.

Although normal B cells also carry EBV, the low number (one carrying the EBV genome per million B cells) allows us to kill EBV-associated tumors by T cell therapy against EBV antigens. Currently, adoptive transfer of EBV-specific T cells has been successfully applied for the prevention of post-transplant lymphoproliferative diseases and the treatment of hodgkin's lymphoma.

Specific T cell immunotherapy refers to a method of killing tumor cells by using specific T cells aiming at tumor antigens, and is a highly personalized tumor immunotherapy method. The function of the autologous T cells in the body of the patient to kill the tumor is limited due to the existence of the tumor local immunosuppressive microenvironment. Therefore, attempts have been made to improve the ability of T cells to kill tumors by genetically modifying them. Both TCR-T and CAR-T are genetically modified cell therapeutic drugs, and after the transferred T Cell Receptor (TCR) or Chimeric Antigen Receptor (CAR) gene is combined with a corresponding target, T cells can be activated, and granzyme, perforin, cytokine and the like released by the T cells are used for eliminating tumor cells; but the significant differences between TCR-T and CAR-T are: the target of CAR-T is a cell surface membrane protein, while the target of TCR-T is the antigen-short peptide-MHC complex (pMHC).

The target of TCR recognition is the "antigen short peptide-MHC molecule complex". Similar to the concept of antibody epitopes, the short peptide-MHC complex of an antigen that can be recognized by a TCR is called a T cell epitope, as is the relationship of antigen and antibody. The TCR is also MHC-restricted and is capable of specifically recognizing short peptides presented by a particular MHC. MHC is polymorphic, the number of currently found human MHC (also known as human leukocyte antigen, HLA) alleles is over 15000, and the frequency of specific HLA occurrence in different populations varies greatly, with the most common HLA type in the chinese population being a 1101. The existing research shows that the complex of EBV LMP2A antigen short peptide SSCSSCPLSK-A1101 can be used as a target point for TCR recognition.

Therefore, those skilled in the art have focused on isolating a TCR specific for the EBV LMP2A antigen short peptide and transducing the TCR into T cells to obtain T cells specific for the EBV LMP2A antigen short peptide, thereby making them useful in T cell immunotherapy.

Disclosure of Invention

The invention aims to provide a T cell receptor for recognizing EBV antigen and application of the T cell receptor. The TCR disclosed by the invention can be specifically combined with the EBV antigen short peptide, and the T cell transduced with the TCR can be specifically activated and has a strong killing effect on a target cell.

The specific technical scheme of the invention is as follows:

1. a T Cell Receptor (TCR), wherein the TCR comprises an alpha chain comprising a variable region comprising complementarity determining region 3(CDR3) having amino acid sequence AVVNNNDMR (SEQ ID NO: 3); and/or

The variable region of the beta chain comprises complementarity determining region 3(CDR3) comprising ASSPGRWYEQY (SEQ ID NO: 6).

2. The T Cell Receptor (TCR) according to claim 1, wherein the TCR is capable of binding to the SSCSSCPLSK-HLAA1101 complex.

3. The T Cell Receptor (TCR) according to claim 1 or 2, wherein the variable region of the alpha chain comprises complementarity determining region 1(CDR1) having the amino acid sequence DSVNN (SEQ ID NO: 1); and/or

The amino acid sequence is complementarity determining region 2(CDR2) of IPSGT (SEQ ID NO: 2).

4. The T Cell Receptor (TCR) according to any of claims 1-3, wherein the variable region of the β chain comprises complementarity determining region 1(CDR1) having the amino acid sequence MGHRA (SEQ ID NO: 4); and/or

The amino acid sequence is the complementarity determining region 2(CDR2) of YSYEKL (SEQ ID NO: 5).

5. The T Cell Receptor (TCR) according to any one of claims 1 to 4, the variable region of the a chain further comprising a first leader sequence; and/or

The variable region of the beta strand further comprises a second leader sequence.

6. The T Cell Receptor (TCR) according to any of claims 1-5, wherein the amino acid sequence of the alpha chain variable region is as shown in SEQ ID NO:9 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:9, and/or the amino acid sequence of the variable region of the beta chain is as shown in SEQ ID NO:10 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10.

7. The T Cell Receptor (TCR) according to any one of claims 1 to 6, wherein the alpha chain further comprises an alpha constant region and/or the beta chain further comprises a beta constant region, preferably the constant region is a mouse constant region or a human constant region.

8. The T Cell Receptor (TCR) according to any one of claims 1 to 7, wherein the TCR is isolated or purified or recombinant.

9. The T Cell Receptor (TCR) according to any one of claims 1 to 8, wherein the TCR is human.

10. The T Cell Receptor (TCR) according to any one of claims 1 to 9, wherein the TCR is monoclonal.

11. The T Cell Receptor (TCR) according to any one of claims 1 to 10, wherein the TCR is single chain.

12. The T Cell Receptor (TCR) according to any one of claims 1 to 11, wherein the TCR comprises two chains.

13. The T Cell Receptor (TCR) according to any one of claims 1 to 12, wherein the TCR is in cell-bound form or in soluble form, preferably in soluble form.

14. A nucleic acid molecule, wherein said nucleic acid molecule comprises an α chain or a β chain encoding a TCR or said TCR of any one of claims 1-13.

15. The nucleic acid molecule according to claim 14, wherein the nucleotide sequence encoding the alpha chain comprises the nucleotide sequence set forth in SEQ ID No. 13; and/or

The nucleotide sequence encoding the beta strand comprises the nucleotide sequence shown in SEQ ID NO. 14.

16. A vector, wherein said vector comprises the nucleic acid molecule of claim 14 or 15.

17. The vector of claim 16, wherein the vector is an expression vector.

18. The vector of claim 16 or 17, wherein the vector is a viral vector, preferably a retroviral vector.

19. The vector of claim 18, wherein the viral vector is a lentiviral vector.

20. An engineered cell comprising a TCR according to any one of claims 1-13, a nucleic acid molecule according to any one of claims 14-15, or a vector according to any one of claims 16-19.

21. The engineered cell of claim 20, wherein the TCR is heterologous to the cell.

22. The engineered cell of claim 20 or 21, wherein the engineered cell is a cell line.

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

24. The engineered cell according to any one of claims 20-23, wherein said engineered cell is a T cell, preferably a T cell isolated from peripheral blood.

25. The engineered cell of claim 24, wherein the T cell is CD8+ or CD4 +.

26. A method of producing an engineered cell of any one of claims 20-25, comprising introducing a nucleic acid molecule of any one of claims 14-15 or a vector of any one of claims 16-19 into a cell in vitro or ex vivo.

