CN112940108B - T cell receptor for identifying EBV antigen and application of T cell receptor - Google Patents

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 a complementarity determining region 3 (CDR 3) having an amino acid sequence AVVNNNDMR (SEQ ID NO: 3); and/or the variable region of the beta strand comprises complementarity determining region 3 (CDR 3) 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 receptors that recognize EBV antigens and applications of the T cell receptors

technical field

The present invention relates to the technical field of medicine, in particular to a T cell receptor that recognizes EBV antigen and the application of the T cell receptor.

Background technique

EBV (Epstein–Barr virus), also known as human herpesvirus 4, is a double-stranded DNA virus. Humans are the sole host for EBV, and approximately >90% of the global adult population carries the virus.

EBV is transmitted through contact with saliva. Most primary infections occur in childhood and usually have no or only nonspecific symptoms, whereas infections in late adolescence or adulthood can lead to infectious mononucleosis. Once infection occurs, EBV usually resides latent in peripheral lymphocytes, making the infected person a lifelong EBV carrier. Carrying EBV has no serious consequences in most cases, as long as the virus remains in a latent, inactive state. However, in rare cases, the latent virus is activated, which can lead to chronic active infection and the occurrence of some epithelial, mesenchymal, and lymphoid malignancies, such as nasopharyngeal cancer, gastric cancer, and Hodgkin lymphoma. (Hodgkin lymphoma), Burkitt lymphoma, etc. Epidemiological studies have shown that 90% of patients with nasopharyngeal carcinoma, 10% of gastric cancer, 40% of Hodgkin's lymphoma, and 95% of epidemiological Burkitt's lymphoma are EBV-positive. It is worth mentioning that the incidence of nasopharyngeal carcinoma is very high in southern China and Southeast Asia (especially in Guangdong males), with 30 cases per 100,000 people, but only per 100,000 people worldwide. 1 case, and 100% of undifferentiated nasopharyngeal carcinomas (the most common type) and most differentiated nasopharyngeal carcinomas are EBV positive.

EBV is usually in a latent state in malignant tumor cells, which refers to a state in which there is persistent viral infection in the body but no active viral replication and release. EBV can be latent in memory B cells as well as epithelial cells. It is currently believed that one in every million B cells in individuals recovering from acute EBV infection carry the EBV genome. EBV expresses only limited proteins and RNAs during the incubation period, which is one of the mechanisms by which it evades immune surveillance. The EBV proteins expressed in the latent period 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), which usually exist in different forms of latency in different cells (or tumors). LMP2A protein is expressed in latent phase II and latent phase III, and the expression of LMP2A can be detected in gastric cancer, nasopharyngeal carcinoma and Hodgkin's lymphoma.

Although normal B cells also carry EBV, the numbers are low (one in every million B cells carry the EBV genome), which allows us to kill EBV-associated tumors with T-cell therapy targeting EBV antigens. At present, the method of adoptive transfer of EBV-specific T cells has been successfully applied to the prevention of post-transplant lymphoproliferative diseases and the treatment of Hodgkin's lymphoma.

Specific T cell immunotherapy refers to the use of specific T cells against tumor antigens to kill tumor cells, which is a highly personalized tumor immunotherapy method. Due to the existence of the tumor's local immunosuppressive microenvironment, the tumor-killing function of the patient's own T cells is limited. Therefore, people try to improve the ability of T cells to kill tumors by genetically modifying them. Both TCR-T and CAR-T are gene-modified cell therapy drugs. After the transferred T cell receptor (TCR) or chimeric antigen receptor (CAR) gene is combined with the corresponding target, the It can activate T cells and use granzymes, perforin, cytokines, etc. released by T cells to clear tumor cells; but the significant difference between TCR-T and CAR-T is that the target of CAR-T is the membrane protein on the cell surface , while the target of TCR-T is the antigen peptide-MHC complex (peptide-major histocompatibility complex, pMHC).

The target recognized by TCR is "antigen peptide-MHC molecule complex". Similar to the concept of antibody epitopes, antigen short peptide-MHC complexes that can be recognized by TCR are called T cell epitopes, just like the relationship between antigens and antibodies. TCR is also MHC-restricted and can specifically recognize short peptides presented by a specific MHC. MHC is polymorphic. The number of human MHC (also known as human leukocyte antigen, HLA) alleles has been found to exceed 15,000, and the frequency of occurrence of specific HLA in different populations is very different. China The most common type of HLA in the population is A1101. Existing studies have shown that the complex of the EBV LMP2A antigen short peptide SSCSS CPLSK-A1101 can be used as a target for TCR recognition.

