CN109306005B - An Epstein-Barr virus-specific T cell antigen receptor and its application - Google Patents

Abstract

An Epstein-Barr virus-specific T cell antigen receptor and its application. The present invention provides a T cell antigen receptor (TCR) that specifically binds to human herpes virus (HHV-4), namely Epstein-Barr Virus (EBV) latency membrane protein LMP2 peptide (comprising the sequence TYGPVFMCL and/or TYGPVFMSL). ) and its application in the preparation of medicines for treating EBV-related diseases or in the preparation of products for killing tumor cells in mice and improving the survival rate of mice. The TCR of the present invention can be specifically activated by virus antigen peptide presenting cells, increase the release level of extracellular cytokines IFNγ and IL2 and the release amount of lactate dehydrogenase, and significantly kill target cells.

Description

An Epstein-Barr virus-specific T cell antigen receptor and its application

technical field

The present invention relates to the technical field of biomedicine, in particular to a high-affinity T cell antigen-specific receptor for EBV LMP2 epitope and a preparation method thereof, as well as its application in the preparation of a medicine for treating EBV-related diseases or in the preparation of the same. Application in products that kill tumor cells in mice and improve the survival rate of mice.

Background technique

T cell receptors (TCRs) are crucial to the cellular immune function of the immune system. TCRs are the only receptors for specific antigenic peptides presented on the major histocompatibility complex (MHC). Through the combination of antigen-specific TCR and pMHC complex, the direct physical contact between T cells and antigen-presenting cells is triggered, and the interaction leads to a series of subsequent cell signaling and other physiological responses, so that T cells with different antigen specificities can interact with their target cells. exert immune effect.

Epstein-Barr Virus (EBV), also known as human herpesvirus type 4, belongs to gamma herpesviruses and is one of the most common viruses that cause human diseases, mainly infecting B cells and nasopharyngeal epithelial cells. EBV infection is divided into proliferative infection and latent infection. The latently expressed proteins mainly include six nuclear proteins EBNA1, 2, 3A, 3B and 3C, EBNA-LP and three latent membrane proteins LMP1, LMP2A and LMP2B, of which LMP2B is a truncated isoform of LMP2A. According to the differences in the latent protein profiles expressed in different diseases, the latent types are divided into stages I, II, and III.

Patent CN108289950A discloses a T cell receptor-like antibody agent specific to EBV LMP2 presented by human HLA, wherein the antigenic peptide epitope recognized by the T cell receptor is CLGGLLTMV. Patent CN1526072A discloses a method for identifying the extracellular domain of EBV tumor-associated latent membrane protein and a method for selecting an antibody reagent reacting therewith, wherein the amino acid sequence of the extracellular domain of EBVLMP2 is any one of 4-9. and numerous T cell CTL epitopes of EBV disclosed in CN1269804A. However, none of the epitopes specifically recognized and bound by the TCR of the present invention are disclosed.

Therefore, the present invention provides a T cell antigen receptor, which can be specifically activated by virus antigen peptide presenting cells, increase the release level of extracellular cytokines IFNγ, IL2 and the release amount of lactate dehydrogenase, and significantly kill target cells.

SUMMARY OF THE INVENTION

The present invention provides a T cell antigen receptor (TCR), which specifically binds to EBV latency membrane protein LMP2, and the EBV latency membrane protein LMP2 comprises SEQ ID NO: 8 and/or SEQ ID NO: 20. amino acid sequence.

Preferably, the T cell antigen receptor specifically binds to the EBV latency membrane protein LMP2 peptide, and the EBV latency membrane protein LMP2 peptide comprises the amino acid sequence shown in SEQ ID NO: 1 and/or SEQ ID NO: 21.

The specific sequence of SEQ ID NO: 1 described in the present invention is: TYGPVFMCL.

The specific sequence of SEQ ID NO: 21 described in the present invention is: TYGPVFMSL.

In a specific embodiment of the present invention, the amino acid sequence of the EBV latency membrane protein LMP2 peptide is shown in SEQ ID NO: 8, or has at least 80% homology with the sequence shown in SEQ ID NO: 8 and has Sequence of antigenic activity.

MGSLEMVPMGAGPPSPGGDPDGYDGGNNSQYPSASGSSGNTPTPPNDEERESNEEPPPPYEDPYWGNGDRHSDYQPLGTQDQSLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEAGRGSMNPVCLPVIVAPYLFWLAAIAASCFTASVSTVVTATGLALSLLLLAAVASSYAAAQRKLLTPVTVLTAVVTFFAICLTWRIEDPPFNSLLFALLAAAGGLQGIYVLVMLVLLILAYRRRWRRLTVCGGIMFLACVLVLIVDAVLQLSPLLGAVTVVSMTLLLLAFVLWLSSPGGLGTLGAALLTLAAALALLASLILGTLNLTTMFLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASALIAGGSILQTNFKSLSSTEFIPNLFCMLLLIVAGILFILAILTEWGSGNRTYGPVFMCLGGLLTMVAGAVWLTVMSNTLLSAWILTAGFLIFLIGFALFGVIRCCRYCCYYCLTLESEERPPTPYRNTV*(SEQ ID NO：8)

Wherein, the underlined sequence in SEQ ID NO: 8 is the amino acid sequence of the EBV latency membrane protein LMP2 peptide recognized by the TCR of the present invention.

In another specific embodiment of the present invention, the amino acid sequence of the EBV latency membrane protein LMP2 peptide is shown in SEQ ID NO: 20, or has at least 80% homology with the sequence shown in SEQ ID NO: 20. Sequences with antigenic activity.

MGSLEMVPMGAGPPSPGGDPDGYDGGNNSQYPSASGSSGNTPTPPNDEERESNEEPPPPYEDPYWGNGDRHSDYQPLGTQDQSLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEAGRGSMNPVCLPVIVAPYLFWLAAIAASCFTASVSTVVTATGLALSLLLLAAVASSYAAAQRKLLTPVTVLTAVVTFFAICLTWRIEDPPFNSLLFALLAAAGGLQGIYVLVMLVLLILAYRRRWRRLTVCGGIMFLACVLVLIVDAVLQLSPLLGAVTVVSMTLLLLAFVLWLSSPGGLGTLGAALLTLAAALALLASLILGTLNLTTMFLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASALIAGGSILQTNFKSLSSTEFIPNLFCMLLLIVAGILFILAILTEWGSGNR TYGPVFMSL GGLLTMVAGAVWLTVMSNTLLSAWILTAGFLIFLIGFALFGVIRCCRYCCYYCLTLESEERPPTPYRNTV*(SEQ ID NO：20)

Wherein, the underlined sequence in SEQ ID NO: 20 is the amino acid sequence of the EBV latency membrane protein LMP2 peptide recognized by the TCR of the present invention.

The T cell antigen receptor of the present invention specifically binds to the LMP2 peptide from the latent membrane protein of EBV through the presentation of the major histocompatibility complex molecule (MHC).

The TCR of the present invention contains at least one α chain variable region and/or β chain variable region.

Preferably, the TCR is an αβ dimer.

The T cell antigen receptor of the present invention comprises α chain and β chain;

The amino acid sequence of the variable region of the α chain comprises the sequence shown in SEQ ID NO: 11, or a sequence having at least 80% homology with the sequence shown in SEQ ID NO: 11.

The amino acid sequence of the variable region of the beta chain comprises the sequence shown in SEQ ID NO: 12, or a sequence having at least 80% homology with the sequence shown in SEQ ID NO: 12.