27. The method of item 26, wherein the vector is a viral vector and the introducing is by transduction.

28. A pharmaceutical composition comprising a T Cell Receptor (TCR) according to any one of claims 1 to 13, a nucleic acid molecule according to any one of claims 14 to 15, a vector according to any one of claims 16 to 19 or an engineered cell according to any one of claims 20 to 25.

29. The pharmaceutical composition of item 28, further comprising a pharmaceutically acceptable carrier or adjuvant.

30. Use of a T Cell Receptor (TCR) according to any one of claims 1 to 13, a nucleic acid molecule according to any one of claims 14 to 15, a vector according to any one of claims 16 to 17, an engineered cell according to any one of claims 20 to 25 or a pharmaceutical composition according to any one of claims 28 to 29 in the preparation of a medicament for the treatment of an EBV-associated disease.

31. The use according to item 30, wherein the EBV-associated disease is nasopharyngeal carcinoma, gastric carcinoma, hodgkin's lymphoma, burkitt's lymphoma, post-transplantation lymphoproliferative disease, nasal extranodal natural killer/T cell lymphoma, B cell lymphoma or follicular dendritic cell sarcoma.

ADVANTAGEOUS EFFECTS OF INVENTION

The TCR disclosed by the invention can be combined with an EBV antigen short peptide complex SSCSSCPLSK-HLAA1101, and meanwhile, T cells transduced with the TCR can be specifically activated and have a strong killing effect on target cells.

Drawings

FIG. 1 is a schematic representation of the flow cytometry used to identify Jurkat-CD8+ T cells in example 3 that were around 100% positive for P2 tetramer transfected with the TCR of the invention;

FIG. 2 is a graph showing the expression level of IL-2 in example 3;

FIG. 3 is a schematic representation of the identification of TCR transfection efficiency using flow cytometry in example 4;

FIG. 4 is a graph showing the expression levels of IFN-. gamma.in example 4.

Detailed Description

The present invention is described in detail in the following description of embodiments with reference to the figures, in which like numbers represent like features throughout the figures. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

It should be noted that certain terms are used throughout the description and claims to refer to particular components. As one skilled in the art will appreciate, various names may be used to refer to a component. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the invention, however, the description is given for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.

The invention provides a T Cell Receptor (TCR), wherein the TCR comprises an alpha chain comprising a variable region and/or a beta chain comprising a variable region, the variable region of the alpha chain comprising a complementarity determining region 3(CDR3) comprising amino acid sequence AVVNNNDMR (SEQ ID NO: 3); and/or

The T cell receptor or TCR is a specific receptor for a specific antigenic peptide presented on the Major Histocompatibility Complex (MHC), and in the immune system, direct physical contact between the T cell and the Antigen Presenting Cell (APC) is initiated by the binding of the antigen-specific TCR to the pMHC complex, and then other cell surface molecules of both the T cell and the APC interact, which causes a series of subsequent cell signaling and other physiological responses, thereby allowing T cells of different antigen specificities to exert an immune effect on the target cell.

The TCR is a molecule containing variable α and β chains or variable γ and δ chains and which is capable of binding to peptide-specific binding of MHC molecules, in some embodiments the TCR is in the α β form. Generally, TCRs in the α β and γ δ forms are generally similar in structure, but T cells expressing them may have different anatomical locations or functions, and TCRs may be found on the cell surface or in soluble form. Generally, a TCR is found on the surface of a T cell (T lymphocyte), where it is generally responsible for recognizing antigens bound to Major Histocompatibility Complex (MHC) molecules.

The variable domain of a TCR contains Complementarity Determining Regions (CDRs), which are typically the major contributors to antigen recognition and binding capacity and specificity of the peptide, MHC and/or MHC-peptide complex, the CDRs or combinations thereof of the TCR forming all or substantially all of the antigen binding site of a given TCR molecule, each CDR within the variable region of the TCR typically being separated by a Framework Region (FR). Where CDR3 is the predominant CDR responsible for antigen binding or specificity, or is most important in three CDRs for antigen recognition and/or for interaction with the processed peptide portion of the peptide-MHC complex on a given TCR variable region, in some embodiments CDR1 of the a chain may interact with the N-terminal portion of certain antigenic peptides; in some embodiments, the CDR1 of the β chain may interact with the C-terminal portion of certain antigenic peptides; in some embodiments, CDR2 has the strongest effect or is the primary responsible CDR on the interaction or recognition of the MHC part of the MHC-peptide complex; in some embodiments, the variable region antigen of the beta chain contains other hypervariable regions (CDR4 or HVR4) that are normally involved in superantigen binding rather than antigen recognition.

In one embodiment, the TCR is capable of binding to the SSCSSCPLSK-HLAA1101 complex.

SSCSSCPLSK-HLAA1101 complex refers to a complex in which HLA-A1101 and polypeptide SSCSSCPLSK bind. Proteins are degraded in cells by proteasomes into polypeptides of different lengths, and a portion of the polypeptides are presented to the cell surface as complexes with HLA. The SSCSSCPLSK-HLAA1101 complex recognized by the TCR can be expressed on a cell membrane and can also exist in solution in the form of a soluble protein.

The amino acid sequence of the HLAA1101 is shown as SEQ ID NO. 16, and the amino acid sequence is as follows:

MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGYYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWELSSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSDRKGGSYTQAASSDSAQGSDVSLTACKVSR

in one embodiment, the variable region of the alpha chain comprises complementarity determining region 1(CDR1) having the amino acid sequence DSVNN (SEQ ID NO: 1); and/or

In one embodiment, the variable region of the beta strand comprises complementarity determining region 1(CDR1) having the amino acid sequence MGHRA (SEQ ID NO: 4); and/or

In one embodiment, the variable region of the alpha chain further comprises a first leader sequence; and/or

The first leader sequence of the variable region of the alpha chain and the second leader sequence of the variable region of the beta chain are well known to those skilled in the art, and for example, the first leader sequence of the variable region of the alpha chain may use a leader sequence having an amino acid sequence shown in SEQ ID NO. 7, and the second leader sequence of the variable region of the beta chain may use a leader sequence having an amino acid sequence shown in SEQ ID NO. 8.

Wherein, the amino acid sequence shown in SEQ ID NO. 7 is:

MKRILGALLGLLSAQVCCVR

the amino acid sequence shown in SEQ ID NO. 8 is:

MGCRLLCCAVLCLLGAVPI。

in one embodiment, the amino acid sequence of the alpha chain variable region is as set forth in SEQ ID NO. 9 or an amino acid sequence having at least 90% sequence identity with SEQ ID NO. 9, and/or the amino acid sequence of the variable region of the beta chain is as set forth in SEQ ID NO. 10 or an amino acid sequence having at least 90% sequence identity with SEQ ID NO. 10.

