T CELL ANTIGEN RECEPTOR, MULTIMERIC COMPLEX THEREOF, AND PREPARATION METHOD THEREFOR AND USE THEREOF

Abstract

Provided is an antibody or an antigen-binding fragment thereof, a T cell antigen receptor, an immune cell expressing the T cell antigen receptor (TCR), and a preparation method therefor and the use thereof. The TCR can specifically recognize corresponding pMHC complexes, activate TCR T cells, and produce high-level cytokines IFNγ, IL2, TNFα, significantly kill target cells and prolong the life of tumor-bearing mice.

Claims

1.-32. (canceled)

33. An antibody or an antigen-binding fragment thereof, wherein the antibody or an antigen-binding fragment thereof specifically binding to an EBV latent membrane protein LMP2, wherein the antibody or the antigen-binding fragment thereof comprises α-chain CDR1α-CDR3α and/or β-chain CDR1β-CDR3β, wherein the CDR1α has an amino acid sequence set forth in any one of SEQ ID NOs: 35-44 or having at least 80% homology to any one of SEQ ID NOs: 35-44, the CDR2α has an amino acid sequence set forth in any one of SEQ ID NOs: 45-54 or having at least 80% homology to any one of SEQ ID NOs: 45-54, the CDR3α has an amino acid sequence set forth in any one of SEQ ID NOs: 55-73 or having at least 80% homology to any one of SEQ ID NOs: 55-73, the CDR1β has an amino acid sequence set forth in any one of SEQ ID NOs: 74-84 or having at least 80% homology to any one of SEQ ID NOs: 74-84, the CDR2β has an amino acid sequence set forth in any one of SEQ ID NOs: 85-96 or having at least 80% homology to any one of SEQ ID NOs: 85-96, the CDR3β has an amino acid sequence set forth in any one of SEQ ID NOs: 97-117 or having at least 80% homology to any one of SEQ ID NOs: 97-117.

34. The antibody or the antigen-binding fragment thereof according to claim 33, wherein the CDR1α-CDR3α and the CDR1β-CDR3β are selected from any one of the following groups: TABLE-US-00011 Binding epitope CDR1α CDR2α CDR3α CDR1β CDR2β CDR3β E23 SEQ ID TSINN (SEQ IRSNERE ATEGDSGYST MNHEY SVGAGI ASSYQGGSSG NO: 29 ID NO: 35) (SEQ ID LT (SEQ ID (SEQ ID (SEQ ID YT (SEQ ID NO: 45) NO: 55) NO: 74) NO: 85) NO: 97) E240 SEQ ID TSINN (SEQ IRSNERE ATVGDSGYST MNHEY SVGAGI ASSGQGGGY NO: 29 ID NO: 35) (SEQ ID LT (SEQ ID (SEQ ID (SEQ ID GYT (SEQ ID NO: 45) NO: 56) NO: 74) NO: 85) NO: 98) E29 SEQ ID SSNFYA MTLNGDE ASTNSNSGYA DFQATT SNEGSKA SARDTSGVNF NO: 30 (SEQ ID (SEQ ID LN (SEQ ID (SEQ ID (SEQ ID YNEQF (SEQ NO: 36) NO: 46) NO: 57) NO: 75) NO: 86) ID NO: 99) E180-1 SEQ ID DSASNY IRSNVGE AARGGGYST LNHDA SQIVND ASAITGGTEA NO: 30 (SEQ ID (SEQ ID LT (SEQ ID (SEQ ID (SEQ ID F (SEQ ID NO: NO: 37) NO: 47) NO: 58) NO: 76) NO: 87) 100) E44 SEQ ID NSAFQY TYSSGN AMFRSTLGRL MNHEY SMNVEV ASTPLPTSSG NO: 31 or (SEQ ID (SEQ ID Y (SEQ ID NO: (SEQ ID (SEQ ID RLGEQY (SEQ 32 NO: 38) NO: 48) 59) NO: 74) NO: 88) ID NO: 101) E141 SEQ ID DSVNN IPSGT (SEQ AVLNNNDMR MGHRA YSYEKL ASSQGRWYE NO: 33 or (SEQ ID ID NO: 49) (SEQ ID NO: (SEQ ID (SEQ ID QY (SEQ ID 34 NO: 39) 60) NO: 77) NO: 89) NO: 102) E149 SEQ ID DSVNN IPSGT (SEQ AVVDNNDMR MGHRA YSYEKL ASSPGRWYE NO: 33 or (SEQ ID ID NO: 49) (SEQ ID NO: (SEQ ID (SEQ ID QF (SEQ ID 34 NO: 39) 61) NO: 77) NO: 89) NO: 103) E168 SEQ ID TTSDR LLSNGAV AVAMNRDDKII SGHKS YYEKEE ASSLDRDRND NO: 33 or (SEQ ID (SEQ ID (SEQ ID NO: (SEQ ID (SEQ ID YGYT (SEQ ID 34 NO: 40) NO: 50) 62) NO: 78) NO: 90) NO: 104) E170 SEQ ID DSASNY IRSNVGE AAREGFYQT KGHSH LQKENI ASSPAPRAGN NO: 33 or (SEQ ID (SEQ ID GANNLF (SEQ (SEQ ID (SEQ ID QPQH (SEQ ID 34 NO: 37) NO: 47) ID NO: 63) NO: 79) NO: 91) NO: 105) E244 SEQ ID DSASNY IRSNVGE AATAGGATN MNHEY SMNVEV ASSLYPPGHS NO: 33 or (SEQ ID (SEQ ID KLI (SEQ ID (SEQ ID (SEQ ID NQPQH (SEQ 34 NO: 37) NO: 47) NO: 64) NO: 74) NO: 88) ID NO: 106) E245 SEQ ID TTSDR LLSNGAV AVELTGNQF SGHKS YYEKEE ASSLEPGWG NO: 33 or (SEQ ID (SEQ ID Y (SEQ ID NO: (SEQ ID (SEQ ID DTQY (SEQ ID 34 NO: 40) NO: 50) 65) NO: 78) NO: 90) NO: 107) E254 SEQ ID DSVNN IPSGT (SEQ AVLNNNDMR SGDLS YYNGEE ASSVGPWYE NO: 33 or (SEQ ID ID NO: 49) (SEQ ID NO: (SEQ ID (SEQ ID QY (SEQ ID 34 NO: 39) 60) NO: 80) NO: 92) NO: 108) E301 SEQ ID DSVNN IPSGT (SEQ AVLNNNDMR MGHRA YSYEKL ASSPGRFYEQ NO: 33 or (SEQ ID ID NO: 49) (SEQ ID NO: (SEQ ID (SEQ ID Y (SEQ ID NO: 34 NO: 39) 60) NO: 77) NO: 89) 109) E304 SEQ ID DSVNN IPSGT (SEQ AVVDNNDMR MGHRA YSYEKL ASSPGRWYE NO: 33 or (SEQ ID ID NO: 49) (SEQ ID NO: (SEQ ID (SEQ ID QY (SEQ ID 34 NO: 39) 61) NO: 77) NO: 89) NO: 110) E305 SEQ ID TTSDR LLSNGAV AVNTGFQKL SNHLY FYNNEI ASSEGPTGTS NO: 33 or (SEQ ID (SEQ ID V (SEQ ID NO: (SEQ ID (SEQ ID YEQY (SEQ ID 34 NO: 40) NO: 50) 66) NO: 81) NO: 93) NO: 111) E307 SEQ ID TRDTTYY RNSFDEQN ALSEPPSGTY SGHVS FQNEAQ ASSQESGGTD NO: 33 or (SEQ ID (SEQ ID KYI (SEQ ID (SEQ ID (SEQ ID TQY(SEQ ID 34 NO: 41) NO: 51) NO: 67) NO: 82) NO: 94) NO: 112) E314 SEQ ID DSVNN IPSGT (SEQ AVLDNNDMR MGHRA YSYEKL ASSQGRWYE NO: 33 or (SEQ ID ID NO: 49) (SEQ ID NO: (SEQ ID (SEQ ID QY (SEQ ID 34 NO: 39) 68) NO: 77) NO: 89) NO: 102) E315 SEQ ID DSAIYN IQSSQRE AGKTSYDKVI SGHAT FQNNGV ASSVFPTSVE NO: 33 or (SEQ ID (SEQ ID (SEQ ID NO: (SEQ ID (SEQ ID QY (SEQ ID 34 NO: 42) NO: 52) 69) NO: 83) NO: 95) NO: 113) E316 SEQ ID TSDQSYG QGSYDEQ AMVSGAGGG LNHDA SQIVND ASSIGVGLSN NO: 33 or (SEQ ID N (SEQ ID ADGLT (SEQ (SEQ ID (SEQ ID TEAF (SEQ ID 34 NO: 43) NO: 53) ID NO: 70) NO: 76) NO: 87) NO: 114) E317 SEQ ID NSASDY IRSNMDK AETPGGYQK MNHEY SMNVEV ASSLWTSNSP NO: 33 or (SEQ ID (SEQ ID VT (SEQ ID (SEQ ID (SEQ ID LH SEQ ID 34 NO: 44) NO: 54) NO: 71) NO: 74) NO: 88) NO: (115) E318 SEQ ID DSASNY IRSNVGE AASNRDDKII SGHNS FNNNVP ASSLGAGHL NO: 33 or (SEQ ID (SEQ ID (SEQ ID NO: (SEQ ID (SEQ ID WGYT (SEQ 34 NO: 37) NO: 47) 72) NO: 84) NO: 96) ID NO: 116) E320 SEQ ID TTSDR LLSNGAV AVDIGTEYGN SGHVS FQNEAQ ASREGVGLYE NO: 33 or (SEQ ID (SEQ ID KLV (SEQ ID (SEQ ID (SEQ ID QY (SEQ ID 34 NO: 40) NO: 50) NO: 73) NO: 82) NO: 94) NO: 117)