The amino acid sequence of the α chain comprising the complementarity determining region CDR3α is the sequence shown in SEQ ID NO: 2, or has at least 80% homology with the sequence shown in SEQ ID NO: 2 and has a peptide epitope with the EBV latency membrane protein LMP2: Sequence of MHC complex binding function, or difference from SEQ ID NO: 2 including substitution, deletion and/or insertion of one or more amino acid residue sequences or at least one N-/C-terminal extension and having a Protein LMP2 peptide epitope: sequence of MHC complex binding function;

The amino acid sequence of the β chain comprising the complementarity determining region CDR3β is the sequence shown in SEQ ID NO: 3, or has at least 80% homology with the sequence shown in SEQ ID NO: 3 and has a peptide epitope with the EBV latency membrane protein LMP2: Sequence of MHC complex binding function, or difference from SEQ ID NO: 3 including substitution, deletion and/or insertion of one or more amino acid residue sequences or at least one N-/C-terminal extension and having a Protein LMP2 Peptide Epitopes: Sequences for MHC Complex Binding Function.

The specific sequence of SEQ ID NO: 2 described in the present invention is: AGERGSTLGRLY.

The specific sequence of SEQ ID NO: 3 described in the present invention is: ASSPLNRGQNEQF.

Preferably, the substitutions are conservative substitutions.

Preferably, the α chain comprises three complementarity determining regions, wherein the amino acid sequence of CDR1α is the sequence shown in SEQ ID NO: 4, or has at least 80% homology with the sequence shown in SEQ ID NO: 4 and has The sequence with the EBV latency membrane protein LMP2 peptide epitope: MHC complex binding function; the amino acid sequence of CDR2α is the sequence shown in SEQ ID NO: 5, or has at least 80% homology with the sequence shown in SEQ ID NO: 5 A sequence with a binding function to the EBV latent membrane protein LMP2 peptide epitope: MHC complex; the amino acid sequence of CDR3α is the sequence shown in SEQ ID NO: 2, or has at least 80% homology with the sequence shown in SEQ ID NO: 2 and has a sequence that binds to the EBV latency membrane protein LMP2 peptide epitope:MHC complex; or,

The β chain comprises three complementarity determining regions, wherein the amino acid sequence of CDR1β is the sequence shown in SEQ ID NO: 6, or has at least 80% homology with the sequence shown in SEQ ID NO: 6 and has an EBV latency membrane Protein LMP2 peptide epitope: the sequence of the binding function of MHC complex; the amino acid sequence of CDR2β is the sequence shown in SEQ ID NO: 7, or has at least 80% homology with the sequence shown in SEQ ID NO: 7 and has the same EBV latency membrane Protein LMP2 peptide epitope: the sequence of the binding function of MHC complex; the amino acid sequence of CDR3β is the sequence shown in SEQ ID NO: 3, or has at least 80% homology with the sequence shown in SEQ ID NO: 3 and has latency with EBV Membrane protein LMP2 peptide epitopes: sequences for MHC complex binding function.

Further preferably, the α chain comprises three complementarity determining regions, wherein:

CDR1α: TTLSN (SEQ ID NO: 4)

CDR2α: LVKSGEV (SEQ ID NO: 5)

CDR3α: AGERGSTLGRLY (SEQ ID NO: 2)

Further preferably, the β chain comprises three complementarity determining regions, wherein:

CDR1β: SEHNR (SEQ ID NO: 6)

CDR2β: FQNEAQ (SEQ ID NO: 7)

CDR3β: ASSPLNRGQNEQF (SEQ ID NO: 3)

In a specific embodiment of the present invention, the amino acid sequence of the variable region of the α chain is shown in SEQ ID NO: 11, or has at least 80% homology with the sequence shown in SEQ ID NO: 11 and has Peptide epitopes of the EBV latent membrane protein LMP2: sequences for the binding function of the MHC complex.

In a specific embodiment of the present invention, the amino acid sequence of the variable region of the beta chain is shown in SEQ ID NO: 12, or has at least 80% homology with the sequence shown in SEQ ID NO: 12 and has Peptide epitopes of the EBV latent membrane protein LMP2: sequences for the binding function of the MHC complex.

In the amino acid sequence of the T cell antigen receptor of the present invention, the β chain and the α chain are connected through a linker sequence. Preferably, the linker sequence is furin-SGSG-p2A sequence (hereinafter abbreviated as fp2A), and the amino acid sequence of fp2A is shown in SEQ ID NO: 13. The fp2A nucleotide sequence is shown in SEQ ID NO:18.

In a specific embodiment of the present invention, in the amino acid sequence of the T cell antigen receptor, the connecting sequence is promoter, β chain, fp2A, and α chain in order. Preferably, to facilitate transfection and expression detection, the IRES and RFP sequences are linked after the alpha chain.

In a specific embodiment of the present invention, the T cell antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 9, or at least 80% homology with the amino acid sequence shown in SEQ ID NO: 9 and Sequence with binding function to the EBV latency membrane protein LMP2 peptide epitope:MHC complex.

MGTSLLCWMALCLLGADHADTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSPLNRGQNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF RRKRSGSGATNFSLLKQAGDVEENPGP MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGERGSTLGRLYFGRGTQLTVWPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS*(SEQIDNO：9)

Wherein, in the amino acid sequence shown in SEQ ID NO: 9, the underlined sequence is the amino acid sequence of fp2A, the upstream amino acid sequence is the β chain, and the downstream amino acid sequence is the α chain.

In a specific embodiment of the present invention, it relates to a nucleotide sequence encoding the above-mentioned T cell antigen receptor, and the nucleotide sequence of the T cell antigen receptor is shown in SEQ ID NO: 10, or A sequence that is at least 80% homologous to the nucleotide sequence set forth in SEQ ID NO:10.

In a specific embodiment of the present invention, each MHC is coupled to a biotin molecule, and the biotin molecule is combined with a streptavidin molecule to form a tetramer, and the tetramer presents the The EBV latency membrane protein LMP2 antigen peptide specifically binds to the T cell antigen receptor of the present invention.

The second aspect of the present invention relates to a nucleotide sequence encoding the α chain of the above-mentioned T cell antigen receptor.

Preferably, the nucleotide sequence of the α chain comprises the sequence shown in SEQ ID NO: 14, or a sequence with at least 80% homology with the sequence shown in SEQ ID NO: 14.

The third aspect of the present invention relates to a nucleotide sequence encoding the β chain of the above-mentioned T cell antigen receptor.

Preferably, the nucleotide sequence of the beta chain comprises the sequence shown in SEQ ID NO: 15, or a sequence with at least 80% homology with the sequence shown in SEQ ID NO: 15.

The nucleotide sequences of the present invention may be codon-optimized. Preferably, the codon optimization includes changing a large number of rare codons used by viruses and the like into corresponding mammalian codons and/or removing mRNA unstable motifs and/or cryptic splice sites.

The fourth aspect of the present invention relates to a vector comprising the above-mentioned T cell antigen receptor nucleotide sequence, alpha chain nucleotide sequence or beta chain nucleotide sequence.

The fifth aspect of the present invention relates to a cell comprising the above-mentioned vector and/or the above-mentioned T cell antigen receptor nucleotide sequence, α-chain nucleotide sequence or β-chain nucleotide sequence.

Preferably, the nucleotide sequence is integrated into the chromosome of the cell.

Preferably, the cells are selected from hematopoietic stem cells or peripheral blood lymphocyte (PBL)-derived T cells.