Wherein, the amino acid sequence shown in SEQ ID NO. 9 is:

MKRILGALLGLLSAQVCCVRGIQVEQSPPDLILQEGANSTLRCNFSDSVNNLQWFHQNPWGQLINLFYIPSGTKQNGRLSATTVATERYSLLYISSSQTTDSGVYFCAVVNNNDMRFGAGTRLTVKPN

the amino acid sequence shown in SEQ ID NO. 10 is:

MGCRLLCCAVLCLLGAVPIDTEVTQTPKHLVMGMTNKKSLKCEQHMGHRAMYWYKQKAKKPPELMFVYSYEKLSINESVPSRFSPECPNSSLLNLHLHALQPEDSALYLCASSPGRWYEQYFGPGTRLTVT。

the amino acid sequence of the alpha chain variable region having at least 90% sequence identity to SEQ ID No. 9 may be an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% sequence identity to SEQ ID No. 9; the amino acid sequence of the variable region of the beta strand may have at least 90% sequence identity with SEQ ID No. 10 and may be an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% sequence identity with SEQ ID No. 9.

In one embodiment, the alpha chain further comprises an alpha constant region and/or the beta chain further comprises a beta constant region, preferably, the constant region is a mouse constant region or a human constant region,

for example, the amino acid sequence of the mouse α constant region is shown in SEQ ID NO. 11 and/or the amino acid sequence of the mouse β constant region is shown in SEQ ID NO. 12.

The amino acid sequence shown in SEQ ID NO. 11 is:

IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS

the amino acid sequence shown in SEQ ID NO. 12 is:

EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS

the constant region of the TCR may contain a short linking sequence in which cysteine residues form a disulfide bond, thereby linking the two chains of the TCR. The TCR may have additional cysteine residues in each of the α and β chains, such that the TCR contains two disulfide bonds in the constant region.

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

In one embodiment, the TCR is isolated or purified or recombinant.

In one embodiment, the TCR is human.

In one embodiment, the TCR is monoclonal.

In one embodiment, the TCR is single chain.

In one embodiment, the TCR comprises two chains.

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, e.g., the TCRs can be derived from one of a number of animal species, such as humans, mice, rats, or other mammals, such as typically from humans.

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

The TCR in soluble form refers to a TCR in which the hydrophobic core region is mutated, preferably such that the mutation of the hydrophobic core region is such that it results in an increased stability of the soluble TCR of the invention.

The invention provides a nucleic acid molecule comprising an alpha or beta chain encoding a TCR or TCR as described above.

In one embodiment, wherein the nucleotide sequence encoding the alpha chain comprises the nucleotide sequence set forth in SEQ ID NO 13; and/or

Wherein, the nucleotide sequence shown in SEQ ID NO. 13 is:

ATGAAAAGAATCCTGGGAGCTCTGCTGGGCCTGCTCTCCGCCCAGGTGTGCTGTGTGCGGGGCATCCAGGTGGAACAGAGCCCTCCAGACCTGATTCTGCAGGAGGGCGCCAACAGCACCCTGAGATGCAACTTCAGCGACTCCGTGAACAACCTGCAATGGTTCCACCAGAACCCCTGGGGCCAGCTGATCAACCTGTTCTACATCCCTAGCGGAACCAAGCAGAATGGCCGCCTGTCTGCCACCACCGTGGCCACAGAGAGATACAGCCTGCTGTATATCAGCTCTAGCCAGCTGACAGATAGCGGCGTGTACTTCTGCGCCGTGGTCAACAACAATGACATGCGGTTTGGCGCTGGCACCAGACTGACAGTGAAGCCTAACATCCAGAATCCAGAGCCCGCCGTGTATCAGCTGAAGGACCCAAGGAGCCAGGATTCCACCCTGTGCCTGTTCACAGACTTTGATAGCCAGATCAACGTGCCCAAGACCATGGAGTCCGGCACCTTCATCACAGACAAGTGCGTGCTGGATATGAAGGCCATGGACTCTAAGAGCAACGGCGCCATCGCCTGGAGCAATCAGACCTCCTTCACATGCCAGGATATCTTTAAGGAGACCAATGCCACATATCCTTCCTCTGACGTGCCATGTGATGCCACCCTGACAGAGAAGTCCTTCGAGACCGACATGAACCTGAATTTTCAGAACCTGTCTGTGATGGGCCTGCGCATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGAGGCTGTGGAGCTCC

the nucleotide sequence shown in SEQ ID NO. 14 is:

ATGGGATGTAGACTGCTGTGCTGCGCCGTGCTGTGCCTGCTGGGCGCTGTGCCAATCGACACCGAGGTGACACAGACCCCTAGATACCTGGTCATGGGCATGACCAACAAGAAGTCCCTGAAGTGCGAGCAACACATGGGCCACCGGGCCATGTACTGGTACAAGCAGAAAGCCAAGAAACCCCCCGAACTGATGTTCGTGTACAGCTACGAGAAGCTGAGCATCAACGAGAGCGTGCCTAGCCGGTTCAGCCCCGAGTGCCCTAATAGCTCTCTGCTCAACCTGCATCTGCACGCCCTGCAGCCTGAAGATAGCGCCCTGTACCTGTGTGCTTCTTCCCCTGGCAGATGGTATGAACAGTACTTTGGCCCTGGAACAAGACTGACCGTGACCGAGGATCTGAGGAACGTGACACCCCCTAAGGTGTCTCTGTTCGAGCCCAGCAAGGCCGAGATCGCCAATAAGCAGAAGGCCACCCTGGTGTGCCTGGCAAGGGGCTTCTTTCCTGATCACGTGGAGCTGTCTTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGCGTGTGCACCGACCCACAGGCCTACAAGGAGTCCAATTACTCTTATTGTCTGAGCTCCCGGCTGAGAGTGTCCGCCACATTTTGGCACAACCCTAGAAATCACTTCAGGTGCCAGGTGCAGTTTCACGGCCTGAGCGAGGAGGATAAGTGGCCAGAGGGATCCCCAAAGCCTGTGACCCAGAACATCTCTGCCGAGGCATGGGGAAGGGCAGACTGTGGAATCACATCCGCCTCTTATCACCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTATGCCGTGCTGGTGAGCGGCCTGGTGCTGATGGCCATGGTGAAGAAGAAGAACTCC

the nucleic acid molecules may include those comprising naturally and/or non-naturally occurring nucleotides and bases, for example including those with backbone modifications, which refers to polymers of nucleotides, such polymers of nucleotides may contain natural and/or non-natural nucleotides, and include, but are not limited to, DNA, RNA and PNA. Nucleotide sequence refers to the linear sequence that constitutes a nucleic acid molecule.