35. The antibody or the antigen-binding fragment thereof according to claim 33, wherein the antibody or the antigen-binding fragment thereof is a single domain antibody or a single chain antibody scFv.

36. The antibody or the antigen-binding fragment thereof according to claim 35, wherein the antibody or the antigen-binding fragment thereof has an amino acid sequence selected from any one of SEQ ID NOs: 5-26 or having at least 80% homology to any one of SEQ ID NOs: 5-26.

37. A T cell antigen receptor, wherein the T cell antigen receptor specifically binding to an EBV latent membrane protein LMP2, wherein the T cell antigen receptor comprises α-chain CDR1α-CDR3α and/or β-chain CDR1β-CDR3β, wherein the CDR1α has an amino acid sequence set forth in any one of SEQ ID NOs: 35-44 or having at least 80% homology to any one of SEQ ID NOs: 35-44, the CDR2α has an amino acid sequence set forth in any one of SEQ ID NOs: 45-54 or having at least 80% homology to any one of SEQ ID NOs: 45-54, the CDR3α has an amino acid sequence set forth in any one of SEQ ID NOs: 55-73 or having at least 80% homology to any one of SEQ ID NOs: 55-73, the CDR1β has an amino acid sequence set forth in any one of SEQ ID NOs: 74-84 or having at least 80% homology to any one of SEQ ID NOs: 74-84, the CDR2β has an amino acid sequence set forth in any one of SEQ ID NOs: 85-96 or having at least 80% homology to any one of SEQ ID NOs: 85-96, the CDR3β has an amino acid sequence set forth in any one of SEQ ID NOs: 97-117 or having at least 80% homology to any one of SEQ ID NOs: 97-117.

38. The T cell antigen receptor according to claim 37, wherein the LMP2 has a binding epitope comprising one of or a combination of two or more of SEQ ID NOs: 29-34.

39. The T cell antigen receptor according to claim 37, wherein the CDR1α-CDR3α and the CDR1β-CDR3β are selected from any one of the following groups: TABLE-US-00012 Binding TCR epitope CDR1α CDR2α CDR3α CDR1β CDR2β CDR3β E23 SEQ ID TSINN (SEQ IRSNERE ATEGDSGYST MNHEY SVGAGI ASSYQGGSSG NO: 29 ID NO: 35) (SEQ ID LT (SEQ ID (SEQ ID (SEQ ID YT (SEQ ID NO: 45) NO: 55) NO: 74) NO: 85) NO: 97) E240 SEQ ID TSINN (SEQ IRSNERE ATVGDSGYST MNHEY SVGAGI ASSGQGGGY NO: 29 ID NO: 35) (SEQ ID LT (SEQ ID (SEQ ID (SEQ ID GYT (SEQ ID NO: 45) NO: 56) NO: 74) NO: 85) NO: 98) E29 SEQ ID SSNFYA MTLNGDE ASTNSNSGYA DFQATT SNEGSKA SARDTSGVNF NO: 30 (SEQ ID (SEQ ID LN (SEQ ID (SEQ ID (SEQ ID YNEQF (SEQ NO: 36) NO: 46) NO: 57) NO: 75) NO: 86) ID NO: 99) E180-1 SEQ ID DSASNY IRSNVGE AARGGGYST LNHDA SQIVND ASAITGGTEA NO: 30 (SEQ ID (SEQ ID LT (SEQ ID (SEQ ID (SEQ ID F (SEQ ID NO: NO: 37) NO: 47) NO: 58) NO: 76) NO: 87) 100) E44 SEQ ID NSAFQY TYSSGN AMFRSTLGRL MNHEY SMNVEV ASTPLPTSSG NO: 31 or (SEQ ID (SEQ ID Y (SEQ ID NO: (SEQ ID (SEQ ID RLGEQY (SEQ 32 NO: 38) NO: 48) 59) NO: 74) NO: 88) ID NO: 101) E141 SEQ ID DSVNN IPSGT (SEQ AVLNNNDMR MGHRA YSYEKL ASSQGRWYE NO: 33 or (SEQ ID ID NO: 49) (SEQ ID NO: (SEQ ID (SEQ ID QY (SEQ ID 34 NO: 39) 60) NO: 7) NO: 89) NO: 102) E149 SEQ ID DSVNN IPSGT (SEQ AVVDNNDMR MGHRA YSYEKL ASSPGRWYE NO: 33 or (SEQ ID ID NO: 49) (SEQ ID NO: (SEQ ID (SEQ ID QF (SEQ ID 34 NO: 39) 61) NO: 77) NO: 89) NO: 103) E168 SEQ ID TTSDR LLSNGAV AVAMNRDDKII SGHKS YYEKEE ASSLDRDRND NO: 33 or (SEQ ID (SEQ ID (SEQ ID NO: (SEQ ID (SEQ ID YGYT (SEQ ID 34 NO: 40) NO: 50) 62) NO: 78) NO: 90) NO: 104) E170 SEQ ID DSASNY IRSNVGE AAREGFYQT KGHSH LQKENI ASSPAPRAGN NO: 33 or (SEQ ID (SEQ ID GANNLF (SEQ (SEQ ID (SEQ ID QPQH (SEQ ID 34 NO: 37) NO: 47) ID NO: 63) NO: 79) NO: 91) NO: 105) E244 SEQ ID DSASNY IRSNVGE AATAGGATN MNHEY SMNVEV ASSLYPPGHS NO: 33 or (SEQ ID (SEQ ID KLI (SEQ ID (SEQ ID (SEQ ID NQPQH (SEQ 34 NO: 37) NO: 47) NO: 64) NO: 74) NO: 88) ID NO: 106) E245 SEQ ID TTSDR LLSNGAV AVELTGNQF SGHKS YYEKEE ASSLEPGWG NO: 33 or (SEQ ID (SEQ ID Y (SEQ ID NO: (SEQ ID (SEQ ID DTQY (SEQ ID 34 NO: 40) NO: 50) 65) NO: 78) NO: 90) NO: 107) E254 SEQ ID DSVNN IPSGT (SEQ AVLNNNDMR SGDLS YYNGEE ASSVGPWYE NO: 33 or (SEQ ID ID NO: 49) (SEQ ID NO: (SEQ ID (SEQ ID QY (SEQ ID 34 NO: 39) 60) NO: 80) NO: 92) NO: 108) E301 SEQ ID DSVNN IPSGT (SEQ AVLNNNDMR MGHRA YSYEKL ASSPGRFYEQ NO: 33 or (SEQ ID ID NO: 49) (SEQ ID NO: (SEQ ID (SEQ ID Y (SEQ ID NO: 34 NO: 39) 60) NO: 77) NO: 89) 109) E304 SEQ ID DSVNN IPSGT (SEQ AVVDNNDMR MGHRA YSYEKL ASSPGRWYE NO: 33 or (SEQ ID ID NO: 49) (SEQ ID NO: (SEQ ID (SEQ ID QY (SEQ ID 34 NO: 39) 61) NO: 77) NO: 89) NO: 110) E305 SEQ ID TTSDR(SEQ LLSNGAV) AVNTGFQKL SNHLY FYNNEI ASSEGPTGTS NO: 33 or ID NO: 40) SEQ ID NO: V (SEQ ID NO: (SEQ ID  (SEQ ID YEQY (SEQ ID 34 50) 66) NO: 81) NO: 93) NO: 111) E307 SEQ ID TRDTTYY RNSFDEQN ALSEPPSGTY SGHVS FQNEAQ ASSQESGGTD NO: 33 or (SEQ ID (SEQ ID KYI (SEQ ID (SEQ ID (SEQ ID TQY (SEQ ID 34 NO: 41) NO: 51) NO: 67) NO: 82) NO: 94) NO: 112) E314 SEQ ID DSVNN IPSGT (SEQ AVLDNNDMR MGHRA YSYEKL ASSQGRWYE NO: 33 or (SEQ ID ID NO: 49) (SEQ ID NO: (SEQ ID (SEQ ID QY (SEQ ID 34 NO: 39) 68) NO: 77) NO: 89) NO: 102) E315 SEQ ID DSAIYN IQSSQRE AGKTSYDKVI SGHAT FQNNGV ASSVFPTSVE NO: 33 or (SEQ ID (SEQ ID (SEQ ID NO: (SEQ ID (SEQ ID QY (SEQ ID 34 NO: 42) NO: 52) 69) NO: 83) NO: 95) NO: 113) E316 SEQ ID TSDQSYG QGSYDEQ AMVSGAGGG LNHDA SQIVND ASSIGVGLSN NO: 33 or (SEQ ID N (SEQ ID ADGLT (SEQ (SEQ ID (SEQ ID TEAF (SEQ ID 34 NO: 43) NO: 53) ID NO: 70) NO: 76) NO: 87) NO: 114) E317 SEQ ID NSASDY IRSNMDK AETPGGYQK MNHEY SMNVEV ASSLWTSNSP NO: 33 or (SEQ ID (SEQ ID VT (SEQ ID (SEQ ID (SEQ ID LH SEQ ID 34 NO: 44) NO: 54) NO: 71) NO: 74) NO: 88) NO: (115) E318 SEQ ID DSASNY IRSNVGE AASNRDDKII SGHNS FNNNVP ASSLGAGHL NO: 33 or (SEQ ID (SEQ ID (SEQ ID NO: (SEQ ID (SEQ ID WGYT (SEQ 34 NO: 37) NO: 47) 72) NO: 84) NO: 96) ID NO: 116) E320 SEQ ID TTSDR LLSNGAV AVDIGTEYGN SGHVS FQNEAQ ASREGVGLYE NO: 33 or (SEQ ID (SEQ ID KLV (SEQ ID (SEQ ID (SEQ ID QY (SEQ ID 34 NO: 40) NO: 50) NO: 73) NO: 82) NO: 94) NO: 117)