The sixth aspect of the present invention relates to an isolated cell expressing the above-mentioned T cell antigen receptor.

The MHC molecules of the present invention may be class I MHC molecules. The complex can be expressed on the surface of almost all nucleated cells, or it can be immobilized by coating on beads or plates after purification by recombinant expression.

The HLA described in the present invention is the name of human MHC, including class I HLA antigens (A, B or C) or class II HLA antigens (DP, DQ or DR).

The TCR of the present invention may be HLA-A*01, A*02, A*03, A*11 or A*24 restricted. Preferably, the TCR is HLA-A*2402.

A seventh aspect of the present invention relates to a method for preparing a recombinant T cell, comprising the following steps:

1) Obtain a nucleotide sequence encoding the α chain and/or β chain of the T cell antigen receptor of the present invention from a positive T cell clone;

2) Isolation and culture of primary T cells;

3) delivering the nucleotide sequence of the α chain and/or β chain described in step 1) to the primary T cell described in step 2) to obtain the recombination comprising the T cell antigen receptor of the present invention T cells.

Preferably, the preparation method of cells expressing T cell antigen receptors includes the following steps:

1) separate the above-mentioned cells, and reverse transcription to obtain cDNA;

2) the nucleotide sequence of the α chain and/or the β chain described in the cloning step 1);

3) The nucleotide sequences of the α chain and the β chain described in step 2) are constructed into an expression plasmid through homologous recombination;

4) The expression plasmid of the α chain and/or the β chain described in step 3) is delivered to the cells to obtain the cells expressing the T cell antigen receptor.

The eighth aspect of the present invention relates to the above-mentioned T cell antigen receptor, the nucleotide sequence of the T cell antigen receptor, the nucleotide sequence of the α chain or the nucleotide sequence of the β chain, the above-mentioned vector or the above-mentioned Use of cells or isolated cells in the manufacture of a medicament for the treatment of EBV-related diseases.

Preferably, the EBV-related disease is selected from infectious mononucleosis, linked lymphoproliferative syndrome, viral hemophagocytic syndrome, oral hairy leukoplakia, chronic active EB virus infection, viral meninges inflammation, peripheral neuritis, viral pneumonia, viral myocarditis, nasopharyngeal carcinoma, Hodgkin lymphoma, Burkitt lymphoma, gastric cancer, hepatocellular carcinoma, lymphoepithelioid sarcoma, salivary gland tumor, breast cancer, thymoma, primary tumor Exudative lymphoma or B/T/NK cell lymphoma.

Preferably, the drug comprises the TCR or multivalent TCR complex of the present invention, the nucleotide sequence of the present invention, a vector containing the nucleotide sequence of the present invention, or the cell of the present invention and isolated cells, and a pharmaceutically acceptable carrier.

The ninth aspect of the present invention relates to the above-mentioned T cell antigen receptor, the nucleotide sequence of the T cell antigen receptor, the nucleotide sequence of the alpha chain or the nucleotide sequence of the beta chain, the above-mentioned vector or the above-mentioned Use of cells or isolated cells in the manufacture of products for the diagnosis or treatment of tumors or in the delivery of vaccines.

The tenth aspect of the present invention relates to a method for diagnosing or treating tumors, the method comprising administering to a patient an effective amount of the above-mentioned TCR or multivalent TCR complex or the above-mentioned cells.

The treatment or diagnosis of tumors according to the present invention includes localizing the TCRs of the present invention in the vicinity of tumors or metastatic tumors to increase the efficacy of toxins or immunostimulants.

The vaccine delivery of the present invention includes positioning the vaccine antigen near antigen-presenting cells to increase the efficacy of the antigen.

The multivalent complex of the present invention is to couple the TCR of the present invention to biotin or streptavidin.

The T cell antigen receptor of the present invention can be specifically activated by EBV LMP2 antigen peptide presenting cells, increase the release level of extracellular cytokines IFNγ, IL2 and the release amount of lactate dehydrogenase, and significantly kill target cells; The function test in the model mice shows that the T cell antigen receptor prepared by the present invention can obviously kill tumor cells and improve the survival rate of mice.

The "T cell receptor" of the present invention is a molecule capable of recognizing a peptide when presented by an MHC molecule, preferably a class I MHC molecule.

The "vector" in the present invention includes an expression vector, ie a construct capable of in vivo or in vitro/ex vivo expression; preferably, the vector is continuously expressed at a high level in cells in vivo.

The references herein to "and/or" include any number of combinations of the listed items in alternative.

In the present invention, "comprising" is an open-ended description, containing the specified elements or steps described, as well as other specified elements or steps that do not materially affect.

"Conservative substitutions" in the present invention include, but are not limited to, substitutions between alanine and serine, glycine, threonine, valine, proline or glutamic acid; and/or, aspartic acid Substitutions with glycine, asparagine, or glutamic acid; and/or, substitutions between serine and glycine, asparagine, or threonine; and/or, leucine with isoleucine or valine Substitutions between acids; and/or, substitutions between valine and leucine, isoleucine; and/or, substitutions between tyrosine and phenylalanine; and/or, lysine Substitution between acid and arginine. The above-mentioned substitutions do not substantially alter the activity of the amino acid sequences of the present invention.

"Prevention" as used in the present invention means avoiding, delaying, resisting or hindering the infection of the disease; preferably, preventing and/or reducing the possibility of EBV infection and/or reactivation.

"Treatment" as used herein means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily relate to Complete elimination of all disease-related signs, symptoms, disorders, or disorders.

The "homology" in the present invention means that in terms of using protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs, so that the used sequences are compared with those obtained in the prior art. , with (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31% , 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48 %, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% homology.

Description of drawings

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:

Figure 1: Schematic diagram of the connection of TCR β chain and α chain in pHAGE vector, wherein the sequence of connection is promoter, β chain, fp2A, α chain, IRES and RFP sequence;

Figure 2: The E9 fragment was obtained by overlapping PCR amplification;

Figure 3: Expression of EBV antigen-specific TCR-E9 in Jurkat clone 5 (Jurkat cell line with TCR α chain and β chain knockout) and its display outside the cell membrane;

Figure 4: Affinity detection of Jurkat clone 5 of EBV antigen-specific TCR-E9 prepared in Example 2 binding to EBV LMP2HLA-A*2402 tetramer probe;

Figure 5: Jurkat clone 5 of EBV antigen-specific TCR-E9 prepared in Example 2 can recognize EBVLMP2TYGPVFMCL and its variant TYGPVFMSL;

Figure 6: The detection result of the release level of TCR-T extracellular cytokine IL2, wherein, E9 represents the TCR-E9 prepared in Example 2, 1G4 represents the control TCR that can recognize the antigen EY-ESO-1, and Targets only represents the blank control of target cells;

Figure 7: Graph of the detection results of the release level of TCR-T extracellular cytokine IFNγ, wherein, E9 represents the TCR-E9 prepared in Example 2, 1G4 represents the control TCR that can recognize the antigen EY-ESO-1, and Targets only represents the blank control of target cells;

Figure 8: Lactate dehydrogenase assay for the killing of target cells by TCR-T cells, where E9 represents the TCR-E9 prepared in Example 2, 1G4 represents the control TCR that can recognize the antigen EY-ESO-1, and Targets only represents the blank control of target cells;

Figure 9: Detection of target cells dead and alive, wherein, E9 represents the TCR-E9 prepared in Example 2, 1G4 represents the control TCR that can recognize the antigen EY-ESO-1, and Targets only represents the blank control with only target cells;

Figure 10: Experimental scheme for animal model construction;

Figure 11: In vivo tumor growth of NSG mice in no T cell injection group (PBS), control TCR-T cell injection group (TCR-1G4), and EBV TCR-T injection group (TCR-E9);

Figure 12: The expansion of TCR-T cells in NSG mice in the control TCR-T cell injection group (TCR-1G4) and the EBV TCR-T injection group (TCR-E9);

Figure 13: The statistical results of the survival rate of NSG mice in the no T cell injection group (PBS), the control TCR-T cell injection group (TCR-1G4), and the EBV TCR-T injection group (TCR-E9).