Therefore, those skilled in the art are devoted to isolating TCRs specific for EBV LMP2A antigenic short peptides, and transducing the TCRs into T cells to obtain T cells specific for EBV LMP2A antigenic short peptides, so that they can be used in T cells. role in cellular immunotherapy.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a T cell receptor that recognizes EBV antigens and applications of the T cell receptor. The TCR of the present invention can specifically bind to the EBV antigen short peptide, and the T cells transduced with the TCR can be specifically activated and have a strong killing effect on the target cells.

The specific technical scheme of the present invention is as follows:

1. a T cell receptor (TCR), wherein, the TCR comprises an α chain containing a variable region and/or a β chain containing a variable region, and the variable region of the α chain comprises an amino acid sequence that is AVVNNNDMR (SEQ ID NO: 3) complementarity determining region 3 (CDR3); and/or

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

2. The T cell receptor (TCR) according to item 1, wherein the TCR is capable of binding to the SSCSS CPLSK-HLAA1101 complex.

3. The T cell receptor (TCR) according to item 1 or 2, wherein the variable region of the alpha chain comprises a complementarity determining region 1 (CDR1) whose amino acid sequence is DSVNN (SEQ ID NO: 1); and /or

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

4. The T cell receptor (TCR) according to any one of items 1 to 3, wherein the variable region of the beta chain comprises the complementarity determining region 1 ( CDR1); 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 items 1-4, the variable region of the alpha chain further comprising a first leader sequence; and/or

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

6. The T cell receptor (TCR) according to any one of items 1-5, wherein the amino acid sequence of the α chain variable region is as shown in SEQ ID NO:9 or has the same value as SEQ ID NO:9 The amino acid sequence having at least 90% sequence identity, 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 with SEQ ID NO: 10.

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

8. The T cell receptor (TCR) according to any one of items 1-7, wherein the TCR is isolated or purified or recombinant.

9. The T cell receptor (TCR) according to any one of items 1-8, wherein the TCR is human.

10. The T cell receptor (TCR) according to any one of items 1-9, wherein the TCR is monoclonal.

11. The T cell receptor (TCR) according to any one of items 1-10, wherein the TCR is single chain.

12. The T cell receptor (TCR) according to any one of items 1-11, wherein the TCR comprises two chains.

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

14. A nucleic acid molecule comprising encoding the TCR of any one of items 1-13 or the alpha chain or beta chain of the TCR.

15. The nucleic acid molecule according to item 14, wherein the nucleotide sequence encoding the alpha chain comprises the nucleotide sequence shown in SEQ ID NO: 13; and/or

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

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

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

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

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

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.

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

22. The engineered cell according to item 20 or 21, wherein the engineered cell is a cell line.

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 the subject is a mammalian subject , preferably human.

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.

25. The engineered cell according to item 24, wherein the T cells are CD8+ or CD4+.

26. A method of producing the engineered cell of any one of items 20-25, comprising in vitro or ex vivo producing the nucleic acid molecule of any one of items 14-15 or of items 16-19 The vector of any one is introduced into a cell.

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

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, any of items 16-19 The vector of item or the engineered cell of any one of items 20-25.

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

30. 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 17, and the item 20 Use of the engineered cell of any one of 25 or the pharmaceutical composition of any one of Items 28 to 29 in the manufacture of a medicament for the treatment of EBV-related diseases.

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

effect of invention

The TCR of the present invention can bind to the EBV antigen short peptide complex SSCSSCPLSK-HLAA1101, and the T cells transduced with the TCR can be specifically activated and have a strong killing effect on target cells.

Description of drawings

1 is a schematic diagram of identifying Jurkat-CD8+ T cells with about 100% positive P2 tetramer transfected with the TCR of the present invention using flow cytometry in Example 3;

Fig. 2 is the expression level curve of IL-2 in Example 3;

Fig. 3 is the schematic diagram of using flow cytometer to identify TCR transfection efficiency in Example 4;

FIG. 4 is the expression level curve of IFN-γ in Example 4. FIG.

Detailed ways

The present invention will be described in detail below with reference to the embodiments described in the accompanying drawings, wherein like numerals represent like features throughout the drawings. While specific embodiments of the present invention are shown in the drawings, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that the present invention will be more thoroughly understood, and will fully convey the scope of the present invention to those skilled in the art.

It should be noted that certain terms are used in the description and claims to refer to specific components. It should be understood by those skilled in the art that the same component may be referred to by different nouns. The present specification and claims do not take the difference in terms as a way to distinguish components, but take the difference in function of the components as a criterion for distinguishing. As referred to throughout the specification and claims, "comprising" or "including" are open-ended terms and should be interpreted as "including but not limited to". Subsequent descriptions in the specification are preferred embodiments for implementing the present invention, however, the descriptions are for the purpose of general principles of the specification and are not intended to limit the scope of the present invention. The scope of protection of the present invention should be determined by the appended claims.