40. The T cell antigen receptor according to claim 39, wherein the T cell antigen receptor has an amino acid sequence selected from any one of SEQ ID NOs: 5-26 or having at least 80% homology to any one of SEQ ID NOs: 5-26.

41. A nucleic acid, wherein the nucleic acid encodes the antibody or the antigen-binding fragment thereof according to claim 33.

42. An immune cell, wherein the immune cell expresses the antibody or the antigen-binding fragment thereof according to claim 33.

43. A method for preparing a recombinant T cell, comprising the following steps: 1) obtaining the nucleic acid sequence according to claim 41 from a positive T cell clone; 2) separating and culturing a primary T cell; 3) delivering the nucleic acid sequence obtained in the step 1) to the primary T cell in the step 2) to obtain a recombinant T cell expressing the T cell antigen receptor.

44. A method for preparing an antibody or an antigen-binding fragment thereof or a T cell antigen receptor, comprising the following steps: (1) obtaining the nucleic acid sequence according to claim 41 from a positive T cell clone; (2) connecting the nucleic acid sequence obtained in the step (1) to a vector backbone to obtain an expression vector; (3) transforming the expression vector obtained in the step (2) into a host cell, and then inducing the expression of the host cell; (4) obtaining the antibody or the antigen-binding fragment thereof or the T cell antigen receptor.

45. A multimeric complex, wherein the multimeric complex comprises the T cell antigen receptor according to claim 37.

46. The multimeric complex according to claim 45, further comprising a monomer, a biotin molecule, and a streptavidin or avidin molecule, wherein the monomer comprises an α-chain extracellular domain of an MHC molecule, a β2m chain and an antigen peptide, the monomer is conjugated to the biotin molecule binding to the streptavidin or avidin molecule.

47. The multimeric complex according to claim 46, wherein the antigen peptide comprises any one of or a combination of two or more of SEQ ID NOs: 29-34.

48. The multimeric complex according to claim 46, wherein the MHC molecule is selected from HLA-A*0201, HLA-A*2402 and HLA-A*1101.

49. A method for preparing the multimeric complex according to claim 45, comprising the following steps: I) expressing and purifying an α-chain extracellular domain of an MHC molecule connected with an avi-tag sequence at the C terminus and a (32m chain; II) refolding an antigen peptide, the α-chain extracellular domain of the MHC molecule connected with the avi-tag sequence at the C terminus and the (32m chain obtained in the step I) to prepare a monomer; III) biotinylating the monomer prepared in the step II) to obtain a biotinylated monomer; IV) subjecting the biotinylated monomer obtained in the step III) to a reaction with fluorescently labeled streptavidin or avidin to prepare an antigen peptide-MHC molecule tetramer; V) co-incubating the antigen peptide-MHC molecule tetramer obtained in the step IV) with T cells to form a complex of a T cell antigen receptor and the antigen peptide-MHC molecule tetramer to fish for a specific T cell antigen receptor.

50. A method for treating an EBV-related disease, comprising administering the antibody or the antigen-binding fragment thereof according to claim 33 or the T cell antigen receptor, the nucleic acid, the immune cell or the multimeric complex.

51. The method according to claim 50, wherein the EBV-related disease is selected from infectious mononucleosis, linked lymphoproliferative syndrome, viral hemophagocytic syndrome, oral hairy leukoplakia, viral meningitis, peripheral neuritis, viral pneumonia, viral myocarditis, nasopharyngeal carcinoma, Hodgkin's lymphoma, Burkitt's lymphoma, gastric carcinoma, hepatocellular carcinoma, lymphoepithelioid sarcoma, salivary gland tumor, breast cancer, thymoma, primary effusion lymphoma, or B/T/NK cell lymphoma.

52. A pharmaceutical composition or kit, wherein the pharmaceutical composition or kit comprises any one of the following groups: i) the antibody or the antigen-binding fragment thereof according to claim 33; ii) the T cell antigen receptor; iii) the nucleic acid; iv) the immune cell; or v) the multimeric complex.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0149] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0150] FIG. 1: SDS PAGE detection results of monomers, wherein M is protein Marker, and bands 1 and 2 are monomers;

[0151] FIG. 2: staining results of tetramers, wherein FIG. 2A shows staining results of A0201-FLYALALLL-tetramer and A1101-SSCSSCPLSK-tetramer, and FIG. 2B shows the comparison of A2402-TYGPVFMSL-tetramer with commercial tetramer in volume gradient assays;

[0152] FIG. 3: staining results of tetramers, wherein FIG. 3A shows staining results of A2402-TYGPVFMCL-tetramer and A2402-TYGPVFMSL-tetramer; FIG. 3B shows staining results of A1101-SSCSSCPLSK-tetramer, and staining results of A1101-SSCSSCPLTK-tetramer; and FIG. 3C shows staining results of A2402-PYLFWLAAI-tetramer.