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1 Construction of pHAGE-TCR-RFP vector

1. Obtain β and α gene fragments of TCR specific for EBV HLA-A*2402-TYGPVFMCL epitope

1) The tetramer of commercial HLA-A*2402TYGPVFMCL (SEQ ID NO: 1) was purchased from MBL Company, and the T cells with positive tetramer staining were subjected to flow single cell sorting according to the product instructions and peripheral blood staining. , reverse transcription to obtain cDNA ( IV Reverse Transcriptase, Invitrogen). According to the principle of rapid amplification of cDNA ends (RACE), the variable region (SEQ ID NO: 15) fragment of TCRβ gene was amplified by two rounds of PCR (KOD-Plus-Neo, TOYOBO).

The reverse transcription primer is: TCAGGCAGTATCTGGAGTCATTG (SEQ ID NO: 16)

The anchor primer is: AAGCAGTGGTATCAACGCAGAGTACATrGrG+G (SEQ ID NO: 17)

Where rG represents ribonucleotides, +G represents O2' and C4' dehydrated self-linked locked nucleotides

PCR amplification primers are:

Upstream primer 1: AAGCAGTGGTATCAACGCAGAGT (SEQ ID NO: 19)

Upstream primer 2: GTGGTATCAACGCAGAGTACATGG (SEQ ID NO: 22)

Downstream Primer 1: GCACCTCCTTCCCATTCACC (SEQ ID NO: 23)

Downstream Primer 2: GCTTCTGATGGCTCAAACACAG (SEQ ID NO: 24)

Specifically, according to the product specification of the PCR polymerase KOD-Plus-Neo, one round of PCR system is 50 μL, the annealing temperature is 60° C., and the reaction is carried out for 30 cycles. Take 1 μL of the product of the first round of PCR reaction as the template of the second round of PCR, the second round of PCR system is 50 μL, the annealing temperature is 60°C, and the reaction is carried out for 30 cycles. The second round of PCR products were then run on agarose gel, and the bands of the corresponding size were cut and recovered (Tiangengum recovery kit), and sent for sequencing, and the sequencing primer was the downstream primer 2.

The obtained TCRβ gene sequence is shown in SEQ ID NO:15.

2) As above, reverse transcription of HLA-A*2402-TYGPVFMCL tetramer positive T cells to obtain cDNA ( IV Reverse Transcriptase, Invitrogen). According to the product instructions, the TCRα gene (SEQ ID NO: 14) fragment was amplified by two rounds of PCR (KOD-Plus-Neo, TOYOBO),

The reverse transcription primer is: CGACCAGCTTGACATCACAG (SEQ ID NO: 25)

The anchor primer is: AAGCAGTGGTATCAACGCAGAGTACATrGrG+G (SEQ ID NO: 17)

Where rG represents ribonucleotides, +G represents O2' and C4' dehydrated self-linked locked nucleotides

PCR amplification primers are:

Upstream primer 1: AAGCAGTGGTATCAACGCAGAGT (SEQ ID NO: 19)

Upstream primer 2: GTGGTATCAACGCAGAGTACATGG (SEQ ID NO: 22)

Downstream primer 3: GTTGCTTCTTGAAGTCCATAGACCTC (SEQ ID NO: 29)

Downstream Primer 4: AATCGGTGAATAGGCAGACAGAC (SEQ ID NO: 30)

Specifically, according to the product specification of the PCR polymerase KOD-Plus-Neo, one round of PCR system is 50 μL, the annealing temperature is 60° C., and the reaction is carried out for 30 cycles. Take 1 μL of the product of the first round of PCR reaction as the template of the second round of PCR, the second round of PCR system is 50 μL, the annealing temperature is 60°C, and the reaction is carried out for 30 cycles. Then run the second round of PCR products on agarose gel, and cut the band of the corresponding size to recover the gel (Tiangen gum recovery kit), and send it for sequencing. The sequencing primer is the downstream primer 4.

The obtained TCRα gene sequence is shown in SEQ ID NO:14.

2. Construction of pHAGE-TCR-E9 vector

TCRβ, fp2A and TCRα were amplified by overlap-PCR with long primers (including fp2A sequence) (KOD-Plus-Neo, TOYOBO) to obtain a TCRβ-fp2A-TCRα fragment, named E9 (see Figure 1).

The primers for amplification are:

Upstream primer 5: atttcaggtgtcgtgaagcggccgcgccaccATGGGCACCAGCCTCCT (SEQ ID NO: 31) Downstream primer 5:

TCTCCAGCCTGCTTCAGCAGGCTGAAGTTAGTAGCTCCGCTTCCGCTccgtttccgccgGAAATCCTTTCTCTTGACCATG (SEQ ID NO: 26)

Upstream primer 6:

TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGCTACTCATCACATCAATGTTG (SEQ ID NO: 27)

Downstream primer 6: agggatcctctagactcgagctagcTCAGCTGGACCACAGCCGCA (SEQ ID NO: 28)

Specifically, primer 5 and primer 6 were used to amplify TCRβ and TCRα respectively, the PCR system was 50 μL, the annealing temperature was 60° C., and the reaction was performed for 30 cycles. The PCR product was run and recovered (Tiangengum recovery kit), 1 μL of the recovered product was taken as a template, and the upstream primer 5 and the downstream primer 6 were used for overlap PCR, the PCR system was 50 μL, the annealing temperature was 60 ° C, and the reaction was carried out for 30 cycles. . Run agarose gel to obtain a band of about 1800bp (as shown in Figure 2), and perform gel cutting and recovery.

The lentiviral vector pHAGE-IRES-RFP was double digested with NotI and NheI. The digestion system was 40 μL, containing 1.5 μL of NotI and NheI respectively, 2-3 μg of plasmid, digested at 37°C for 6 hours, and then added 1 μL of alkaline phosphatase (NEB) to the system for one hour to reduce plasmid self-ligation. The digested plasmid was recovered by gel running, and the concentration was measured by nanodrop, which was used as the backbone for constructing the plasmid.

According to the product instructions of Clone Express II One Step Cloning kit, the E9 was ligated with the linearized pHAGE-IRES-RFP vector after enzymatic digestion through the overlap, and the Stbl3 strain was transformed, and cultured on the LB plate containing ampicillin for 12-16 hours. The monoclonal clones were sequenced, and the sequencing primers were selected from the primers seq-pHAGE-F and seq-pHAGE-R on the pHAGE carrier, and the downstream primer 4.

Example 2 Membrane expression and affinity detection of EBV TCR-E9

1. Lentiviral Packaging

The aseptically extracted pHAGE-TCR-E9 plasmid and the packaging plasmids psPAX2 and pMD2.G were transfected into 293T cells with the transfection reagent PEI according to a certain ratio, and the cell culture supernatants for 48 hours and 72 hours were collected. After mixing with PEG8000 and standing at 4°C overnight, centrifugation at 3500 rpm for 30 min, discarding the supernatant, and resuspending with a small volume of medium to obtain the concentrated lentivirus.