The present invention provides a T cell receptor (TCR), wherein the TCR comprises an α chain containing a variable region and/or a β chain containing a variable region, and the variable region of the α chain contains the amino acid sequence AVVNNNDMR Complementarity Determining Region 3 (CDR3) of (SEQ ID NO: 3); and/or

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

The T cell receptor or TCR is a specific receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC), in the immune system, through the binding of antigen-specific TCRs to the pMHC complex Initiating direct physical contact between T cells and antigen-presenting cells (APCs), and then interacting with other cell surface molecules of both T cells and APCs, which leads to a series of subsequent cell signaling and other physiological responses, resulting in T cells with different antigen specificities exert immune effects on target cells.

The TCR is a molecule containing variable alpha and beta chains or variable gamma and delta chains and the molecule is capable of peptide-specific binding to an MHC molecule, in some embodiments the TCR is in the alpha beta form. In general, TCRs in αβ and γδ forms are generally similar in structure, but the T cells expressing them can have different anatomical locations or functions, and TCRs can be found on the cell surface or in soluble forms. Typically, the TCR is found on the surface of T cells (T lymphocytes), where it is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

The variable domains of TCRs contain complementarity determining regions (CDRs), which are typically major contributors to antigen recognition, as well as binding capacity and specificity of peptides, MHCs and/or MHC-peptide complexes, the CDRs of TCRs, or a combination thereof, form the To define all or substantially all of the antigen-binding site of a TCR molecule, the individual CDRs within the variable region of the TCR are typically separated by framework regions (FRs). Of these, CDR3 is the primary CDR responsible for antigen binding or specificity, or is the most important of the 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 Importantly, in some embodiments, the CDR1 of the alpha chain can interact with the N-terminal portion of certain antigenic peptides; in some embodiments, the CDR1 of the beta chain can interact with the C-terminal portion of certain antigenic peptides; in some In embodiments, CDR2 has the strongest effect on interaction or recognition of the MHC portion of the MHC-peptide complex or is primarily responsible for the CDR; in some embodiments, the variable region antigen of the beta chain contains other hypervariable regions ( CDR4 or HVR4), which are normally involved in superantigen binding rather than antigen recognition.

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

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

The amino acid sequence of the HLAA1101 is shown in SEQ ID NO: 16, and its amino acid sequence is:

MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGYYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWELSSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSDRKGGSYTQAASSDSAQGSDVSLTACKVSR

In one embodiment, the variable region of the alpha chain comprises Complementarity Determining Region 1 (CDR1) having the amino acid sequence of DSVNN (SEQ ID NO: 1); and/or

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

In one embodiment, the variable region of the beta chain comprises the complementarity determining region 1 (CDR1) of 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).

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

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

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 be The leader sequence of the amino acid sequence shown in SEQ ID NO: 7 can be used, and the second leader sequence of the variable region of the beta chain can be the leader sequence of the 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 shown in SEQ ID NO: 9 or has at least 90% sequence identity with SEQ ID NO: 9, and/or the beta chain can be The amino acid sequence of the variable region is set forth in SEQ ID NO:10 or an amino acid sequence having at least 90% sequence identity to 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 with SEQ ID NO:9 may be 90%, 91%, 92%, 93%, 94%, 90%, 91%, 92%, 93%, 94%, Amino acid sequences with 95%, 96%, 97%, 98% sequence identity; the amino acid sequence of the variable region of the beta chain having at least 90% sequence identity with SEQ ID NO: 10 may be with SEQ ID NO: 9 Amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% sequence identity.

In one embodiment, 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,

For example, the amino acid sequence of the mouse alpha constant region is set forth in SEQ ID NO:11 and/or the amino acid sequence of the mouse beta constant region is set forth 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 linker sequence in which the cysteine residues form a disulfide bond linking the two chains of the TCR. The TCR may have additional cysteine residues in each of the alpha and beta 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 constant region of the alpha chain and beta chain of the TCR, and the positions of the 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-stranded.

In one embodiment, the TCR comprises two chains.

TCRs can be obtained from biological sources, such as from cells (eg, from T cells (eg, cytotoxic T cells)), T-cell hybridomas, or other publicly available sources, eg, TCRs can be derived from one of a number of animal species, such as Human, mouse, rat or other mammal, such as usually from humans.

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

The TCR in a soluble form refers to a TCR with mutations in its hydrophobic core region, and these mutations in the hydrophobic core region are preferably mutations capable of improving the stability of the soluble TCR of the present invention.

The present invention provides a nucleic acid molecule comprising an α chain or a β chain encoding the above-mentioned TCR or TCR.

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

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

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, including, for example, those with backbone modifications, the nucleic acid molecules refer to polymers of nucleotides, nucleotides of Such polymers 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 makes up a nucleic acid molecule.