[0153] FIG. 4: a schematic diagram of the linking of TCR β and α chains in a pHAGE vector, wherein the linking is performed by the following order: a promoter, the β chain, furin-p2A, the α chain, IRES and RFP sequences;

[0154] FIG. 5: the expression of HLA-A*A0201 FLYALALLL-specific TCRs (E23 and E240), HLA-A*A2402 TYGPVFMSL/TYGPVFMCL-specific TCR (E44) and PYLFWLAAI-specific TCRs (E29 and E180-1) on a membrane surface as assayed by flow cytometry, wherein BV421 is one of fluoresceins, and BV stands for Brilliant Violet;

[0155] FIG. 6: the affinity of HLA-A*A0201 FLYALALLL-specific TCRs (E23 and E240), HLA-A*A2402 TYGPVFMSL/TYGPVFMCL-specific TCR (E44) and PYLFWLAAI-specific TCRs (E29 and E180-1) for binding to the EBV LMP2 tetramer probe as assayed by flow cytometry;

[0156] FIG. 7: the affinity of HLA-A*1101 epitope SSCSSCPLSK (LSK)- and SSCSSCPLTK (LTK)-specific TCRs for binding to tetramer probes as assayed by flow cytometry;

[0157] FIG. 8: IL-2 production by JC5-TCR cells stimulated by LSK peptide fragments at different concentrations;

[0158] FIG. 9: EC.sub.50 statistics for LSK epitope-specific TCRs, wherein public TCR represents the TCR conserved in the CDR3 motif and private TCR represents the TCR without conserved sequences in the CDR3 region;

[0159] FIG. 10: IL-2 production by JC5-TCR cells stimulated by LTK peptide fragments at different concentrations;

[0160] FIG. 11: EC.sub.50 statistics for LTK epitope-specific TCRs, wherein public TCR represents the TCR conserved in the CDR3 motif and private TCR represents the TCR without conserved sequences in the CDR3 region;

[0161] FIG. 12: IL2 release level after incubation of LSK-specific TCR T cells with EBV-LCL cells;

[0162] FIG. 13: IFNγ release level after incubation of LSK-specific TCR T cells and EBV-LCL cells;

[0163] FIG. 14: proliferation results of TCR T cells under long-term stimulation by excess target cells in vitro;

[0164] FIG. 15: assay results of killing ability of TCR T cells under long-term stimulation by excess target cells in vitro;

[0165] FIG. 16: assay results of the release levels of cytokines IL2, TNFα and IFNγ of E23-TCRT and E240-TCRT and the luciferase level of target cells, wherein E23 and E240 represent the TCRs prepared in Example 2, NE represents the blank control group, NT represents the T cell-only group, Ctrl is Raji cells untransfected with LMP2, and their effector-to-target ratios are 0.5:1, 1:1 and 2:1, respectively;

[0166] FIG. 17: assay results of the release levels of cytokines IL2, TNFα and IFNγ of E29-TCRT and E180-1-TCRT, wherein E29 and E180-1 represent the TCRs prepared in Example 2, 1G4 represents the control TCR capable of recognizing the antigen EY-ESO-1, NE represents the blank control group, and NT represents the T cell-only group;

[0167] FIG. 18: assay results of the release levels of cytokines IL2, TNFα and IFNγ of E44-TCRT, wherein E44 represents the TCR prepared in Example 2, E9 represents the positive control TCR capable of recognizing the antigen LMP2, RFP represents the negative control group, NE represents the blank control group, and NT represents the T cell-only group;

[0168] FIG. 19: structural affinity results of public TCRs as assayed by a BFP method;

[0169] FIG. 20: contribution of each amino acid in the E141-TCR CDR3 region to antigen recognition and target cell killing ability as analyzed by an Alanine Scanning method;

[0170] FIG. 21: contribution of each amino acid in the E141-TCR CDR3 region to antigen recognition and T cell activation ability as analyzed by an Alanine Scanning method;

[0171] FIG. 22: evaluation of the inhibition of E23-TCR and E240-TCR on tumor growth in mice in a lymphoma animal model;

[0172] FIG. 23: evaluation of the inhibition of E29-TCR and E44-TCR on tumor growth in mice in a lymphoma animal model;

[0173] FIG. 24: evaluation of the inhibition of E141-TCR on tumor growth in mice in a lymphoma animal model;

[0174] FIG. 25: evaluation of the inhibition of E141-TCR on tumor growth in mice in a solid tumor animal model;

[0175] FIG. 26: statistics for tumor growth in mice in the solid tumor model, the T cell-free injection group (PBS), the control TCR-T cell injection group (TCR-1G4), and the EBV TCR-T injection group (E141-TCR);

[0176] FIG. 27: statistics for TCR T cell-specific proliferation in mice in the solid tumor model, the T cell-free injection group (PBS), the control TCR-T cell injection group (TCR-1G4), and the EBV TCR-T injection group (E141-TCR).

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0177] Technical solutions in the embodiments of the present invention will be described clearly and completely below with reference to the drawings. It is apparent that the described embodiments are only a 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 making creative work shall fall within the protection scope of the present invention.

Example 1. Construction and Effect Assay on EBV Antigenic Epitope Tetramers

[0178] I. Construction of EBV Antigenic Epitope Tetramers

[0179] 1) An α chain and a β2m chain (with the amino acid sequence set forth in SEQ ID NO: 4, and the nucleotide sequence set forth in SEQ ID NO: 121) of HLA-A*0201 (with the amino acid sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 118), HLA-A*2402 (with the amino acid sequence set forth in SEQ ID NO: 2, and the nucleotide sequence set forth in SEQ ID NO: 119) and HLA-A*1101 (with the amino acid sequence set forth in SEQ ID NO: 3, and the nucleotide sequence set forth in SEQ ID NO: 120) with optimized expression sequences were provided. The structure of the α chain was as follows: the extracellular domain sequence of the α chain of the corresponding HLA type was connected with an Avi-tag sequence, with a BamHI enzyme cutting site as a spacer to provide a biotinylation site. The β2m chain was depleted of the signal peptide sequence, with two amino acids (M and A) added before a mature peptide sequence. The expression vector was PET28a+, and the expression strain was transetta or BL21. IPTG was at a concentration of 0.5 mM, and the expression was induced for 4 h. The protein inclusion bodies of the α chain and β2m chain were extracted.

[0180] 2) Selection of EBV epitopes (antigen peptides): HLA-A*0201 type corresponds to an antigenic epitope FLYALALLL (SEQ ID NO: 29); HLA-A*2402 type corresponds to antigenic epitopes PYLFWLAAI (SEQ ID NO: 30), TYGPVFMSL (SEQ ID NO: 31) and TYGPVFMCL (SEQ ID NO: 32); and HLA-A*1101 type corresponds to antigenic epitopes SSCSSCPLSK (SEQ ID NO: 33) and SSCSSCPLTK (SEQ ID NO: 34).

[0181] 3) Folding and purification of pMHC I monomer: the antigen peptides in the step 2), and the corresponding β2m chain renaturation proteins and the α chain proteins in the step 1) were added into a reduction system in order according to the molar ratio of 40:2:1, and the folding reaction was performed for 72 h. The resulting products were purified on a Superdex75 10/300GL column. The purified products were collected, biotinylated using an avidity kit, and purified again to obtain biotinylated monomers, which were determined for the purity by gel electrophoresis.

[0182] 4) The biotinylated monomers in the step 3) were subjected to a binding reaction with APC-labeled streptavidin to obtain corresponding tetramers, which were named as A0201-FLYALALLL-tetramer, A2402-PYLFWLAAI-tetramer, A2402-TYGPVFMSL-tetramer, A2402-TYGPVFMCL-tetramer, A1101-SSCSSCPLSK-tetramer and A1101-SSCSSCPLTK-tetramer, respectively.

[0183] II. Assay on the Effect of EBV Antigenic Epitope Tetramers

[0184] 1. Human peripheral blood mononuclear cells (PBMCs) were isolated, and a cell suspension was prepared at a cell density of 1×10.sup.6 cells/mL.

[0185] 2. The cells were centrifuged at 3000 rpm for 5 min. The supernatant was removed and resuspended in 50 μL of PBS containing 1% serum.

[0186] 3. 2 μL of tetramer was added, and the mixture was incubated at room temperature for 30 min.

[0187] 4. 2 μL of CD8 antibody was added, and the mixture was incubated on ice for 20 min.

[0188] 5. 1 mL of PBS was added, and the mixture was centrifuged at 3000 rpm for 5 min.

[0189] 6. The supernatant was removed, 1 mL of PBS was added, and the mixture was centrifuged at 3000 rpm for 5 min.

[0190] 7. The supernatant was removed, the cells were resuspended in 500 μL of 4% paraformaldehyde, and the cell suspension was filtered through a filter membrane.

[0191] 8. Positive cells were detected by a flow cytometer.

[0192] III. Experimental Results

[0193] The SDS PAGE detection results of the monomers are shown in FIG. 1. As shown in the figure, after refolding and HPLC purification, for the resulting monomers, the proteins with sizes corresponding to those of the heavy chain (the α-chain extracellular domain connected with the Avi-tag sequence at the C-terminus) and the light chain (the β2m chain depleted of the signal peptide region), respectively, and having higher purity are clearly showed.