2. Lentiviral Infection of Human T Cell Lines

Jurkat clone 5 cells in logarithmic growth phase were infected with lentivirus carrying TCR-E9. The concentrated lentivirus and the sensitizer Polybrene were added to the T cell culture medium and infected by centrifugation at 1500 rpm at 32°C for 2 hours. Three days after infection, fluorescent reporter genes can be observed and target protein expression can be detected.

3. TCR-E9 membrane condition and affinity detection

72 hours after infection, T cells were collected and stained with Brilliant Violet 421 ^™ anti-human TCRα/β (Biolegend) and EBV LMP2HLA-A*2402 tetramer-APC (Tetramer-APC), followed by flow cytometry. The experimental results are shown in Figures 3 and 4. Figure 3 shows that E9 can be stained by TCRα/β antibody, which indicates that the α chain and β chain of TCR-E9 can be correctly expressed and displayed outside the cell membrane. Figure 4 shows that TCRα/β antibody-positive cells can be stained by Tetramer-APC, indicating that the α chain and β chain of TCR-E9 prepared in Example 1 can correctly form pairings, and probe with EBVLMP2HLA-A*2402 tetramer Needles have a certain affinity.

Example 3 EBV TCR-E9 recognizes mutant epitopes

TCR-E9 can specifically recognize the EBV LMP2 epitope sequence TYGPVFMCL. According to literature reports, a variant of this epitope, TYGPVFMSL virus strain, is mainly distributed in the southeast coastal population of my country. Therefore, the tetramer of commercial HLA-A*2402TYGPVFMSL was purchased from MBL Company, and the recognition effect of TCR-E9 was detected. As shown in Figure 5, the results show that TCR-E9 can recognize both TYGPVFMCL and TYGPVFMSL.

Example 4 Construction of EBV TCR-E9T cells and in vitro functional detection

1. Isolation, Culture and Lentiviral Infection of Human Primary T Cells

In order to verify the recognition and killing function of the screened TCR-E9 on EBV LMP2 antigen, according to the product manual of EasySepHuman T cell isolation kit (stem cell technologies), T cells were obtained from human peripheral blood by negative selection, and then T cells were isolated from human peripheral blood. The cells were cultured in petri dishes coated with anti-CD3/CD28 antibodies. After 48 hours of activation, T cells were infected with TCR-E9-loaded virus particles using a lentiviral system. The infection method was centrifugation at 1500 rpm at 32°C for 2 hours. T cells stably expressing the TCR-E9 gene were constructed and cultured to a sufficient number in RPMI 1640 medium containing 20% serum and 100 U/mL IL-2, and the fluorescently labeled RFP-positive cells were separated by flow cytometry. selected.

2. Construction of Target Cells

Raji cells in logarithmic growth phase were infected with virus particles loaded with LMP2-RFP, HLA-A*2402-BSD and Luciferase-GFP, respectively, using a lentiviral system. Through drug screening and flow sorting, Raji cells stably expressing LMP2, HLA-A*2402 and Luciferas-GFP were obtained and named Raji_HLA matched.

3. In vitro functional validation of E9 in human primary T cells

Raji_HLA matched and LMP2-untransfected Raji cells (named Raji_Ctrl) were co-cultured with TCR-E9T cells 1G4T cells at a ratio of 1:3. Activation of TCR-E9 T cells and death of target cells. Judging from the release levels of extracellular cytokines IL2 (see Figure 6) and IFNγ (see Figure 7), after co-incubating TCR-E9T with target cells, compared with the control group 1G4T, it can significantly induce T cell activation. And the amount of lactate dehydrogenase in the cell supernatant and the effector-target ratio of flow analysis can reflect the death of target cells (see Figure 8, Figure 9). The experimental results prove that the TCR-E9T cells constructed in the examples of the present invention can be specifically activated by EBV LMP2 antigen peptide-presenting cells, and can significantly kill target cells.

Example 5 Animal model construction and in vivo function detection of EBV TCR-T

Raji_HLA matched tumor cell line was established in NSG mice as an animal model (see Figure 10) to further evaluate the function of EBV TCR-E9T cells to kill tumor cells in vivo. On day 0, 3e5 Raji_HLA matched cells were injected into the tail vein of each NSG mouse; after 3 days, the mice were divided into three groups, A: no T cell injection group (injected with equal volume of PBS); B: control TCR-T cell injection group (TCR-1G4T cells); C: EBV TCR-T injection group (TCR-E9T cells); mice in group B/C were injected with 5e6T cells via tail vein, and group A was injected with an equal volume of 200 μL PBS. Tumor cell growth, T cell proliferation in vivo, and mouse survival were monitored in different groups of mice over the next few weeks. As shown in Figure 11, Figure 12 and Figure 13, compared with the control group, the EBV-specific TCR-E9T cells constructed in the examples of the present invention can significantly kill tumor cells in mice and improve the survival rate of mice.

The preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the specific details of the above-mentioned embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.

In addition, it should be noted that the specific technical features described in the above-mentioned specific embodiments can be combined in any suitable manner unless they are inconsistent. In order to avoid unnecessary repetition, the present invention provides The combination method will not be specified otherwise.

sequence listing

<110> Tsinghua University, Huaxia Yingtai (Beijing) Biotechnology Co., Ltd., Shanghai Saiao Biotechnology Co., Ltd.