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, sequences can be designed to contain terminal restriction site sequences for purposes of cloning into vectors.

In some cases, a nucleic acid molecule encoding a TCR can be obtained from a variety of sources, such as by a nucleic acid-encoding polymerase chain within or isolated from one or more given cells PCR amplification was obtained.

In one embodiment, the nucleotide sequence encoding the alpha chain and/or the nucleotide sequence encoding the beta chain is codon optimized. Generally, codon optimization involves balancing the percentage of selected codons with the published abundance of human transfer RNAs so that neither is overloaded or limited. 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 the protein expression and immunogenicity of the nucleic acid construct. Typically, for codon optimization, codons are chosen to select those codons that are in balance with the frequency of human usage. In general, the redundancy of amino acid codons is such that different codons encode one amino acid. In some embodiments, in selecting codons for substitution, it may be desirable that the resulting mutations be silent mutations such that the codon changes do not affect the amino acid sequence. In general, the last nucleotide of a codon can be left unchanged without affecting the amino acid sequence.

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

For example, one or more nucleic acids encoding one or both strands of the TCR described above are cloned into a suitable expression vector or vectors, which can be any suitable recombinant expression vector, and can be used for transformation or transfection infection with 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 transcriptional and translational initiation and termination codons) that are specific to the type of host into which the vector is to be introduced (e.g., bacterial, fungal, plant, or animal), as appropriate and considering that the vector is DNA-based Still based on RNA. 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, SV40 promoter, RSV promoter and promoters found in the long terminal repeats of murine stem cell virus, also contemplated Other promoters known to the skilled artisan.

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 present invention provides a host cell comprising such a nucleic acid. For recombinant production of a TCR, the nucleic acid encoding the TCR can 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 (eg, by using oligonucleotide probes capable of binding specifically to genes encoding the alpha and beta chains of the TCR). In some embodiments, a method of preparing a TCR is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding a TCR provided above under conditions suitable for expression of the TCR molecule, and optionally extracting a TCR from the host cell (or host cell culture medium) to recover TCR.

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

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

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 cells are primary cells obtained from a subject, preferably 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 cells can be, for example, a population of cells or genetically engineered cells expressing a TCR, typically eukaryotic cells, such as mammalian cells, and typically human cells. In some embodiments, the cells are derived from blood, bone marrow, lymphoid, or lymphoid organs and are cells of the immune system, such as cells of innate or adaptive immunity, such as myeloid or lymphoid cells (including lymphocytes, typically T cells and and/or NK cells). Other exemplary cells include stem cells, such as pluripotent stem cells and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells are typically primary cells, such as those isolated directly from the subject and/or isolated from the subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as the entire T cell population, CD+ cells, CD8+ cells, and subsets 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 their subtypes (eg stem cell memory T (T _SCM ), central memory T ( _TCM ), effector memory T ( _TEM ) or terminally differentiated effector memory T cells), tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, Cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, etc.

In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils and/or basophils.

The present invention provides a method for producing the above-mentioned engineered cells, which comprises introducing the above-mentioned nucleic acid molecules or the above-mentioned vectors into cells in vitro or ex vivo.

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

The present invention provides a pharmaceutical composition comprising the above-mentioned T cell receptor (TCR), the above-mentioned nucleic acid molecule, the above-mentioned vector or the above-mentioned engineered cell.

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

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

The pharmaceutical compositions can utilize timed-release, delayed-release, and sustained-release delivery systems such that delivery of the composition occurs prior to and sufficient time to induce sensitization at the site to be treated. Many types of release delivery systems are available and known. Such systems can be used to avoid repeated administration of the composition, thereby increasing convenience for both the subject and the physician.

The present invention provides 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 in the preparation and treatment of EBV-related Use in medicines for diseases.

In one embodiment, the disease associated with EBV is nasopharyngeal carcinoma, gastric cancer, 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, etc.

Example

The present invention generally and/or specifically describes the materials and test methods used in the test. In the following examples, unless otherwise specified, % represents wt%, that is, weight percentage. The reagents or instruments used without the manufacturer's indication are all conventional reagent products that can be obtained from the market.