[0194] The constructed tetramers were separately co-incubated with cells infected with TCRs of corresponding HLA types. Illustratively, the constructed A0201-FLYALALLL-tetramer, A2402-TYGPVFMSL-tetramer, A2402-TYGPVFMCL-tetramer, A2402-PYLFWLAAI-tetramer, A1101-SSCSSCPLTK-tetramer, and A1101-SSCSSCPLSK-tetramer were separately co-incubated with cells infected with TCRs of corresponding HLA types (the LLL tetramer corresponds to TCR E23; the AAI tetramer corresponds to TCR E29; the MSL/MCL tetramer corresponds to TCR E44; and the LSK/LTK tetramer corresponds to TCR E141). By comparing with commercial tetramers from MBL, the percentage of positive cells detected for the A0201-FLYALALLL-tetramer was 70.5%, which was much higher than that of the commercial FLYALALLL tetramer (59.7%), and the percentage of positive cells detected for the A1101-SSCSSCPLSK-tetramer was 20.3%, which was significantly higher than that of the commercial SSCSSCPLSK-tetramer (18.1%), as shown in FIG. 2A. This fully suggests that the tetramers of the present invention exhibit a high degree of specificity and staining effect in detecting the positive rate of cells. In a further volume gradient assay, the independently developed A2402-TYGPVFMSL tetramer was found to have a specific binding effect much higher than the commercial tetramer (see FIG. 2B).

[0195] The TCRs fished by the tetramers constructed by the present invention can simultaneously recognize the wild-type antigenic epitope and the mutant-type antigenic epitope to prevent immune escape. As shown in FIG. 3-A, both A2402 HLA wild-type antigenic epitope (MCL) and mutant-type antigenic epitope (MSL) tetramers were able to be recognized by TCR E44, which was fished by the MSL tetramer. As shown in FIG. 3-B, both A1101 HLA wild-type antigenic epitope (LSK) and mutant-type antigenic epitope (LTK) tetramers were able to be recognized by TCR E141, which was fished by the LSK tetramer. The TCR with corresponding specificity can be fished by different antigenic epitope tetramers of the same HLA type. As shown in FIG. 3-C, positive cells corresponding to TCR E29 was able to be detected by the AAI tetramer.

Example 2. Construction of pHAGE-TCR-RFP Vector

[0196] I. Acquisition off and a gene fragments of EBV LMP2 epitope-specific TCRs

[0197] 1) The A0201-FLYALALLL-tetramer, A2402-PYLFWLAAI-tetramer, A2402-TYGPVFMSL-tetramer, A2402-TYGPVFMCL-tetramer, A1101-SSCSSCPLSK-tetramer and A1101-SSCSSCPLTK-tetramer prepared in Example 1 were stained with peripheral blood, T cells positive for tetramer staining were sorted by flow cytometry to obtain single cells, and reverse transcription was performed to obtain cDNA (SuperScript® IV Reverse Transcriptase, Invitrogen). The variable region fragments of the TCRβ gene were obtained by amplification by two rounds of PCR (KOD-Plus-Neo, TOYOBO) based on the principle of multiplex PCR.

TABLE-US-00005 Reverse transcription primer: (SEQ ID NO: 144) TRBC1-TCAGGCAGTATCTGGAGTCATTG

[0198] PCR Amplification Primers:

TABLE-US-00006 Upstream primer 1: (see SEQ ID NOs: 147-185 and 366) TRBV_F1 Upstream primer 2: (see SEQ ID NOs: 186-225) TRBV_F2 Downstream primer 1: (SEQ ID NO: 145) TRBC2-GCACCTCCTTCCCATTCACC Downstream primer 2: (SEQ ID NO: 146) TRBC3-GCTTCTGATGGCTCAAACACAG

[0199] Specifically, according to the product instructions of the PCR polymerase KOD-Plus-Neo, the PCR system of the first round was at 20 μL, the annealing temperature was 60° C., and the reaction was performed for 30 cycles. 1 μL of the product from the first round of PCR reaction was taken as a template of the second round of PCR, wherein the PCR system of the second round was at 30 μL, the annealing temperature was 60° C., and the reaction was performed for 30 cycles. The product from the second round of PCR was subjected to agarose gel electrophoresis, and the band with the corresponding size was extracted from gel (TIANGEN Gel Extraction Kit) and sent for sequencing, wherein the sequencing primer was the downstream primer 2. The TCRβ gene sequences were obtained, wherein the specific TCRβ gene sequences for E23, E240, E29, E180-1, E44, E141, E149, E168, E170, E244, E245, E254, E301, E304, E305, E307, E314, E315, E316, E317, E318 and E320 were shown as “double underlined” nucleotide sequences in SEQ ID NOs: 122-143, respectively.

[0200] 2) As above, reverse transcription was performed on T cells positive for tetramer staining to obtain cDNA (SuperScript® IV Reverse Transcriptase, Invitrogen). The TCRα gene fragments were obtained by amplification by two rounds of PCR (KOD-Plus-Neo, TOYOBO) according to the product instructions.

TABLE-US-00007 Reverse transcription primer: (SEQ ID NO: 226) TRAC1-CGACCAGCTTGACATCACAG

[0201] PCR Amplification Primers:

TABLE-US-00008 Upstream primer 3: (see SEQ ID NOs: 229-273) TRAV_F1 Upstream primer 4: (see SEQ ID NOs: 274-315) TRAV_F2 Downstream primer 3: (SEQ ID NO: 227) TRAC2-GTTGCTCTTGAAGTCCATAGACCTC Downstream primer 4: (SEQ ID NO: 228) TRAC3-CAGGGTCAGGGTTCTGGATA

[0202] Specifically, according to the product instructions of the PCR polymerase KOD-Plus-Neo, the PCR system of the first round was at 20 μL, the annealing temperature was 60° C., and the reaction was performed for 30 cycles. 1 μL of the product from the first round of PCR reaction was taken as a template of the second round of PCR, wherein the PCR system of the second round was at 30 μL, the annealing temperature was 60° C., and the reaction was performed for 30 cycles. The product from the second round of PCR was subjected to agarose gel electrophoresis, and the band with the corresponding size was extracted from gel (TIANGEN Gel Extraction Kit) and sent for sequencing, wherein the sequencing primer was the downstream primer 4. The TCRα gene sequences were obtained, wherein the specific TCRα gene sequences for E23, E240, E29, E180-1, E44, E141, E149, E168, E170, E244, E245, E254, E301, E304, E305, E307, E314, E315, E316, E317, E318 and E320 were shown as “wavy underlined” nucleotide sequences in SEQ ID NOs: 122-143, respectively.

[0203] II. Construction of pHAGE-TCR Vector

[0204] TCRβ, fp2A and TCRα were amplified by overlap-PCR (KOD-Plus-Neo, TOYOBO) with long primer (containing fp2A sequence) to obtain TCRβ-fp2A-TCRα fragments, which were named as pHAGE-TCR plasmids for E23, E240, E29, E180-1, E44, E141, E149, E168, E170, E244, E245, E254, E301, E304, E305, E307, E314, E315, E316, E317, E318 and E320, respectively.

[0205] Amplification Primers:

[0206] Upstream primer 5 is shown in Table 1.