<120> An Epstein-Barr virus-specific T cell antigen receptor and its application

<130> 1

<160> 31

<170> PatentIn version 3.5

<210> 1

<211> 9

<212> PRT

<213> Epstein-Barr Virus latent membrane protein LMP2

<400> 1

Thr Tyr Gly Pro Val Phe Met Cys Leu

1 5

<210> 2

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 2

Ala Gly Glu Arg Gly Ser Thr Leu Gly Arg Leu Tyr

1 5 10

<210> 3

<211> 13

<212> PRT

<213> Artificial Sequence

<400> 3

Ala Ser Ser Pro Leu Asn Arg Gly Gln Asn Glu Gln Phe

1 5 10

<210> 4

<211> 5

<212> PRT

<213> Artificial Sequence

<400> 4

Thr Thr Leu Ser Asn

1 5

<210> 5

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 5

Leu Val Lys Ser Gly Glu Val

1 5

<210> 6

<211> 5

<212> PRT

<213> Artificial Sequence

<400> 6

Ser Glu His Asn Arg

1 5

<210> 7

<211> 6

<212> PRT

<213> Artificial Sequence

<400> 7

Phe Gln Asn Glu Ala Gln

1 5

<210> 8

<211> 497

<212> PRT

<213> Epstein-Barr Virus latent membrane protein LMP2

<400> 8

Met Gly Ser Leu Glu Met Val Pro Met Gly Ala Gly Pro Pro Ser Pro

1 5 10 15

Gly Gly Asp Pro Asp Gly Tyr Asp Gly Gly Asn Asn Ser Gln Tyr Pro

20 25 30

Ser Ala Ser Gly Ser Ser Gly Asn Thr Pro Thr Pro Pro Asn Asp Glu

35 40 45

Glu Arg Glu Ser Asn Glu Glu Pro Pro Pro Pro Tyr Glu Asp Pro Tyr

50 55 60

Trp Gly Asn Gly Asp Arg His Ser Asp Tyr Gln Pro Leu Gly Thr Gln

65 70 75 80

Asp Gln Ser Leu Tyr Leu Gly Leu Gln His Asp Gly Asn Asp Gly Leu

85 90 95

Pro Pro Pro Pro Tyr Ser Pro Arg Asp Asp Ser Ser Gln His Ile Tyr

100 105 110

Glu Glu Ala Gly Arg Gly Ser Met Asn Pro Val Cys Leu Pro Val Ile

115 120 125

Val Ala Pro Tyr Leu Phe Trp Leu Ala Ala Ile Ala Ala Ser Cys Phe

130 135 140

Thr Ala Ser Val Ser Thr Val Val Thr Ala Thr Gly Leu Ala Leu Ser

145 150 155 160

Leu Leu Leu Leu Ala Ala Val Ala Ser Ser Tyr Ala Ala Ala Gln Arg

165 170 175

Lys Leu Leu Thr Pro Val Thr Val Leu Thr Ala Val Val Thr Phe Phe

180 185 190

Ala Ile Cys Leu Thr Trp Arg Ile Glu Asp Pro Pro Phe Asn Ser Leu

195 200 205

Leu Phe Ala Leu Leu Ala Ala Ala Gly Gly Leu Gln Gly Ile Tyr Val

210 215 220

Leu Val Met Leu Val Leu Leu Ile Leu Ala Tyr Arg Arg Arg Trp Arg

225 230 235 240

Arg Leu Thr Val Cys Gly Gly Ile Met Phe Leu Ala Cys Val Leu Val

245 250 255

Leu Ile Val Asp Ala Val Leu Gln Leu Ser Pro Leu Leu Gly Ala Val

260 265 270

Thr Val Val Ser Met Thr Leu Leu Leu Leu Ala Phe Val Leu Trp Leu

275 280 285

Ser Ser Pro Gly Gly Leu Gly Thr Leu Gly Ala Ala Leu Leu Thr Leu

290 295 300

Ala Ala Ala Leu Ala Leu Leu Ala Ser Leu Ile Leu Gly Thr Leu Asn

305 310 315 320

Leu Thr Thr Met Phe Leu Leu Met Leu Leu Trp Thr Leu Val Val Leu

325 330 335

Leu Ile Cys Ser Ser Cys Ser Ser Cys Pro Leu Ser Lys Ile Leu Leu

340 345 350

Ala Arg Leu Phe Leu Tyr Ala Leu Ala Leu Leu Leu Leu Ala Ser Ala

355 360 365

Leu Ile Ala Gly Gly Ser Ile Leu Gln Thr Asn Phe Lys Ser Leu Ser

370 375 380

Ser Thr Glu Phe Ile Pro Asn Leu Phe Cys Met Leu Leu Leu Ile Val

385 390 395 400

Ala Gly Ile Leu Phe Ile Leu Ala Ile Leu Thr Glu Trp Gly Ser Gly

405 410 415

Asn Arg Thr Tyr Gly Pro Val Phe Met Cys Leu Gly Gly Leu Leu Thr

420 425 430

Met Val Ala Gly Ala Val Trp Leu Thr Val Met Ser Asn Thr Leu Leu

435 440 445

Ser Ala Trp Ile Leu Thr Ala Gly Phe Leu Ile Phe Leu Ile Gly Phe

450 455 460

Ala Leu Phe Gly Val Ile Arg Cys Cys Arg Tyr Cys Cys Tyr Tyr Cys

465 470 475 480

Leu Thr Leu Glu Ser Glu Glu Arg Pro Pro Thr Pro Tyr Arg Asn Thr

485 490 495

Val

<210> 9

<211> 609

<212> PRT

<213> Artificial Sequence

<400> 9

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr

20 25 30

Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His

35 40 45

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

50 55 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu

65 70 75 80

Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu

85 90 95

Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala

100 105 110

Ser Ser Pro Leu Asn Arg Gly Gln Asn Glu Gln Phe Phe Gly Pro Gly

115 120 125

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

130 135 140

Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys

145 150 155 160

Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu

165 170 175

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr

180 185 190

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

195 200 205

Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro

210 215 220

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

225 230 235 240

Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser

245 250 255

Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr

260 265 270

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

275 280 285

Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala

290 295 300

Met Val Lys Arg Lys Asp Phe Arg Arg Lys Arg Ser Gly Ser Gly Ala

305 310 315 320

Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro

325 330 335

Gly Pro Met Leu Leu Ile Thr Ser Met Leu Val Leu Trp Met Gln Leu

340 345 350

Ser Gln Val Asn Gly Gln Gln Val Met Gln Ile Pro Gln Tyr Gln His

355 360 365

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

370 375 380

Leu Ser Asn Ile Gln Trp Tyr Lys Gln Arg Pro Gly Gly His Pro Val

385 390 395 400

Phe Leu Ile Gln Leu Val Lys Ser Gly Glu Val Lys Lys Gln Lys Arg

405 410 415

Leu Thr Phe Gln Phe Gly Glu Ala Lys Lys Asn Ser Ser Leu His Ile

420 425 430

Thr Ala Thr Gln Thr Thr Asp Val Gly Thr Tyr Phe Cys Ala Gly Glu

435 440 445

Arg Gly Ser Thr Leu Gly Arg Leu Tyr Phe Gly Arg Gly Thr Gln Leu

450 455 460

Thr Val Trp Pro Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu

465 470 475 480

Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe

485 490 495

Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile

500 505 510

Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn

515 520 525

Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala

530 535 540

Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu

545 550 555 560

Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr

565 570 575

Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu

580 585 590

Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser

595 600 605

Ser

<210> 10

<211> 1830

<212> DNA/RNA

<213> Artificial Sequence