Example 1: Cloning of EBV LMP2A antigen short peptide-specific T cells and acquisition of TCR gene

Peripheral blood lymphocytes from healthy volunteers with HLA-A1101 genotype were stimulated with the synthetic short peptide SSCSSPCLSK (SEQ ID NO: 15; Jiangsu GenScript Biotechnology Co., Ltd.). The SSCSS CPLSK peptide was renatured with biotin-labeled HLA-A1101 to prepare pHLA monomer. These monomers were combined with PE-labeled streptavidin (BD Company) to form a PE-labeled tetramer, the tetramer and anti-CD8-FITC double-positive cells were enriched, and the double-positive cells obtained were subjected to flow cytometry Single cells were obtained by sorting, and the single-step RT-PCR kit (QIAGEN, catalog number 210212) was used to amplify the TCR α chain and β chain of the single cells obtained by sorting, and the PCR products were sequenced. By comparing the sequencing results with the sequences in the public database of IMGT (International Immunogenetics Information System), the nucleotide sequences of the variable region sequence of the α chain and the variable region of the β chain of TCR and their CDR1, CDR2, and CDR3 sequences can be obtained. , wherein the nucleotide sequence (SEQ ID NO: 13) of the variable region of the alpha chain is:

ATGAAAAGAATCCTGGGAGCTCTGCTGGGCCTGCTCTCCGCCCAGGTGTGCTGTGTGCGGGGCATCCAGGTGGAACAGAGCCCTCCAGACCTGATTCTGCAGGAGGGCGCCAACAGCACCCTGAGATGCAACTTCAGCGACTCCGTGAACAACCTGCAATGGTTCCACCAGAACCCCTGGGGCCAGCTGATCAACCTGTTCTACATCCCTAGCGGAACCAAGCAGAATGGCCGCCTGTCTGCCACCACCGTGGCCACAGAGAGATACAGCCTGCTGTATATCAGCTCTAGCCAGCTGACAGATAGCGGCGTGTACTTCTGCGCCGTGGTCAACAACAATGACATGCGGTTTGGCGCTGGCACCAGACTGACAGTGAAGCCTAAC

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

MKRILGALLGLLSAQVCCVRGIQVEQSPPDLILQEGANSTLRCNFSDSVNNLQWFHQNPWGQLINLFYIPSGTKQNGRLSATTVATERYSLLYISSSQTTDSGVYFCAVVNNNDMRFGAGTRLTVKPN, where,

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 variable region of the translated beta chain (SEQ ID NO: 9) is:

MGCRLLCCAVLCLLGAVPIDTEVTQTPKHLVMGMTNKKSLKCEQHMGHRAMYWYKQKAKKPPELMFVYSYEKLSINESVPSRFSPECPNSSLLNLHLHALQPEDSALYLCASSPGRWYEQYFGPGTRLTVT, where,

Complementarity determining region 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: Construction of EBV LMP2A antigen short peptide-specific TCR lentiviral vector and lentiviral packaging

(1) Construction of TCR lentiviral vector (named pLKO-TCR048)

The EBV LMP2A TCR α and β chain variable region sequences were cloned into a pLKO-based expression plasmid (Addgene), and the α or β variable domains were cloned by a multi-fragment recombination cloning kit (Novizymes, Cat. No. C113). To a PLKO-based expression plasmid containing an α or β constant region, the ligated plasmid was transformed into competent E. coli strain Stbl3 cells (Shanghai Weidi Biotechnology Co., Ltd.) and inoculated in LB/ml containing 100 μg/ml ampicillin. on agar plates. After overnight incubation at 37°C, individual colonies were picked and grown overnight at 37°C with shaking in 10 ml of LB containing 100 μg/ml ampicillin. The cloned plasmid was purified and sequenced to yield pLKO-TCR048 using a mini-pump kit (TIANGEN Cat# DP118-02).

(2) Lentiviral packaging

reagent

Assay medium: 10% FBS (Lonsera, cat. no. S711-001), DMEM (cytiva, cat. no. SH30243.01)

Prepare 293T cells (the depository is the American Type Culture Collection or ATCC, and the depository number is CRL-1573) to culture in a 10cm dish, and start plasmid transfection when not exceeding 80% full, viral packaging plasmid and pLKO- The ratio of TCR048 plasmid was 1:1, totaling 10 μg. The above plasmids were mixed with PEI (polyethylenimine, polyethyleneimine) in serum-free DMEM medium, and then the mixed solution was added to 293T cells and cultured at 37°C. After 72h, the supernatant of the cells was concentrated with a 100kd ultrafiltration tube to collect the viral vector.

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

Use T2-A11 cells (T2 cells are deposited in ATCC, the deposit catalog number is CRL-1992, T2-A11 cells are constructed on the basis of T2 cells with reference to Cancer Biology & Therapy, 8:21, 2025-2032) to verify the transduction of the TCR of the present invention function of effector cells

ELISA protocol

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

(1) Reagents

Assay Media: 10% FBS (ThermoFisher, Cat. No. 10099-044), RPMI1640 (ThermoFisher, Cat. No. C11875500BT)

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

Human IL-2 uncoated ELISA kit (ThermoFisher, cat. no. 88-7316-88) contains all other required reagents (capture and detection antibodies, streptavidin-HRP and human IL- 2 Substrate solution for ELISA 96-well plate)

(2) Method

target cell preparation

The target cells used in this experiment were T2-A11 cells. The target cells were prepared in the experimental medium, the concentration of the target cells was adjusted to 3.2×10 ⁶ cells/ml, and 50 μl were taken from each well to obtain 1.6×10 ⁵ cells/well.