In some cases, the nucleic acid molecule comprises cDNA, and in some cases, the nucleic acid molecule can be modified for use in the constructs of the invention, e.g., for codon optimization. In some cases, the sequences may be designed to contain terminal restriction site sequences for cloning into a vector.

In some cases, the nucleic acid molecule encoding the TCR may be obtained from a variety of sources, such as by Polymerase Chain Reaction (PCR) amplification of the encoding nucleic acid within or isolated from one or more given cells.

In one embodiment, the nucleotide sequence encoding the alpha chain and/or the nucleotide sequence encoding the beta chain is codon optimized. In general, codon optimization involves balancing the percentage of codons selected with the abundance of human transfer RNA disclosed, such that none 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. Differences in codon usage between the transfected gene and the host cell may affect protein expression and immunogenicity of the nucleic acid construct. Typically, for codon optimization, codons are selected to select those that are balanced with human frequency of use. Typically, the redundancy of amino acid codons is such that different codons encode one amino acid. In some embodiments, in selecting codons for replacement, it may be desirable that the resulting mutation be a silent mutation, such that the codon change does not affect the amino acid sequence. In general, the last nucleotide of a codon can remain unchanged without affecting the amino acid sequence.

The present invention provides a vector comprising the nucleic acid molecule as described above.

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

The vector may contain regulatory sequences (such as transcription and translation initiation and termination codons) that are specific for the type of host into which the vector is to be introduced (e.g., bacterial, fungal, plant or animal), as appropriate and taking into account whether the vector is DNA-based or RNA-based. The vector may also contain a non-native 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 promoters found in the long terminal repeats of murine stem cell viruses, although other promoters known to the skilled artisan are also contemplated.

In one embodiment, the vector is an expression vector.

In one embodiment, the vector is a viral vector, preferably a retroviral vector.

In one embodiment, the viral vector is a lentiviral vector.

The invention provides a host cell comprising such a nucleic acid, for recombinant production of the TCR the nucleic acid encoding the TCR may be isolated and inserted into one or more vectors for further cloning and/or expression in the host cell. Such nucleic acids can be readily isolated and sequenced using conventional techniques (e.g., by using oligonucleotide probes that are capable of specifically binding to the genes encoding the α and β chains of the TCR). In some embodiments, methods of making a TCR are provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding a TCR, as provided above, under conditions suitable for expression of a TCR molecule, and optionally recovering the TCR from the host cell (or host cell culture medium).

The host cell refers to a cell into which an exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include transformants and transformed cells, including primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations.

The invention provides an engineered cell comprising the TCR described above, the nucleic acid molecule described above or the vector described above.

In one embodiment, the TCR is heterologous to the cell.

In one embodiment, the engineered cell is a cell line.

In one embodiment, the engineered cell is a primary cell obtained from a subject, preferably, the subject is a mammalian subject, preferably a human.

In one embodiment, the engineered cells are T cells, preferably T cells isolated from peripheral blood.

In one embodiment, the T cells are CD8+ or CD4 +.

The engineered cell may be, for example, a population of cells or a genetically engineered cell expressing a TCR, which is typically a eukaryotic cell, such as a mammalian cell, and typically a human cell. In some embodiments, the cell is derived from blood, bone marrow, lymph or lymphoid organs, and is a cell of the immune system, such as a cell of innate or adaptive immunity, e.g., 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). The cells are typically primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells comprise one or more subsets of T cells or other cell types, such as the entire T cell population, CD + cells, CD8+ cells, and subpopulations thereof.

Subtypes and subpopulations 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 subtypes thereof (e.g., 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 (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated constant T (mait) cells, naturally occurring and adaptive regulatory T (treg) cells, and the like.

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.

The present invention provides a method for producing the above engineered cell comprising introducing the above nucleic acid molecule or the above vector into a cell in vitro or ex vivo.

In one embodiment, the vector is a viral vector and the introducing is by transduction.

The invention provides a pharmaceutical composition comprising the T Cell Receptor (TCR) described above, the nucleic acid molecule described above, the vector described above or the engineered cell described above.

In one embodiment, it further comprises a pharmaceutically acceptable carrier or adjuvant.

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

The pharmaceutical compositions may utilize timed release, delayed release, and sustained release delivery systems such that delivery of the composition occurs prior to sensitization of the site to be treated and sufficient time is allowed to cause sensitization. Many types of delivery systems are available and known. Such systems can avoid repeated administrations of the composition, thereby increasing convenience for the subject and the physician.

The invention provides the use of the T Cell Receptor (TCR), the nucleic acid molecule, the vector, the engineered cell or the pharmaceutical composition in the preparation of a medicament for treating EBV-related diseases.

In one embodiment, the EBV-associated disease is nasopharyngeal carcinoma, gastric carcinoma, hodgkin's lymphoma, burkitt's lymphoma, post-transplant lymphoproliferative disease, nasal extranodal natural killer/T cell lymphoma, B cell lymphoma, follicular dendritic cell sarcoma, or the like.

Examples

The invention is described generally and/or specifically for the materials used in the tests and the test methods, in the following examples,% means wt%, i.e. percent by weight, unless otherwise specified. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

Example 1: cloning EBV LMP2A antigen short peptide specific T cell and obtaining TCR gene

Peripheral blood lymphocytes from healthy volunteers of genotype HLA-A1101 were stimulated with synthetic short peptide SSCSSCPLSK (SEQ ID NO: 15; Kingstony Biotech, Inc.). The SSCSSCPLSK short peptide was renatured with HLA-A1101 having a biotin label to prepare a pHLA monomer. These monomers and streptavidin (BD) labeled with PE were combined into a PE-labeled tetramer, the tetramer and anti-CD 8-FITC double positive cells were enriched, the obtained double positive cells were subjected to flow sorting to obtain single cells, the single cells obtained by sorting were amplified respectively for TCR α chain and β chain with one-step RT-PCR kit (QIAGEN, catalog No. 210212), and the PCR products were sequenced. Comparing the sequencing result with the sequences in the public database of IMGT (International immunogenetics information System), the nucleotide sequences of TCR alpha chain variable region sequence and beta chain variable region and the information of CDR1, CDR2 and CDR3 thereof can be obtained, wherein, the nucleotide sequence of the variable region of the alpha chain (SEQ ID NO:13) is:

ATGAAAAGAATCCTGGGAGCTCTGCTGGGCCTGCTCTCCGCCCAGGTGTGCTGTGTGCGGGGCATCCAGGTGGAACAGAGCCCTCCAGACCTGATTCTGCAGGAGGGCGCCAACAGCACCCTGAGATGCAACTTCAGCGACTCCGTGAACAACCTGCAATGGTTCCACCAGAACCCCTGGGGCCAGCTGATCAACCTGTTCTACATCCCTAGCGGAACCAAGCAGAATGGCCGCCTGTCTGCCACCACCGTGGCCACAGAGAGATACAGCCTGCTGTATATCAGCTCTAGCCAGCTGACAGATAGCGGCGTGTACTTCTGCGCCGTGGTCAACAACAATGACATGCGGTTTGGCGCTGGCACCAGACTGACAGTGAAGCCTAAC

the amino acid sequence of the translated variable region of the alpha chain (SEQ ID NO:9) is:

MKRILGALLGLLSAQVCCVRGIQVEQSPPDLILQEGANSTLRCNFSDSVNNLQWFHQNPWGQLINLFYIPSGTKQNGRLSATTVATERYSLLYISSSQTTDSGVYFCAVVNNNDMRFGAGTRLTVKPN, wherein the first and second end caps are, among others,

complementarity determining region 1(CDR1) is DSVNN (SEQ ID NO:1),

complementarity determining region 2(CDR2) is IPSGT (SEQ ID NO:2),

complementarity determining region 3(CDR3) is AVVNNNDMR (SEQ ID NO: 3).

The nucleotide sequence of the beta chain variable region (SEQ ID NO:14) is:

ATGGGATGTAGACTGCTGTGCTGCGCCGTGCTGTGCCTGCTGGGCGCTGTGCCAATCGACACCGAGGTGACACAGACCCCTAGATACCTGGTCATGGGCATGACCAACAAGAAGTCCCTGAAGTGCGAGCAACACATGGGCCACCGGGCCATGTACTGGTACAAGCAGAAAGCCAAGAAACCCCCCGAACTGATGTTCGTGTACAGCTACGAGAAGCTGAGCATCAACGAGAGCGTGCCTAGCCGGTTCAGCCCCGAGTGCCCTAATAGCTCTCTGCTCAACCTGCATCTGCACGCCCTGCAGCCTGAAGATAGCGCCCTGTACCTGTGTGCTTCTTCCCCTGGCAGATGGTATGAACAGTACTTTGGCCCTGGAACAAGACTGACCGTGACC

the amino acid sequence of the translated variable region of the beta strand (SEQ ID NO:9) is:

MGCRLLCCAVLCLLGAVPIDTEVTQTPKHLVMGMTNKKSLKCEQHMGHRAMYWYKQKAKKPPELMFVYSYEKLSINESVPSRFSPECPNSSLLNLHLHALQPEDSALYLCASSPGRWYEQYFGPGTRLTVT, wherein the first and second end caps are, among others,

CDR 1(CDR1) is MGHRA (SEQ ID NO:4),

complementarity determining region 2(CDR2) is YSYEKL (SEQ ID NO:5),

complementarity determining region 3(CDR3) is ASSPGRWYEQY (SEQ ID NO: 6).

Example 2: EBV LMP2A antigen short peptide specific TCR slow virus vector construction and slow virus package

(1) TCR Lentiviral vector construction (named pLKO-TCR048)

EBV LMP2A TCR alpha and beta variable region sequences were cloned into pLKO-based expression plasmid (Addgene), alpha or beta variable domains were cloned into PLKO-based expression plasmid containing alpha or beta constant regions by a multi-fragment recombinant cloning kit (Nonuoz Biotech, Cat. No. C113), the ligated plasmid was transformed into competent E.coli strain Stbl3 cells (Shanghai Weidi Biotech, Ltd.) and plated on LB/agar plates containing 100. mu.g/ml ampicillin. After overnight incubation at 37 ℃, single colonies were picked and grown overnight at 37 ℃ with shaking in 10ml of LB containing 100. mu.g/ml ampicillin. Cloned plasmids were purified using a miniprep kit (Tiangen Biochemical Technology Inc. (TIANGEN) catalog # DP118-02) and the plasmids were sequenced to yield pLKO-TCR 048.

(2) Lentiviral packaging

Reagent

Test medium: 10% FBS (Lonsera, Cat. No. S711-001), DMEM (Situofen (cytiva), Cat. No. SH30243.01)

293T cells (deposited with the American type culture Collection ATCC at the depository, accession number CRL-1573) were prepared and cultured in 10cm dishes, plasmid transfection was started at a time of not more than 80% filling, and the ratio of virus-packaging plasmid to pLKO-TCR048 plasmid was 1: 1, total 10. mu.g. The above plasmid was mixed with PEI (polyethyleneimine) in serum-free DMEM medium, and the mixture was added to 293T cells and cultured at 37 ℃. After 72h, the cell supernatant was concentrated using a 100kd ultrafiltration tube to collect viral vectors.

Example 3: construction and functional identification of Jurkat cell line expressing EBV LMP2A antigen short peptide specific TCR

Function of effector cells transducing the TCR of the invention was verified using T2-A11 cells (T2 cells deposited in ATCC with accession number CRL-1992, T2-A11 cells constructed on the basis of T2 cells with reference to Cancer Biology & Therapy,8:21,2025-

ELISA protocol

The following assay was performed to demonstrate the specific activation response of TCR-transduced T cells to target cells. The amount of IL-2 secretion detected by ELISA assay was used as a readout for T cell activation.

(1) Reagent

Test medium: 10% FBS (Sermomeffei, catalog No. 10099-

Wash buffer (PBST): 1xPBS (Sigma, No. P3813) containing 0.05% Tween-20, prepared in deionized water.

Human IL-2 uncoated ELISA kit (Sermer Fei company (ThermoFisher), catalog number 88-7316-88) contains all other required reagents (capture and detection antibody, streptavidin-HRP and substrate solution of human IL-2ELISA 96-well plate)

(2) Method of producing a composite material

Target cell preparation

The target cells used in this experiment were T2-A11 cells. Preparing target cells in experimental culture medium, adjusting the concentration of target cells to 3.2 × 10⁶One/ml, 50. mu.l/well to obtain 1.6X 10⁵Individual cells/well.

Effector cell preparation

The effector cells (T cells) of this experiment were Jurkat-CD8+ T cells transduced with the TCR of the invention, and Jurkat-CD8+ T cells not transfected with the TCR of the invention were used as a control.

Jurkat-CD8+ T cells (Jurkat cells deposited in ATCC under accession No. TIB-152, Jurkat-CD8 cells were constructed on the basis of Jurkat cells under reference to Cancer Res 2006; 66(23): 11455-61) were added to the lentivirus carrying the TCR gene of the invention obtained in example 2 at an MOI (multiplicity of infection) of 10, and the transfection positivity was confirmed at about 100% by flow cytometry 72 hours later (the results are shown in FIG. 1). The effector cell concentration after the expanded culture was adjusted to 3.2X 10⁶One/ml, 50. mu.l/well to obtain 1.6X 10⁵Individual cells/well.