<400> 10

atgggcacca gcctcctatg ctggatggcc ctgtgtctcc tgggggcaga tcacgcagat 60

actggagtct cccaggaccc cagacacaag atcacaaaga ggggacagaa tgtaactttc 120

aggtgtgatc caatttctga acacaaccgc ctttattggt accgacagac cctggggcag 180

ggcccagagt ttctgactta cttccagaat gaagctcaac tagaaaaatc aaggctgctc 240

agtgatcggt tctctgcaga gaggcctaag ggatctttct ccaccttgga gatccagcgc 300

acagagcagg gggactcggc catgtatctc tgtgccagca gccctttaaa cagggggcag 360

aatgagcagt tcttcgggcc agggacacgg ctcaccgtgc tagaggacct gaaaaacgtg 420

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 480

gccacactgg tgtgcctggc cacaggcttc ttccccgacc acgtggagct gagctggtgg 540

gtgaatggga aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag 600

cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 660

tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 720

gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 780

ggtagagcag actgtggctt tacctcggtg tcctaccagc aaggggtcct gtctgccacc 840

atcctctatg agatcctgct agggaaggcc accctgtatg ctgtgctggt cagcgccctt 900

gtgttgatgg ccatggtcaa gagaaaggat ttccggcgga aacggagcgg aagcggagct 960

actaacttca gcctgctgaa gcaggctgga gacgtggagg agaaccctgg acctatgcta 1020

ctcatcacat caatgttggt cttatggatg caattgtcac aggtgaatgg acaacaggta 1080

atgcaaattc ctcagtacca gcatgtacaa gaaggagagg acttcaccac gtactgcaat 1140

tcctcaacta ctttaagcaa tatacagtgg tataagcaaa ggcctggtgg acatcccgtt 1200

ttttttgatac agttagtgaa gagtggagaa gtgaagaagc agaaaagact gacatttcag 1260

tttggagaag caaaaaagaa cagctccctg cacatcacag ccacccagac tacagatgta 1320

ggaacctact tctgtgcagg agagagaggc tcaaccctgg ggaggctata ctttggaaga 1380

ggaactcagt tgactgtctg gcctgatatc cagaaccctg accctgccgt gtaccagctg 1440

agagactcta aatccagtga caagtctgtc tgcctattca ccgattttga ttctcaaaca 1500

aatgtgtcac aaagtaagga ttctgatgtg tatatcacag acaaaactgt gctagacatg 1560

aggtctatgg acttcaagag caacagtgct gtggcctgga gcaacaaatc tgactttgca 1620

tgtgcaaacg ccttcaacaa cagcattatt ccagaagaca ccttcttccc cagcccagaa 1680

agttcctgtg atgtcaagct ggtcgagaaa agctttgaaa cagatacgaa cctaaacttt 1740

caaaacctgt cagtgattgg gttccgaatc ctcctcctga aagtggccgg gtttaatctg 1800

ctcatgacgc tgcggctgtg gtccagctga 1830

<210> 11

<211> 112

<212> PRT

<213> Artificial Sequence

<400> 11

Gly Gln Gln Val Met Gln Ile Pro Gln Tyr Gln His Val Gln Glu Gly

1 5 10 15

Glu Asp Phe Thr Thr Tyr Cys Asn Ser Ser Thr Thr Leu Ser Asn Ile

20 25 30

Gln Trp Tyr Lys Gln Arg Pro Gly Gly His Pro Val Phe Leu Ile Gln

35 40 45

Leu Val Lys Ser Gly Glu Val Lys Lys Gln Lys Arg Leu Thr Phe Gln

50 55 60

Phe Gly Glu Ala Lys Lys Asn Ser Ser Leu His Ile Thr Ala Thr Gln

65 70 75 80

Thr Thr Asp Val Gly Thr Tyr Phe Cys Ala Gly Glu Arg Gly Ser Thr

85 90 95

Leu Gly Arg Leu Tyr Phe Gly Arg Gly Thr Gln Leu Thr Val Trp Pro

100 105 110

<210> 12

<211> 115

<212> PRT

<213> Artificial Sequence

<400> 12

Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly

1 5 10 15

Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu

20 25 30

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

35 40 45

Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg

50 55 60

Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln

65 70 75 80

Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro

85 90 95

Leu Asn Arg Gly Gln Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu

100 105 110

Thr Val Leu

115

<210> 13

<211> 27

<212> PRT

<213> Artificial Sequence

<400> 13

Arg Arg Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys

1 5 10 15

Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro

20 25

<210> 14

<211> 816

<212> DNA/RNA

<213> Artificial Sequence

<400> 14

atgctactca tcacatcaat gttggtctta tggatgcaat tgtcacaggt gaatggacaa 60

caggtaatgc aaattcctca gtaccagcat gtacaagaag gagaggactt caccacgtac 120

tgcaattcct caactacttt aagcaatata cagtggtata agcaaaggcc tggtggacat 180

cccgtttttt tgatacagtt agtgaagagt ggagaagtga agaagcagaa aagactgaca 240

tttcagtttg gagaagcaaa aaagaacagc tccctgcaca tcacagccac ccagactaca 300

gatgtaggaa cctacttctg tgcaggagag agaggctcaa ccctggggag gctatacttt 360

ggaagaggaa ctcagttgac tgtctggcct gatatccaga accctgaccc tgccgtgtac 420

cagctgagag actctaaatc cagtgacaag tctgtctgcc tattcaccga ttttgattct 480

caaacaaatg tgtcacaaag taaggattct gatgtgtata tcacagacaa aactgtgcta 540

gacatgaggt ctatggactt caagagcaac agtgctgtgg cctggagcaa caaatctgac 600

tttgcatgtg caaacgcctt caacaacagc attattccag aagacacctt cttccccagc 660

ccagaaagtt cctgtgatgt caagctggtc gagaaaagct ttgaaacaga tacgaaccta 720

aactttcaaa acctgtcagt gattgggttc cgaatcctcc tcctgaaagt ggccgggttt 780

aatctgctca tgacgctgcg gctgtggtcc agctga 816

<210> 15

<211> 933

<212> DNA/RNA

<213> Artificial Sequence

<400> 15

atgggcacca gcctcctatg ctggatggcc ctgtgtctcc tgggggcaga tcacgcagat 60

actggagtct cccaggaccc cagacacaag atcacaaaga ggggacagaa tgtaactttc 120

aggtgtgatc caatttctga acacaaccgc ctttattggt accgacagac cctggggcag 180

ggcccagagt ttctgactta cttccagaat gaagctcaac tagaaaaatc aaggctgctc 240

agtgatcggt tctctgcaga gaggcctaag ggatctttct ccaccttgga gatccagcgc 300

acagagcagg gggactcggc catgtatctc tgtgccagca gccctttaaa cagggggcag 360

aatgagcagt tcttcgggcc agggacacgg ctcaccgtgc tagaggacct gaaaaacgtg 420

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 480

gccacactgg tgtgcctggc cacaggcttc ttccccgacc acgtggagct gagctggtgg 540

gtgaatggga aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag 600

cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 660

tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 720

gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 780

ggtagagcag actgtggctt tacctcggtg tcctaccagc aaggggtcct gtctgccacc 840

atcctctatg agatcctgct agggaaggcc accctgtatg ctgtgctggt cagcgccctt 900

gtgttgatgg ccatggtcaa gagaaaggat ttc 933

<210> 16

<211> 23

<212> DNA/RNA

<213> Artificial Sequence

<400> 16

tcaggcagta tctggagtca ttg 23

<210> 17

<211> 32

<212> DNA/RNA

<213> Artificial Sequence

<400> 17

aagcagtggt atcaacgcag agtacatrgr gg 32

<210> 18

<211> 81

<212> DNA/RNA

<213> Artificial Sequence

<400> 18

cggcggaaac ggagcggaag cggagctact aacttcagcc tgctgaagca ggctggagac 60

gtggaggaga accctggacc t 81

<210> 19

<211> 23

<212> DNA/RNA

<213> Artificial Sequence

<400> 19

aagcagtggt atcaacgcag agt 23

<210> 20

<211> 497

<212> PRT