Effector cell preparation

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

Jurkat-CD8+ T cells (Jurkat cells are deposited in ATCC, the catalog number is TIB-152, Jurkat-CD8 cells are constructed on the basis of Jurkat cells with reference to Cancer Res 2006; 66(23): 11455-61) according to MOI (multiplicity of infection)=10 The lentivirus carrying the TCR gene of the present invention obtained in Example 2 was added, and the positive rate of transfection was identified by flow cytometry 72 hours later (the results are shown in Figure 1). The effector cell concentration after the expanded culture was adjusted to 3.2×10 ⁶ cells/ml, and 50 microliters were taken from each well to obtain 1.6×10 ⁵ cells/well.

Preparation of short peptide solutions

The corresponding short peptide (SSCSS CPLSK) was added to the experimental group of the corresponding target cells (T2-A11), so that the final concentration of the short peptide in the ELISA plate was 100 μg/ml, 10 μg/ml, 1 μg/ml, 0.1 μg/ml, 0.01 μg/ml, respectively. μg/ml, 0.001 μg/ml, 0.0001 μg/ml, 0.0001 μg/ml. Then, different gradients of short peptide concentrations of 50 μl/well were sequentially added to the 96-well plate from well A1 to well H1, and finally the plate was incubated overnight.

ELISA

Following the manufacturer's instructions, prepare the well plates as follows: Dilute the anti-human IFN-γ capture antibody 1:250 in 5 mL of sterile 1xPBS per plate, then aliquot 100 microliters of the diluted capture antibody into each well . Plates were blocked overnight by incubating at 4°C. After incubation, the plate was washed the next day to remove excess capture antibody. Add 200 microliters/well of 1x ELISA/ELISPORT dilution and incubate the plate for 1 hour at room temperature to block the plate. The 1x ELISA/ELISPORT dilution was then washed from the plate, removing any residual wash buffer by flicking and tapping the ELISA plate on the paper. Add 100μl of 1x ELISA/ELISPORT diluent to the wells of columns A/B except A1/A2, then add 200μl of the diluted standard to wells A1/A2, and aspirate 100μl from well A1/A2 and add it to B1/ In well B2, after mixing evenly, aspirate 100 μl into well C1/C2, and so on, dilute to well G1/G2, H1/H2 as blank control, add only 1x ELISA/ELISPORT dilution solution; The experimental groups were added to the ELISA plate:

⁵⁰ microliters of target cells (3.2 x ¹⁰⁶ cells/ml (resulting in a total of about 1.6 x 105 target cells/well).

⁵⁰ microliters of effector cells (1.6 x 105 control effector cells/well and TCR positive T cells/well).

All wells were prepared and added in duplicate.

Plates were then incubated at 4°C for 2 hours to block, after incubation, anti-human IFN-γ detection antibody was diluted 1:250 in 5 ml of 1x ELISA/ELISPORT dilution per plate, and then 100 μl of diluted detection antibody was aliquoted Each well was added and the plate was incubated for 1 hour at room temperature to block the plate. Then wash 3 times with wash buffer and tap on paper towel to remove residual wash buffer. Dilute the Streptavidin-HRP provided by the kit at a 1:100 dilution with 1x ELISA/ELISPORT diluent, add 100 μl/well to each well, incubate the plate for half an hour at room temperature, and then wash 6 times with washing buffer. Tap on the plate to remove excess wash buffer. After washing, 100 μl/well of 1xTMB solution provided by the kit was added for development. Cover the well plate with foil during development to protect from light and let stand for 15 minutes. Routinely check the spots of the developing plate during this period to determine the optimal time to terminate the reaction. Subsequently, 50 microliters of sulfuric acid solution was used to stop the development reaction, and the OD value was measured using a microplate reader (CTL; Cellular Technology Limited).

result

IL-2 release by TCR-transduced T cells of the invention in response to target cells loaded with P2 antigenic short peptides was examined by ELISA experiments (as described above). The expression level of IL-2 was plotted using Graphpad prism8, and the results are shown in Figure 2.

It can be seen from Fig. 2 that the T cells transduced with the TCR of the present invention have a good activation response to the target cells loaded with their specific short peptides.