TABLE-US-00009 TABLE 1 TCR ID Upstream primer 5 E23 atttcaggtgtcgtgaagcggccgcgccaccATGAGCATCGGCCTCCT (SEQ ID NO: 316) E240 atttcaggtgtcgtgaagcggccgcgccaccATGAGCATCGGCCTCCT (SEQ ID NO: 317) E29 atttcaggtgtcgtgaagcggccgcgccaccATGCTGCTGCTTCTGCT (SEQ ID NO: 318) E180-1 atttcaggtgtcgtgaagcggccgcgccaccATGAGCAACCAGGTGCT (SEQ ID NO: 319) E44 atttcaggtgtcgtgaagcggccgcgccaccATGGGCCCCCAGCTCCT (SEQ ID NO: 320) E141 atttcaggtgtcgtgaagcggccgcgccaccATGGGCTGCAGGCTGCTC (SEQ ID NO: 321) E149 atttcaggtgtcgtgaagcggccgcgccaccATGGGCTGCAGGCTGCTC (SEQ ID NO: 322) E168 atttcaggtgtcgtgaagcggccgcgccaccATGGGCCCTGGGCTCCT (SEQ ID NO: 323) E170 atttcaggtgtcgtgaagcggccgcgccaccATGGACACCAGAGTACTCTGCTG (SEQ ID NO: 324) E244 atttcaggtgtcgtgaagcggccgcgccaccATGGGCCCCCAGCTCC (SEQ ID NO: 325) E245 atttcaggtgtcgtgaagcggccgcgccaccATGGGCCCTGGGCTCCT (SEQ ID NO: 326) E254 atttcaggtgtcgtgaagcggccgcgccaccATGGGCTTCAGGCTCCTCTG (SEQ ID NO: 327) E301 atttcaggtgtcgtgaagcggccgcgccaccATGGGCTGCAGGCTGCTC (SEQ ID NO: 328) E304 atttcaggtgtcgtgaagcggccgcgccaccATGGGCTGCAGGCTGCTC (SEQ ID NO: 329) E305 atttcaggtgtcgtgaagcggccgcgccaccATGGATACCTGGCTCGTATGC (SEQ ID NO: 330) E307 atttcaggtgtcgtgaagcggccgcgccaccATGGGCACCAGGCTCCTC (SEQ ID NO: 331) E314 atttcaggtgtcgtgaagcggccgcgccaccATGGGCTGCAGGCTGCTC (SEQ ID NO: 332) E315 atttcaggtgtcgtgaagcggccgcgccaccATGGGCACCAGGCTCCTC (SEQ ID NO: 333) E316 atttcaggtgtcgtgaagcggccgcgccaccATGAGCAACCAGGTGCTCTG (SEQ ID NO: 334) E317 atttcaggtgtcgtgaagcggccgcgccaccATGGGCCCCCAGCTCC (SEQ ID NO: 335) E318 atttcaggtgtcgtgaagcggccgcgccaccATGGACTCCTGGACC (SEQ ID NO: 336) E320 atttcaggtgtcgtgaagcggccgcgccaccATGGGCACCAGGCTCCTC (SEQ ID NO: 337) Downstream primer 5: TCTCCAGCCTGCTTCAGCAGGCTGAAGTTAGTAGCTCCGCTTCCGCTccgtttccgccgGAAATCCTTTC TCTTGACCATG (SEQ ID NO: 338)

[0207] Upstream primer 6 is shown in Table 2.

TABLE-US-00010 TABLE 2 TCR ID Upstream primer 6 E23 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC T ATGGAAACTCTCCTGGGAGTGTCT (SEQ ID NO: 339) E240 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC T ATGGAAACTCTCCTGGGAGTGTCT (SEQ ID NO: 340) E29 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC T ATGGAGAAGAATCCTTTGGCAGCC (SEQ ID NO: 341) E180-1 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC T ATGACATCCATTCGAGCTGTATTT (SEQ ID NO: 342) E44 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC T ATGATGAAATCCTTGAGAGTTTTA (SEQ ID NO: 343) E141 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC TATGAAGAGGATATTGGGAGCTCTG (SEQ ID NO: 344) E149 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC TATGAAGAGGATATTGGGAGCTCTG (SEQ ID NO: 345) E168 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC TATGAAGAAGCTACTAGCAATGATTCTG (SEQ ID NO: 346) E170 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC TATGACATCCATTCGAGCTGTATTTAT (SEQ ID NO: 347) E244 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC TATGACATCCATTCGAGCTGTATTTAT (SEQ ID NO: 348) E245 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC TATGAAGAAGCTACTAGCAATGATTCTG (SEQ ID NO: 349) E254 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC TATGAAGAGGATATTGGGAGCTCTG (SEQ ID NO: 350) E301 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC TATGAAGAGGATATTGGGAGCTCTG (SEQ ID NO: 351) E304 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC TATGAAGAGGATATTGGGAGCTCTG (SEQ ID NO: 352) E305 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC TATGAAGAAGCTACTAGCAATGATTCTG (SEQ ID NO: 353) E307 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC TATGCTGACTGCCAGCCTGT (SEQ ID NO: 354) E314 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC TATGAAGAGGATATTGGGAGCTCTG (SEQ ID NO: 355) E315 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC TATGGAGACCCTCTTGGGCCT (SEQ ID NO: 356) E316 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC TATGTCACTTTCTAGCCTGCTGAAG (SEQ ID NO: 357) E317 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC TATGGCAGGCATTCGAGCTT (SEQ ID NO: 358) E318 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC TATGACATCCATTCGAGCTGTATTTAT (SEQ ID NO: 359) E320 TCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC TATGAAGAAGCTACTAGCAATGATTCTG (SEQ ID NO: 360) Downstream primer 6: agggatcctctagactcgagctagcTCAGCTGGACCACAGCCGCA (SEQ ID NO: 361)

[0208] Specifically, the TCRβ and the TCRα were firstly obtained by amplification by using a primer 5 and a primer 6, respectively, wherein the PCR system was at 50 μL, the annealing temperature was 60° C., and the reaction was performed for 30 cycles. The PCR products were subjected to gel electrophoresis and extracted (TIANGEN Gel Extraction Kit), and the extracted products were taken as templates, each at 1 μL, and subjected to overlap PCR by using an upstream primer 5 and a downstream primer 6, respectively, wherein the PCR system was at 50 μL, the annealing temperature was 60° C., and the reaction was performed for 30 cycles. The product was subjected to agarose gel electrophoresis to obtain a band of about 1800 bp, which was then extracted from gel. The lentiviral vector pHAGE-IRES-RFP was double digested with NotI and NheI, wherein the enzyme digestion system was at 40 μL, wherein the NotI and NheI were each at 1.5 μL, the plasmid was at 2-3 μg, and the enzyme digestion was performed at 37° C. for 6 h. Then 1 μL of alkaline phosphatase (NEB) was added into the system and treated for 1 h to reduce the self-ligation of the plasmid, and the plasmid after the enzyme digestion was subjected to gel electrophoresis and extracted, determined for the concentration using nanodrop, and used as a backbone for constructing the plasmid.

[0209] According to the product instructions of Clone Express II One Step Cloning kit, the TCR was connected with a linearized pHAGE-IRES-RFP vector after enzyme digestion through overlap (see FIG. 4), and transformed into Stb13 strain, which was then cultured in an ampicillin-containing LB plate for 12-16 h. The monoclonal strain was picked for sequencing, wherein the sequencing primers selected were primers seq-pHAGE-F and seq-pHAGE-R on the pHAGE vector and a downstream primer 4. Corresponding TCRs were obtained, abbreviated as E23, E240, E29, E180-1, E44, E141, E149, E168, E170, E244, E245, E254, E301, E304, E305, E307, E314, E315, E316, E317, E318 and E320, respectively.

Example 3. Assay on Membrane Expression and Affinity of TCRs by a pMHC Tetramer Staining Method

[0210] 1. Construction of Endogenous TCR Knockout Jurkat T Cell Lines

[0211] Based on the sequence characteristics of the Jurkat cell TCR, guide sequences (TRA_oligo1-CACCGTCTCTCAGCTGGTACACGGC (SEQ ID NO: 362), TRA_oligo2-AAACGCCGTGTACCAGCTGAGAGAC (SEQ ID NO: 363), TRB_oligo1-CACCGGGCTCAAACACAGCGACCTC (SEQ ID NO: 364), TRB_oligo2-AAACGAGGTCGCTGTGTTTGAGCCC (SEQ ID NO: 365)) were designed in the constant regions of the α chain and the β chain.

[0212] The synthesized guide sequences of the α chain and the β chain were constructed into sgRNA-LentiCRISPR-puro and sgRNA-LentiCRISPR-BSD lentiviral vectors, respectively, and the vectors were co-transfected with packaging plasmids psPAX2 and pMD2.G and a PEI transfection reagent into 293T cells according to a certain ratio. The cell culture supernatants were harvested at 48 h and 72 h and concentrated, and the two viruses after concentration were simultaneously used to infect a human Jurkat T cell line. 48 h after the infection, killing was performed using puromycin and blasticidin at appropriate concentrations until all cells in the control group for each of the two drugs were dead. Surviving cells were sorted by flow cytometry to obtain single cells, which were added into a 96-well plate for culturing. For the obtained monoclonal cell line, its expression was separately identified using antibodies of the TCRα chain and the TCRβ chain, and the cell strain defective in both chains was the obtained endogenous TCR knockout Jurkat T cell, which was named as JC5.