Preparation of short peptide solution

The corresponding short peptide (SSCSSCPLSK) was added to the corresponding target cell (T2-A11) group to give final concentrations of 100. mu.g/ml, 10. mu.g/ml, 1. mu.g/ml, 0.1. mu.g/ml, 0.01. mu.g/ml, 0.001. mu.g/ml, 0.0001. mu.g/ml, and 0.0001. mu.g/ml, respectively, in the ELISA well plate. Different gradients of short peptide concentration 50 μ l/well were then added sequentially from a1 well to H1 well into 96 well plates, and the plates were finally incubated overnight.

ELISA

The well plate was prepared as follows according to the manufacturer's instructions: 5 ml of sterile 1xPBS per plate was prepared at 1: anti-human IFN-. gamma.capture antibody was diluted 250, and 100. mu.l of the diluted capture antibody was added in aliquots to each well. The plates were incubated at 4 ℃ overnight and sealed. Following incubation, the plates were washed the next day to remove excess capture antibody. 200 microliter/well 1x ELISA/ELISPORT dilution was added and the well plate was incubated at room temperature for 1 hour to close the well plate. The 1x ELISA/elispot dilution was then washed from the well plate, and any residual wash buffer was removed by flicking and tapping the ELISA well plate on paper. Adding 100 ul of 1x ELISA/ELISPORT diluent into two rows of A/B wells except A1/A2, then adding 200 ul of each diluted standard into the A1/A2 well, sucking 100 ul of the diluted standard from the A1/A2 well, adding the diluted standard into the B1/B2 well, uniformly mixing, sucking 100 ul of the diluted standard into the C1/C2 well, and so on, diluting to the G1/G2 well, taking the H1/H2 as a blank control, and only adding 1x ELISA/ELISPORT diluent; the assay components were then added to the ELISA plate in the following order:

50 microliter of target cells (3.2X 10)⁶One cell/ml (about 1.6X 10 in total was obtained)⁵Individual target cells/well).

50 microliter of effector cells (1.6X 10)⁵Individual control effector cells/well and TCR positive T cells/well).

All wells were prepared in duplicate for addition.

The plates were then incubated at 4 ℃ for 2 hours, and after incubation, the plates were incubated at 5 ml per plate in 1 × ELISA/ELISPORT dilutions at 1: the anti-human IFN- γ detection antibody was diluted 250, then 100 μ l of the diluted detection antibody was added in equal portions to each well, and the well plates were incubated at room temperature for 1 hour to close the well plates. Then washed 3 times with wash buffer and tapped on a paper towel to remove residual wash buffer. Dilutions were run at 1x ELISA/elispot as 1: Streptavidin-HRP provided in the kit was diluted 100, added to each well at 100 μ l/well, the wells incubated for half an hour at room temperature, washed 6 times with wash buffer, and the wells tapped on a paper towel to remove excess wash buffer. After washing, 100. mu.l/well of 1XTMB solution provided by the kit was added for development. The plates were covered with tinfoil paper and kept in the dark for 15 minutes during development. Spots on the developing plate were routinely detected during this period to determine the optimum time for terminating the reaction. The development reaction was then stopped with 50. mu.l of sulfuric acid solution, and the OD value was measured using a microplate reader (CTL; cell Technology Limited).

Results

The TCR transduced T cells of the invention were tested for IL-2 release in response to target cells loaded with the P2 antigen short peptide by ELISA assay (as described above). The expression level of IL-2 was plotted using Graphpad prism8, and the results are shown in FIG. 2.

As can be seen from FIG. 2, T cells transduced with the TCR of the invention were very responsive to activation of target cells loaded with their specific short peptides.

Example 4: construction and functional identification of primary T cells expressing EBV LMP2A antigen short peptide specific TCR

Validation of the function of effector cells transducing the TCR of the invention Using T2-A11 cells

ELISA protocol

The following assay was performed to demonstrate the specific activation response of TCR-transduced T cells to target cells. The amount of IFN- γ secretion detected by ELISA assay was used as a readout for T cell activation.

Reagent

Test medium: 10% FBS (Sermomeffei, catalog # 10099-

Human IFN γ uncoated ELISA kit (ThermoFisher, catalog # 88-7316-88) contains all other required reagents (capture and detection antibody, streptavidin-HRP and substrate solution of human IFN- γ ELISA 96 well plate)

Method

Target cell preparation

The target cells used in this experiment were T2-A11 cells. Preparing target cells in experimental culture medium, adjusting the concentration of target cells to 8.0 × 10⁵One/ml, 50. mu.l/well to obtain 4.0X 10⁴Individual cells/well.

Effector cell preparation

The effector cells (T cells) of this experiment were T cells transduced with the TCR of the present invention, and the same volunteer did not transfect the TCR of the present invention as a control.

Subjecting peripheral blood of volunteers to density gradient centrifugation to obtain peripheral blood mononuclear cells, and subjecting the peripheral blood mononuclear cells to 24-well plate with 5.0 × 10 per well⁵Mu.l/500 well, 1E6 cells were collected, T cells were stimulated with anti-CD 3/CD28 magnetic beads and placed at 37 ℃ in 5% CO₂Culturing in an incubator. After 24 hours, cell clumping was observed, and after the lentivirus carrying the TCR gene of the present invention obtained in example 2 was added at an MOI of 3 and transduced, the TCR transfection efficiency was identified by flow cytometry after amplification in 1640 medium containing 200IU/ml IL-2 containing 10% FBS until 3 to 4 days after transduction (the results are shown in fig. 3).

Preparation of short peptide solution

The corresponding short peptide (SSCSSCPLSK) was added to the corresponding target cell (T2) test group to give a final concentration of 1. mu.g/ml, 0.1. mu.g/ml, 0.01. mu.g/ml, 0.001. mu.g/ml, 0.0001. mu.g/ml, 0.00001. mu.g/ml, 0.000001. mu.g/ml, 0.0000001. mu.g/ml in the ELISA well plate. Different gradients of short peptide concentration 50 μ l/well were then added sequentially from a1 well to H1 well into 96 well plates, and the plates were finally incubated overnight.

ELISA

50 microliter target cell 8.0X 10⁵Individual cells/ml (giving a total of about 4X 10⁴Individual target cells/well).

50 microliter of effector cells (2X 10)³Individual control effector cells/well and TCR positive T cells/well).

All wells were prepared in duplicate for addition.