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

Using T2-A11 cells to verify the function of effector cells transduced with the TCR of the present invention

ELISA protocol

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

reagent

Assay Medium: 10% FBS (ThermoFisher, Cat. No. 10099-044), RPMI1640 (ThermoFisher, Cat. No. C11875500BT)

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

Human IFNγ uncoated ELISA kit (ThermoFisher, cat. no. 88-7316-88) contains all other required reagents (capture and detection antibodies, streptavidin-HRP and human IFN-γ ELISA 96 well plate substrate solution)

method

target cell preparation

The target cells used in this experiment were T2-A11 cells. The target cells were prepared in the experimental medium, the concentration of the target cells was adjusted to 8.0×10 ⁵ cells/ml, and 50 μl was taken from each well to obtain 4.0×10 ⁴ cells/well.

Effector cell preparation

The effector cells (T cells) in this experiment were T cells transduced with the TCR of the present invention, and the T cells of the same volunteer who were not transfected with the TCR of the present invention were used as a control group.

The peripheral blood of volunteers was subjected to density gradient centrifugation to obtain peripheral blood mononuclear cells. The peripheral blood mononuclear cells were placed in each well of 24-well plate at 5.0×10 ⁵ /500 μl, and a total of 1E6 cells were collected. T cells were stimulated with CD28 magnetic beads and cultured in a 37°C 5% CO ₂ incubator. After 24 hours, the cells were observed to form clusters. After adding the lentivirus carrying the TCR gene of the present invention obtained in Example 2 at MOI (multiplicity of infection) = 3, the cells were transduced in 1640 cells containing 10% FBS containing 200IU/ml IL-2. After the medium was expanded until 3-4 days after transduction, the TCR transfection efficiency was identified by flow cytometry (the results are shown in Figure 3).

Preparation of short peptide solutions

The corresponding short peptide (SSCSS CPLSK) was added to the corresponding target cell (T2) experimental group, so that the final concentration of the short peptide in the ELISA plate was 1 μg/ml, 0.1 μg/ml, 0.01 μg/ml, 0.001 μg/ml, 0.0001 μg/ml, 0.00001 μg/ml, 0.000001 μg/ml, 0.0000001 μg/ml. Then, different gradients of short peptide concentrations of 50 μl/well were sequentially added to the 96-well plate from well A1 to well H1, and finally the plate was incubated overnight.

ELISA

Following the manufacturer's instructions, prepare the well plates as follows: Dilute the anti-human IFN-γ capture antibody 1:250 in 5 mL of sterile 1xPBS per plate, then aliquot 100 microliters of the diluted capture antibody into each well . Plates were blocked overnight by incubating at 4°C. After incubation, the plate was washed the next day to remove excess capture antibody. Add 200 microliters/well of 1x ELISA/ELISPORT dilution and incubate the plate for 1 hour at room temperature to block the plate. The 1x ELISA/ELISPORT dilution was then washed from the plate, removing any residual wash buffer by flicking and tapping the ELISA plate on the paper. Add 100μl of 1x ELISA/ELISPORT diluent to the wells of columns A/B except A1/A2, then add 200μl of the diluted standard to wells A1/A2, and aspirate 100μl from well A1/A2 and add it to B1/ In well B2, after mixing evenly, aspirate 100 μl into well C1/C2, and so on, dilute to well G1/G2, H1/H2 as blank control, add only 1x ELISA/ELISPORT dilution solution; The experimental groups were added to the ELISA plate:

⁵⁰ microliters of target cells 8.0 x 105 cells/ml (resulting in a total of about ⁴ x 104 target cells/well).

50 microliters of effector cells (2 x ¹⁰³ control effector cells/well and TCR positive T cells/well).

All wells were prepared and added in duplicate.

Plates were then incubated at 4°C for 2 hours to block, after incubation, anti-human IFN-γ detection antibody was diluted 1:250 in 5 ml of 1x ELISA/ELISPORT dilution per plate, and then 100 μl of diluted detection antibody was aliquoted Each well was added and the plate was incubated for 1 hour at room temperature to block the plate. Then wash 3 times with wash buffer and tap on paper towel to remove residual wash buffer. Dilute the Streptavidin-HRP provided by the kit at a 1:100 dilution with 1x ELISA/ELISPORT diluent, add 100 μl/well to each well, incubate the plate for half an hour at room temperature, and then wash 6 times with washing buffer. Tap on the plate to remove excess wash buffer. After washing, 100 μl/well of 1xTMB solution provided by the kit was added for development. Cover the well plate with foil during development to protect from light and let stand for 15 minutes. Routinely check the spots of the developing plate during this period to determine the optimal time to terminate the reaction. Then, 50 microliters of 2N sulfuric acid solution was used to stop the development reaction, and the OD value was measured using a microplate reader (CTL; Cellular Technology Limited).

result

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

It can be seen from Figure 4 that the T cells transduced with the TCR of the present invention have a good activation response to the target cells loaded with their specific short peptides.

To sum up, the TCR of the present invention can bind to the EBV antigen short peptide, and the T cells transduced with the TCR can be specifically activated and have a strong killing effect on target cells.