<213> Epstein-Barr Virus latent membrane protein LMP2

<400> 20

Met Gly Ser Leu Glu Met Val Pro Met Gly Ala Gly Pro Pro Ser Pro

1 5 10 15

Gly Gly Asp Pro Asp Gly Tyr Asp Gly Gly Asn Asn Ser Gln Tyr Pro

20 25 30

Ser Ala Ser Gly Ser Ser Gly Asn Thr Pro Thr Pro Pro Asn Asp Glu

35 40 45

Glu Arg Glu Ser Asn Glu Glu Pro Pro Pro Pro Tyr Glu Asp Pro Tyr

50 55 60

Trp Gly Asn Gly Asp Arg His Ser Asp Tyr Gln Pro Leu Gly Thr Gln

65 70 75 80

Asp Gln Ser Leu Tyr Leu Gly Leu Gln His Asp Gly Asn Asp Gly Leu

85 90 95

Pro Pro Pro Pro Tyr Ser Pro Arg Asp Asp Ser Ser Gln His Ile Tyr

100 105 110

Glu Glu Ala Gly Arg Gly Ser Met Asn Pro Val Cys Leu Pro Val Ile

115 120 125

Val Ala Pro Tyr Leu Phe Trp Leu Ala Ala Ile Ala Ala Ser Cys Phe

130 135 140

Thr Ala Ser Val Ser Thr Val Val Thr Ala Thr Gly Leu Ala Leu Ser

145 150 155 160

Leu Leu Leu Leu Ala Ala Val Ala Ser Ser Tyr Ala Ala Ala Gln Arg

165 170 175

Lys Leu Leu Thr Pro Val Thr Val Leu Thr Ala Val Val Thr Phe Phe

180 185 190

Ala Ile Cys Leu Thr Trp Arg Ile Glu Asp Pro Pro Phe Asn Ser Leu

195 200 205

Leu Phe Ala Leu Leu Ala Ala Ala Gly Gly Leu Gln Gly Ile Tyr Val

210 215 220

Leu Val Met Leu Val Leu Leu Ile Leu Ala Tyr Arg Arg Arg Trp Arg

225 230 235 240

Arg Leu Thr Val Cys Gly Gly Ile Met Phe Leu Ala Cys Val Leu Val

245 250 255

Leu Ile Val Asp Ala Val Leu Gln Leu Ser Pro Leu Leu Gly Ala Val

260 265 270

Thr Val Val Ser Met Thr Leu Leu Leu Leu Ala Phe Val Leu Trp Leu

275 280 285

Ser Ser Pro Gly Gly Leu Gly Thr Leu Gly Ala Ala Leu Leu Thr Leu

290 295 300

Ala Ala Ala Leu Ala Leu Leu Ala Ser Leu Ile Leu Gly Thr Leu Asn

305 310 315 320

Leu Thr Thr Met Phe Leu Leu Met Leu Leu Trp Thr Leu Val Val Leu

325 330 335

Leu Ile Cys Ser Ser Cys Ser Ser Cys Pro Leu Ser Lys Ile Leu Leu

340 345 350

Ala Arg Leu Phe Leu Tyr Ala Leu Ala Leu Leu Leu Leu Ala Ser Ala

355 360 365

Leu Ile Ala Gly Gly Ser Ile Leu Gln Thr Asn Phe Lys Ser Leu Ser

370 375 380

Ser Thr Glu Phe Ile Pro Asn Leu Phe Cys Met Leu Leu Leu Ile Val

385 390 395 400

Ala Gly Ile Leu Phe Ile Leu Ala Ile Leu Thr Glu Trp Gly Ser Gly

405 410 415

Asn Arg Thr Tyr Gly Pro Val Phe Met Ser Leu Gly Gly Leu Leu Thr

420 425 430

Met Val Ala Gly Ala Val Trp Leu Thr Val Met Ser Asn Thr Leu Leu

435 440 445

Ser Ala Trp Ile Leu Thr Ala Gly Phe Leu Ile Phe Leu Ile Gly Phe

450 455 460

Ala Leu Phe Gly Val Ile Arg Cys Cys Arg Tyr Cys Cys Tyr Tyr Cys

465 470 475 480

Leu Thr Leu Glu Ser Glu Glu Arg Pro Pro Thr Pro Tyr Arg Asn Thr

485 490 495

Val

<210> 21

<211> 9

<212> PRT

<213> Epstein-Barr Virus latent membrane protein LMP2

<400> 21

Thr Tyr Gly Pro Val Phe Met Ser Leu

1 5

<210> 22

<211> 24

<212> DNA/RNA

<213> Artificial Sequence

<400> 22

gtggtatcaa cgcagagtac atgg 24

<210> 23

<211> 20

<212> DNA/RNA

<213> Artificial Sequence

<400> 23

gcacctcctt cccattcacc 20

<210> 24

<211> 22

<212> DNA/RNA

<213> Artificial Sequence

<400> 24

gcttctgatg gctcaaacac ag 22

<210> 25

<211> 20

<212> DNA/RNA

<213> Artificial Sequence

<400> 25

cgaccagctt gacatcacag 20

<210> 26

<211> 81

<212> DNA/RNA

<213> Artificial Sequence

<400> 26

tctccagcct gcttcagcag gctgaagtta gtagctccgc ttccgctccg tttccgccgg 60

aaatcctttc tcttgaccat g 81

<210> 27

<211> 71

<212> DNA/RNA

<213> Artificial Sequence

<400> 27

tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacctatg ctactcatca 60

catcaatgtt g 71

<210> 28

<211> 45

<212> DNA/RNA

<213> Artificial Sequence

<400> 28

agggatcctc tagactcgag ctagctcagc tggaccacag ccgca 45

<210> 29

<211> 25

<212> DNA/RNA

<213> Artificial Sequence

<400> 29

gttgctcttg aagtccatag acctc 25

<210> 30

<211> 23

<212> DNA/RNA

<213> Artificial Sequence

<400> 30

aatcggtgaa taggcagaca gac 23

<210> 31

<211> 48

<212> DNA/RNA

<213> Artificial Sequence

<400> 31

atttcaggtg tcgtgaagcg gccgcgccac catgggcacc agcctcct 48

Claims (8)

1. A T cell antigen receptor comprising an alpha chain and a beta chain, said alpha chain comprising three complementarity determining regions, wherein the amino acid sequence of CDR1 alpha is set forth in SEQ ID NO: 4; the amino acid sequence of CDR2 α is set forth in SEQ ID NO: 5, and the amino acid sequence of CDR3 alpha is shown in SEQ ID NO: 2; and, the beta chain comprises three complementarity determining regions, wherein the amino acid sequence of CDR1 beta is set forth in SEQ ID NO: 6, and the amino acid sequence of CDR2 beta is shown in SEQ ID NO: 7, and the amino acid sequence of CDR3 beta is shown in SEQ ID NO: 3, and (b) is shown in the specification.

2. A nucleic acid encoding the T cell antigen receptor of claim 1, an alpha chain or a beta chain of said T cell antigen receptor.

3. A vector comprising the nucleic acid of claim 2.

4. A cell expressing the T cell antigen receptor of claim 1, comprising the nucleic acid of claim 2 and/or comprising the vector of claim 3.

5. A method for producing a recombinant T cell, comprising the steps of:

1) obtaining nucleic acid encoding the alpha and/or beta chain of the T cell antigen receptor of claim 1 from a positive T cell clone;

2) separating and culturing primary T cells;

3) delivering the alpha and/or beta chain nucleic acid of step 1) to the primary T cell of step 2) to obtain a recombinant T cell comprising the T cell antigen receptor of claim 1.

6. Use of the T cell antigen receptor of claim 1, the nucleic acid of claim 2, the vector of claim 3, or the cell of claim 4 in the preparation of a medicament for treating an EBV-associated disease.

7. The use according to claim 6, wherein the EBV-associated disease is selected from the group consisting of infectious mononucleosis, linked lymphoproliferative syndrome, viral hemophilus syndrome, oral mucoleukoplakia, viral meningitis, peripheral neuritis, viral pneumonia myocarditis, nasopharyngeal carcinoma, Hodgkin's lymphoma, Burkitt's lymphoma, gastric carcinoma, hepatocellular carcinoma, lymphoepithelioid sarcoma, salivary gland tumor, breast cancer, thymoma, primary effusion lymphoma, and B/T/NK cell lymphoma.

8. Use of the T cell antigen receptor of claim 1, the nucleic acid of claim 2, the vector of claim 3 or the cell of claim 4 for the preparation of a product for the diagnosis or treatment of an EBV-associated disease.