[0213] 2. Construction of JC5 Cell Line Stably Integrating EBV TCR

[0214] The pHAGE-TCR plasmids such as E23 and E240 constructed in Example 2 were separately mixed with packaging plasmids psPAX2 and pMD2.G and a PEI transfection reagent according to a certain ratio, and transfected into 293T cells. The cell culture supernatants were harvested at 48 h and 72 h and concentrated to infect JC5 cells in the logarithmic growth phase (MOI=0.3). 3 days after infection, cells were stained with anti-human CD3 and anti-human TCRαβ flow cytometry antibodies, and the cells with the same TCR expression level were sorted and cultured to obtain the JC5-TCR cell line.

[0215] 3. Assay on Expression-On-Membrane and Affinity of TCRs

[0216] 1×10.sup.6 JC5-TCR cells were taken, stained with Brilliant Violet 421™ anti-human TCRαβ (Biolegend) and the corresponding EBV LMP2 pMHC tetramer-APC (tetramer-APC) and then analyzed by flow cytometry.

[0217] As can be seen from FIGS. 5 and 6, the prepared specific E23-TCR and E240-TCR for EBV LMP2 HLA-A*A0201 FLYALALLL, specific E29-TCR and E180-1-TCR for EBV LMP2HLA-A*A2402 PYLFWLAAI, and specific E44-TCR for EBV LMP2HLA-A*A2402 TYGPVFMSL/TYGPVFMCL were all able to be correctly expressed and displayed on the outer side of cell membrane, and had a certain affinity to the corresponding tetramer probes.

[0218] As can be seen from FIG. 7, among the prepared specific TCRs for EBV LMP2 HLA-A*A1101 SSCSSCPLSK/SSCSSCPLTK, all the constructed TCRs except E244-TCR and E307-TCR showed better binding to the SSCSSCPLSK/SSCSSCPLTK epitope. This result not only suggests that the self-made tetramers can be successfully used to identify specifically bound T cells, but also shows that the obtained TCRs have good affinity.

Example 4. Functional Activity and EC.SUB.50 .of TCRs

[0219] It was taken into consideration that the pMHC tetramers in Example 3 were used to test the structural affinity of TCRs, and the tetramers binds tetravalently to the TCR on the surface of JC5. To further identify the activity of the TCRs, we stably integrated the HLA-A*1101 molecule in T2 cells and constructed a T2-HLA-A*1101 cell line for quantification of the half maximal effect antigen concentration (EC.sub.50) of the TCRs, thus achieving the comparison of the functional activity of the TCRs.

[0220] 1. Construction of T2 Cell Line Stably Integrating HLA-A*1101

[0221] The HLA-A*1101 molecule and β2m molecule (derived from human) were cloned, linked with fp2A, and constructed into a pHAGE-BSD vector, which was co-transfected with packaging plasmids psPAX2 and pMD2.G and a PEI transfection reagent into 293T cells according to a certain ratio for virus encapsulation, thereby infecting the T2 cell line. 48 h after infection, killing was performed on T2 cells using blasticidin at the appropriate concentration until all cells in the control group were dead, so that a T2-HLA-A*1101 cell line was obtained.

[0222] 2. Determination of Functional Activity and EC.sub.50 of TCR

[0223] The synthesized LMP2 antigenic epitopes were diluted with a DMSO solvent to a stock concentration of 4 mg/mL. Then peptide fragments of antigenic epitope were serially diluted at a gradient with a complete medium to obtain LSK and LTK peptide fragment solutions at 2×10.sup.−8-2×10.sup.−4 M, each of which were added to a T2-HLA-A*1101 cell suspension at 1×10.sup.6 cells/mL in a volume ratio of 1:100, and mixed uniformly. The cells were seeded in a 96-well plate at 100 μL/well, 100 μL of JC5-TCR cells at the concentration of 1×10.sup.6 cells/mL were added, and mixed uniformly to obtain a T2 incubation system with the peptide fragment concentration of 1×10.sup.−10-1×10.sup.−6 M. After 24 h of co-incubation, the culture supernatant was collected and assayed for IL2 production by an ELISA kit. The experiment was repeated three times. FIGS. 8 and 10 represent IL2 production by JC5-TCR cells stimulated by different concentrations of LSK and LTK peptides, respectively. Corresponding EC.sub.50 values can be obtained by calculation with prism, and EC.sub.50 values for triplicates were detailed in FIGS. 9 and 11. As can be seen from FIGS. 8-11, TCR E149, E304, E170 and E315 had excellent functional activity for the synthetic LSK antigenic epitope, while E149, E254, E170, E316, E317 and E318 all exhibited better functional activity for the LTK antigenic epitope. The results for E244 and E307 are consistent with the results of tetramer staining, that is, both E244 and E307 have weaker recognition ability for the SSCSSCPLSK/SSCSSCPLTK antigenic epitope.

Example 5. Construction of and In Vitro Functional Assay on Human Primary TCR T Cells

[0224] 1. Isolation, Culture and Lentivirus Infection of Human Primary T Cells

[0225] To further verify the recognition and killing function of the selected TCRs for the EBV LMP2 antigens, mononuclear cells (PBMCs) were isolated from peripheral blood of volunteers using the lymphocyte isolation solution Ficoll, then T cells were obtained from PBMCs by negative selection according to the product instructions of EasySep Human T cell isolation kit (stem cell technologies), resuspended to 1×10.sup.6 cells/mL in a 1640 complete medium containing 100 U/mL IL2, and cultured in an anti-CD3/CD28 antibody coated culture dish for activation. After 48 h of activation, the T cells were infected with the TCR-loaded viral particles (prepared in Example 3) using a lentivirus system by centrifuging at 1500 rpm for 2 h at 32° C., culturing in a 37° C. cell incubator for 10 h and terminating the infection by media exchange, and then cultured in a 37° C. cell incubator. Three days after infection, TCR positive cells were sorted using a flow cytometer to obtain TCRT cells (including E23, E240, E29, E180-1, E44, E141, E149, E168, E170, E244, E245, E254, E301, E304, E305, E307, E314, E315, E316, E317, E318 and E320 described above).

[0226] 2. Construction of Target Cells

[0227] Virus particles separately loaded with LMP2-RFP, HLA-A*0201-BSD/HLA-A*2402-BSD/HLA-A*1101-BSD and Luciferase-GFP were used to infect into Raji cells in the logarithmic growth phase using a lentivirus system. Raji cells simultaneously stably expressing LMP2, HLA-A molecules and Luciferas-GFP were obtained by drug screening and flow cytometry sorting, and named as Raji-HLA-A*A0201/2402/1101-LMP2-luciferase. In addition, virus particles of HLA-A*0201-BSD/HLA-A*2402-BSD/HLA-A*1101-BSD were used to infect EBV-LCL cells in the logarithmic growth phase. EBV-LCL cells stably expressing HLA-A molecules were obtained by drug screening, and named as EBV-LCL-HLA-A*0201, EBV-LCL-HLA-A*2402 and EBV-LCL-HLA-A*1101 cells, respectively.

[0228] 3. In Vitro Functional Verification of TCRs in Human Primary T Cells

[0229] 1) Verification of the Recognition Ability of TCRs to Epitopes at an Endogenous Level

[0230] The EBV-LCL is the immortalized human B cell infected with the EB virus, which more realistically simulates the antigen level in tumor cells in vivo. Thus, TCR T cells recognizing the SSCSSCPLSK/SSCSSCPLTK epitope and EBV-LCL-HLA-A*1101 cells were co-incubated at effector-to-target ratios of 8:1, 4:1, 2:1, 1:1, 0.5:1 and 0.25:1, with the target cells fixed at 1×10.sup.5 cells. After 24 h of co-incubation, supernatants were collected for detection of secreted cytokines IL2 (FIG. 12) and IFN-γ (FIG. 13). In terms of the release level of cytokines, TCRs E141, E170, E254 and E315 can significantly activate T cells after binding to LSK at an endogenous level, particularly the E315 TCR T cell, which not only shows the best IFN-γ level at each effector-to-target ratio, but also has a higher corresponding IL2 value. The results suggest that the TCRs prepared by the present invention can effectively mediate the recognition of tumor endogenous antigens.