The above are only preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any person skilled in the art may use the technical content disclosed above to make changes or modifications to equivalent changes. Example. However, any simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solutions of the present invention still belong to the protection scope of the technical solutions of the present invention.

sequence listing

<110> Henan Cancer Hospital

Guangzhou Medical University

<120> T cell receptors that recognize EBV antigens and applications of the T cell receptors

<130> TPE01381

<160> 16

<170> PatentIn version 3.5

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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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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

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

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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

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

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

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Thr Val Thr

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

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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

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

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

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115 120 125

Met Thr Leu Arg Leu Trp Ser Ser

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

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

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

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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

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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

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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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Ser Ser Cys Ser Ser Cys Pro Leu Ser Lys

1 5 10

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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 (35)

1. A T Cell Receptor (TCR), wherein the TCR comprises an alpha chain comprising a variable region and a beta chain comprising a variable region, the variable region of the alpha chain comprising a complementarity determining region 1 (CDR 1) having an amino acid sequence DSVNN (SEQ ID NO: 1), a complementarity determining region 2 (CDR 2) having an amino acid sequence IPSGT (SEQ ID NO: 2) and a complementarity determining region 3 (CDR 3) having an amino acid sequence AVVNNNDMR (SEQ ID NO: 3); and

the variable region of the beta chain comprises complementarity determining region 1 (CDR 1) having the amino acid sequence MGHRA (SEQ ID NO: 4), complementarity determining region 2 (CDR 2) having the amino acid sequence YSYEKL (SEQ ID NO: 5), and complementarity determining region 3 (CDR 3) of ASSPGRWYEQY (SEQ ID NO: 6).

2. The T Cell Receptor (TCR) according to claim 1, wherein the TCR is capable of binding to the sscslpsk-HLAA 1101 complex.

3. The T Cell Receptor (TCR) according to any of claims 1-2, 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.

4. The T Cell Receptor (TCR) according to any of claims 1-2, wherein the amino acid sequence of the alpha chain variable region is as set forth in SEQ ID No. 9, and/or the amino acid sequence of the beta chain variable region is as set forth in SEQ ID No. 10.

5. The T Cell Receptor (TCR) according to any of claims 1-2, wherein the alpha chain further comprises an alpha constant region and/or the beta chain further comprises a beta constant region.

6. The T Cell Receptor (TCR) according to claim 5, wherein the constant region is a mouse constant region or a human constant region.

7. The T Cell Receptor (TCR) according to any of claims 1-2, wherein the TCR is isolated or purified or recombinant.

8. The T Cell Receptor (TCR) according to any one of claims 1-2, wherein the TCR is human.

9. The T Cell Receptor (TCR) according to any of claims 1-2, wherein the TCR is monoclonal.

10. The T Cell Receptor (TCR) according to any one of claims 1-2, wherein the TCR is single chain.

11. The T Cell Receptor (TCR) according to any of claims 1-2, wherein the TCR comprises two chains.

12. The T Cell Receptor (TCR) according to any one of claims 1-2, wherein the TCR is in cell-bound form or in soluble form.

13. The T Cell Receptor (TCR) according to any one of claims 1-2, wherein the TCR is in soluble form.

14. A nucleic acid molecule, wherein said nucleic acid molecule comprises a nucleic acid molecule encoding a TCR as claimed in any one of claims 1-13.

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

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

16. A vector, wherein the 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.

19. The vector of claim 16 or 17, wherein the vector is a retroviral vector.

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

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

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

23. The engineered cell of claim 21, wherein the engineered cell is a cell line.

24. The engineered cell of claim 21, wherein the engineered cell is a primary cell obtained from a subject.

25. The engineered cell of claim 24, wherein the subject is a mammalian subject.

26. The engineered cell of claim 24, wherein the subject is a human.

27. The engineered cell of any one of claims 21-26, wherein the engineered cell is a T cell.

28. The engineered cell of any one of claims 21-26, wherein the engineered cell is a peripheral blood isolated T cell.

29. The engineered cell of claim 27, wherein the T cell is CD8+ or CD4+.

30. A method of producing an engineered cell of any one of claims 21-29 comprising introducing the nucleic acid molecule of any one of claims 14-15 or the vector of any one of claims 16-20 into a cell in vitro or ex vivo.

31. The method of claim 30, wherein the vector is a viral vector and the introducing is by transduction.

32. 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 20 or an engineered cell according to any one of claims 21 to 29.

33. The pharmaceutical composition of claim 32, further comprising a pharmaceutically acceptable carrier or adjuvant.

34. 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 20, the engineered cell of any one of claims 21 to 29 or the pharmaceutical composition of any one of claims 31 to 32 in the manufacture of a medicament for the treatment of a disease associated with EBV.

35. The use of claim 34, 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.

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