The plates were then incubated at 4 ℃ for 2 hours, and after incubation, the plates were incubated at 5 ml per plate in 1 × ELISA/ELISPORT dilutions at 1: the anti-human IFN- γ detection antibody was diluted 250, then 100 μ l of the diluted detection antibody was added in equal portions to each well, and the well plates were incubated at room temperature for 1 hour to close the well plates. Then washed 3 times with wash buffer and tapped on a paper towel to remove residual wash buffer. Dilutions were run at 1x ELISA/elispot as 1: Streptavidin-HRP provided in the kit was diluted 100, added to each well at 100 μ l/well, the wells incubated for half an hour at room temperature, washed 6 times with wash buffer, and the wells tapped on a paper towel to remove excess wash buffer. After washing, 100. mu.l/well of 1XTMB solution provided by the kit was added for development. The plates were covered with tinfoil paper and kept in the dark for 15 minutes during development. Spots on the developing plate were routinely detected during this period to determine the optimum time for terminating the reaction. The development reaction was then terminated with 50. mu.l of a 2N sulfuric acid solution, and the OD value was measured using a microplate reader (CTL; cell Technology Limited).

Results

The TCR transduced T cells of the invention were tested for IFN- γ release in response to target cells loaded with the P2 antigen short peptide by ELISA assay (as described above). IFN-. gamma.expression level was plotted using Graphpad prism8, and the results are shown in FIG. 4.

As can be seen in FIG. 4, T cells transduced with the TCR of the invention were very responsive to activation of target cells loaded with their specific short peptides.

In conclusion, the TCR of the invention can be bound to EBV antigen short peptides, and the T cell transduced with the TCR can be specifically activated and has strong killing effect on target cells.

The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

Sequence listing

<110> tumor hospital in Henan province

GUANGZHOU MEDICAL University

<120> T cell receptor recognizing EBV antigen and use thereof

<130> TPE01381

<160> 16

<170> PatentIn version 3.5

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Ala Ser Ser Pro Gly Arg Trp Tyr Glu Gln Tyr

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Met Lys Arg Ile Leu Gly Ala Leu Leu Gly Leu Leu Ser Ala Gln Val

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Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly Ala

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Val Pro Ile

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Met Lys Arg Ile Leu Gly Ala Leu Leu Gly Leu Leu Ser Ala Gln Val

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Cys Cys Val Arg Gly Ile Gln Val Glu Gln Ser Pro Pro Asp Leu Ile

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Val Asn Asn Leu Gln Trp Phe His Gln Asn Pro Trp Gly Gln Leu Ile

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Asn Leu Phe Tyr Ile Pro Ser Gly Thr Lys Gln Asn Gly Arg Leu Ser

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Ser Gln Thr Thr Asp Ser Gly Val Tyr Phe Cys Ala Val Val Asn Asn

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Asn Asp Met Arg Phe Gly Ala Gly Thr Arg Leu Thr Val Lys Pro Asn

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Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly Ala

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Val Pro Ile Asp Thr Glu Val Thr Gln Thr Pro Lys His Leu Val Met

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Gly Met Thr Asn Lys Lys Ser Leu Lys Cys Glu Gln His Met Gly His

35 40 45

Arg Ala Met Tyr Trp Tyr Lys Gln Lys Ala Lys Lys Pro Pro Glu Leu

50 55 60

Met Phe Val Tyr Ser Tyr Glu Lys Leu Ser Ile Asn Glu Ser Val Pro

65 70 75 80

Ser Arg Phe Ser Pro Glu Cys Pro Asn Ser Ser Leu Leu Asn Leu His

85 90 95

Leu His Ala Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu Cys Ala Ser

100 105 110

Ser Pro Gly Arg Trp Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu

115 120 125

Thr Val Thr

130

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Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser

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Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn

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Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Cys Val

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Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala Trp

50 55 60

Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr Asn

65 70 75 80

Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr Glu

85 90 95

Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Ser Val

100 105 110

Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu

115 120 125

Met Thr Leu Arg Leu Trp Ser Ser

130 135

<210> 12

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Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro

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Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val Cys Leu

20 25 30

Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

35 40 45

Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Ala Tyr Lys

50 55 60

Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala

65 70 75 80

Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe

85 90 95

His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro Lys Pro

100 105 110

Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly

115 120 125

Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu Ser Ala Thr Ile Leu

130 135 140

Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser

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Gly Leu Val Leu Met Ala Met Val Lys Lys Lys Asn Ser

165 170

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atgaaaagaa tcctgggagc tctgctgggc ctgctctccg cccaggtgtg ctgtgtgcgg 60

ggcatccagg tggaacagag ccctccagac ctgattctgc aggagggcgc caacagcacc 120

ctgagatgca acttcagcga ctccgtgaac aacctgcaat ggttccacca gaacccctgg 180

ggccagctga tcaacctgtt ctacatccct agcggaacca agcagaatgg ccgcctgtct 240

gccaccaccg tggccacaga gagatacagc ctgctgtata tcagctctag ccagctgaca 300

gatagcggcg tgtacttctg cgccgtggtc aacaacaatg acatgcggtt tggcgctggc 360

accagactga cagtgaagcc taacatccag aatccagagc ccgccgtgta tcagctgaag 420

gacccaagga gccaggattc caccctgtgc ctgttcacag actttgatag ccagatcaac 480

gtgcccaaga ccatggagtc cggcaccttc atcacagaca agtgcgtgct ggatatgaag 540

gccatggact ctaagagcaa cggcgccatc gcctggagca atcagacctc cttcacatgc 600

caggatatct ttaaggagac caatgccaca tatccttcct ctgacgtgcc atgtgatgcc 660

accctgacag agaagtcctt cgagaccgac atgaacctga attttcagaa cctgtctgtg 720

atgggcctgc gcatcctgct gctgaaggtg gccggcttca atctgctgat gaccctgagg 780

ctgtggagct cc 792

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atgggatgta gactgctgtg ctgcgccgtg ctgtgcctgc tgggcgctgt gccaatcgac 60

accgaggtga cacagacccc tagatacctg gtcatgggca tgaccaacaa gaagtccctg 120

aagtgcgagc aacacatggg ccaccgggcc atgtactggt acaagcagaa agccaagaaa 180

ccccccgaac tgatgttcgt gtacagctac gagaagctga gcatcaacga gagcgtgcct 240

agccggttca gccccgagtg ccctaatagc tctctgctca acctgcatct gcacgccctg 300

cagcctgaag atagcgccct gtacctgtgt gcttcttccc ctggcagatg gtatgaacag 360

tactttggcc ctggaacaag actgaccgtg accgaggatc tgaggaacgt gacaccccct 420

aaggtgtctc tgttcgagcc cagcaaggcc gagatcgcca ataagcagaa ggccaccctg 480

gtgtgcctgg caaggggctt ctttcctgat cacgtggagc tgtcttggtg ggtgaacggc 540

aaggaggtgc acagcggcgt gtgcaccgac ccacaggcct acaaggagtc caattactct 600

tattgtctga gctcccggct gagagtgtcc gccacatttt ggcacaaccc tagaaatcac 660

ttcaggtgcc aggtgcagtt tcacggcctg agcgaggagg ataagtggcc agagggatcc 720

ccaaagcctg tgacccagaa catctctgcc gaggcatggg gaagggcaga ctgtggaatc 780

acatccgcct cttatcacca gggcgtgctg agcgccacca tcctgtacga gatcctgctg 840

ggcaaggcca cactgtatgc cgtgctggtg agcggcctgg tgctgatggc catggtgaag 900

aagaagaact cc 912

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<400> 15

Ser Ser Cys Ser Ser Cys Pro Leu Ser Lys

1 5 10

<210> 16

<211> 367

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<220>

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<400> 16

Met Ala Val Met Ala Pro Arg Thr Leu Leu Leu Leu Leu Ser Gly Ala

1 5 10 15

Leu Ala Leu Thr Gln Thr Trp Ala Gly Ser His Ser Met Arg Tyr Phe

20 25 30

Tyr Thr Ser Val Ser Arg Pro Gly Arg Gly Glu Pro Arg Phe Ile Ala

35 40 45

Val Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp Ala

50 55 60

Ala Ser Gln Arg Met Glu Pro Arg Ala Pro Trp Ile Glu Gln Glu Gly

65 70 75 80

Pro Glu Tyr Trp Asp Gln Glu Thr Arg Asn Val Lys Ala Gln Ser Gln

85 90 95

Thr Asp Arg Val Asp Leu Gly Thr Leu Arg Gly Tyr Tyr Asn Gln Ser

100 105 110

Glu Asp Gly Ser His Thr Ile Gln Ile Met Tyr Gly Cys Asp Val Gly

115 120 125

Pro Asp Gly Arg Phe Leu Arg Gly Tyr Arg Gln Asp Ala Tyr Asp Gly

130 135 140

Lys Asp Tyr Ile Ala Leu Asn Glu Asp Leu Arg Ser Trp Thr Ala Ala

145 150 155 160

Asp Met Ala Ala Gln Ile Thr Lys Arg Lys Trp Glu Ala Ala His Ala

165 170 175

Ala Glu Gln Gln Arg Ala Tyr Leu Glu Gly Arg Cys Val Glu Trp Leu

180 185 190

Arg Arg Tyr Leu Glu Asn Gly Lys Glu Thr Leu Gln Arg Thr Asp Pro

195 200 205

Pro Lys Thr His Met Thr His His Pro Ile Ser Asp His Glu Ala Thr

210 215 220

Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile Thr Leu Thr

225 230 235 240

Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Thr Glu Leu Val Glu

245 250 255

Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ala Val Val

260 265 270

Val Pro Ser Gly Glu Glu Gln Arg Tyr Thr Cys His Val Gln His Glu

275 280 285

Gly Leu Pro Lys Pro Leu Thr Leu Arg Trp Glu Leu Ser Ser Gln Pro

290 295 300

Thr Ile Pro Ile Val Gly Ile Ile Ala Gly Leu Val Leu Leu Gly Ala

305 310 315 320

Val Ile Thr Gly Ala Val Val Ala Ala Val Met Trp Arg Arg Lys Ser

325 330 335

Ser Asp Arg Lys Gly Gly Ser Tyr Thr Gln Ala Ala Ser Ser Asp Ser

340 345 350

Ala Gln Gly Ser Asp Val Ser Leu Thr Ala Cys Lys Val Ser Arg

355 360 365

Claims

4. A T Cell Receptor (TCR) as claimed in any one of claims 1 to 3 wherein the variable region of the β chain comprises the complementarity determining region 1(CDR1) having the amino acid sequence MGHRA (SEQ ID NO: 4); and/or

6. The T Cell Receptor (TCR) according to any of claims 1-5, wherein the amino acid sequence of the alpha chain variable region is as set forth in SEQ ID NO 9 or an amino acid sequence having at least 90% sequence identity with SEQ ID NO 9, and/or the amino acid sequence of the variable region of the beta chain is as set forth in SEQ ID NO 10 or an amino acid sequence having at least 90% sequence identity with SEQ ID NO 10.

7. The T Cell Receptor (TCR) according to any of claims 1 to 6, wherein the alpha chain further comprises an alpha constant region and/or the beta chain further comprises a beta constant region, preferably the constant region is a mouse constant region or a human constant region.

11. The T Cell Receptor (TCR) according to any one of claims 1 to 10, wherein the TCR is a single chain.

13. The T Cell Receptor (TCR) according to any of claims 1 to 12, wherein the TCR is in a cell-bound form or in a soluble form, preferably in a soluble form.

14. A nucleic acid molecule comprising an alpha or beta chain encoding a TCR according to any one of claims 1 to 13 or a said TCR.

15. The nucleic acid molecule of claim 14, wherein the nucleotide sequence encoding the alpha chain comprises the nucleotide sequence set forth in SEQ ID No. 13; and/or

17. The vector of claim 16, wherein the vector is an expression vector.

18. The vector according to claim 16 or 17, wherein the vector is a viral vector, preferably a retroviral vector.

19. The vector of claim 18, wherein the viral vector is a lentiviral vector.

20. An engineered cell comprising a TCR according to any one of claims 1 to 13, a nucleic acid molecule according to any one of claims 14 to 15 or a vector according to any one of claims 16 to 19.

25. The engineered cell of claim 24, wherein the T cell is CD8+ or CD4 +.

26. A method of producing an engineered cell of any one of claims 20-25 comprising introducing the nucleic acid molecule of any one of claims 14-15 or the vector of any one of claims 16-19 into a cell in vitro or ex vivo.

27. The method of claim 26, wherein the vector is a viral vector and the introducing is by transduction.

29. The pharmaceutical composition of claim 28, further comprising a pharmaceutically acceptable carrier or adjuvant.

30. Use of the T Cell Receptor (TCR) of any one of claims 1 to 13, the nucleic acid molecule of any one of claims 14 to 15, the vector of any one of claims 16 to 17, the engineered cell of any one of claims 20 to 25 or the pharmaceutical composition of any one of claims 28 to 29 in the preparation of a medicament for the treatment of an EBV-associated disease.

31. The use of claim 30, wherein the EBV-associated disease is nasopharyngeal carcinoma, gastric carcinoma, hodgkin's lymphoma, burkitt's lymphoma, post-transplant lymphoproliferative disease, nasal extranodal natural killer/T cell lymphoma, B cell lymphoma, or follicular dendritic cell sarcoma.