[0231] 2) Verification of the Long-Term Killing Ability of TCR T Cells to Tumor Cells

[0232] TCR T cells recognizing the SSCSSCPLSK/SSCSSCPLTK epitope and Raji-HLA-A*1101-LMP2-luciferase cells were initially co-incubated at an effector-to-target of 1:3, recorded as day 0, and then cells were separately collected for flow cytometry analysis on day 1, day 3 and day 5. The culture medium used was a 1640 complete medium without IL2, the TCR T cells were initially at 1×10.sup.5 cells, the samples at each time point were incubated independently, and the remaining co-incubated samples were separately subjected to half medium exchange on day 2 and day 4, and supplemented with target cells. Cells for flow cytometry analysis were firstly stained with the anti-human CD3 antibody, the cells with a specified volume were collected and recorded at the time of loading, and the number of T cells in the system was determined by conversion (see FIG. 14). As can be seen from the proliferation curves of absolute T cell numbers, E315-TCR T cells exhibited the best activation and proliferation after recognizing two antigenic epitopes. In addition, the effector-to-target ratio in the system was further analyzed (see FIG. 15), and E315-TCRT cells exhibited the strongest tumor-clearing ability, as with the proliferation results.

[0233] 3) In Vitro Functional Verification of TCRs in Human Primary T Cells

[0234] Raji-HLA-A*0201-LMP2 and Raji cells untransfected with LMP2 were each separately co-incubated with E23-TCRT cells, E240-TCRT cells and 1G4 T cells according to the ratios of 1:0.5, 1:1 and 1:2. After 24 h of co-incubation, the cells and supernatant were separately collected, and the activation of E23-TCRT cells and E240-TCRT cells and the death of target cells were preliminarily determined. In terms of the release levels of the extracellular cytokines TNFα, IL2 and IFNγ (see FIG. 16), E240-TCRT and E23-TCRT, when co-incubated with target cells, were able to significantly cause the activation of T cells as compared to the control group 1G4 T. Moreover, the amount of luciferase released from the target cells after lysis reflects the death of the target cells (see FIG. 16). Experimental results show that the E23-TCRT cells and E240-TCRT cells constructed in the example of the present invention can be specifically activated by EBV LMP2 antigen peptide presenting cells, and can significantly kill target cells.

[0235] Raji-HLA-A*2402-antigen peptide, Raji cells untransfected with antigen peptide, T2-HLA2402 cells with antigen peptide and T2-HLA2402 cells without antigen peptide were each separately co-incubated with E29-TCRT cells, E180-1-TCRT cells and 1G4 T cells according to the ratio of 1:3. After 24 h of co-incubation, the cells and supernatant were separately collected, and the activation of the E29-TCRT cells and 180-1-TCRT cells were preliminarily determined. In terms of the release levels of the extracellular cytokines IL2 and IFNγ (see FIG. 17), E29-TCRT cells and 180-1-TCRT cells, when co-incubated with target cells, were able to significantly cause the activation of T cells as compared to the control group 1G4 T. In addition, in terms of the release levels of the extracellular cytokines (see FIG. 18), E44-TCRT cells, when co-incubated with target cells, were able to significantly cause the activation of T cells as compared to the control group RFP T. Experimental results show that the E29-TCRT cells, E180-1-TCRT cells and E44-TCRT cells constructed in the example of the present invention can be specifically activated by EBV LMP2 antigen peptide presenting cells, and can significantly kill target cells.

Example 6. Comparison of Function of TCRs Sharing CDR3 Motif

[0236] Since the identified E141, E149, E254, E301, E304 and E314 recognizing the SSCSSCPLSK/SSCSSCPLTK epitope, were very conserved in the CDR3 hypervariable region of both TCR α and β chains, and the sequences were highly similar with only one amino acid difference but were functionally far apart (FIGS. 9 and 11), TCRs with those conserved CDR3 motifs were defined as public TCRs, and TCRs without those conserved sequences were defined as private TCRs. Moreover, in this example, the structural affinity and function of the public TCRs were analyzed.

[0237] 1. Determination of Structural Affinity of Public TCRs by a BFP Method

[0238] Red blood cells were fixed on one side of a micropipette, and beads specifically embedded with pMHC molecules were adsorbed on the surface of the red blood cell surface to form a hypersensitive biomembrane force probe (BFP). Meanwhile, JCR-TCR cells were fixed on the other side of the micropipette, and the contact between the two type of cells was controlled by a piezoelectric transducer, wherein the applied pressure was 10 pN and the contact time was 0.1 s when each cycle of contact was performed, then separated at the speed of 1000 pN/s for the next cycle of contact. The deformation of the red blood cell-bead surface was recorded by a microscope in the whole process, and whether a bond was formed or not and the duration were determined. As can be seen from the bonding duration in FIG. 19, the structural affinity for the LSK epitope varied among public TCRs, with E304 having better function, which was consistent with the results of T2 (FIG. 9).

[0239] 2. Analysis of the Function of Conserved CDR3 Motif by an Alanine Screening Method

[0240] The amino acids after the first position of CDR3 regions of TCR E141 α and β chains were mutated into alanine in sequence, named as a2-a9 and b2-b11, constructed into a pHAGE lentiviral vector, and used to infect human primary T cells (MOI=10) after virus encapsulation. After three days of infection, TCR positive cells could be sorted out by a flow cytometer. The sorted TCR T cells were co-incubated with Raji-LMP2-luciferase target cells according to the effector-to-target of 1:1, wherein the T cells were at 1×10.sup.5 cells. After 24 h of co-incubation, the cells and supernatant were separately collected. The cells were used to determine the amount of luciferase released from the surviving target cells after lysis of the cell pellet (FIG. 20), and the supernatant was used to determine the cytokine IL2 secreted by TCR T cells (FIG. 21). As can be seen from FIGS. 20 and 21, compared with the background values of the control groups HLA unmatched-Raji cells, or HLA matched-Raji cells without TCR-T cells and with pan-T cells untransfected with TCRs, the unmutated E141 was able to efficiently clear the target cells when exposed to HLA matched-Raji and specifically produce a large amount of cytokine IL2. However, at the sites a3, a5 and a6 as well as b6, b7 and b8, a single amino acid mutation is sufficient to completely inactivate TCR T cells. Collectively, these experimental results suggest that although the public TCRs share the same conserved motif, their function may differ significantly from each other by one amino acid difference.

Example 7 Animal Model Construction and In Vivo Functional Assay on EBV TCRT

[0241] EB virus mainly infects nasopharyngeal epithelial cells and B cells, and is closely related to development and progression of nasopharyngeal carcinoma and various B cell lymphomas. In this example, a mouse model of B-cell lymphoma and a solid tumor model of nasopharyngeal carcinoma were constructed to verify the in vivo function of the identified TCRs.

[0242] 1. Lymphoma Model and In Vivo Functional Assay on TCRT

[0243] NOD/Scid IL-2Rγ null (NCG) female mice aged 5-6 weeks were inoculated with 3×10.sup.5 Raji-HLA-A*1101/0201/2402-LMP2-luciferase tumor cells via tail veins to construct a lymphoma model (see FIGS. 22, 23 and 24), which was recorded as day 1. On day 5, the mice were divided into 3 groups, i.e., A: a PBS injection group (with equal volume of PBS injected); B: a control TCRT cell injection group (TCR-1G4 T cells); and C: an EBV TCRT injection group (E141-TCRT cells), wherein the mice in group B/C were injected with 5×10.sup.6 TCR T cells via tail veins, and the mice in group A were injected with equal volume (200 μL) of PBS. The second injection was given on day 8, and the procedure was identical to that on day 5. Specific reinfusion volume and reinfusion time points for other TCR T cells are shown in FIGS. 22 and 23. The mice were monitored for tumor cell growth, T cell proliferation and mouse survival over the next few weeks. As shown in FIG. 24, compared with the control group, the EBV-specific E141-TCRT cells constructed in the example of the present invention were able to significantly kill tumor cells in mice, and increase the survival rate of mice. In addition, as shown in FIGS. 22 and 23, compared with the control group, the EBV-specific E23-TCRT, E240-TCRT, E29-TCRT and E44-TCRT cells constructed in the example of the present invention were also able to significantly kill tumor cells in mice, and increase the survival rate of mice.

[0244] 2. Solid Tumor Model

[0245] NCG female mice aged 5-6 weeks were subcutaneously inoculated with 1×10.sup.6 C666-1-HLA-A*1101-LMP2-luciferase tumor cells to construct a nasopharyngeal carcinoma solid tumor model (see FIG. 25), which was recorded as day 0. After 7 days, the mice were divided into 3 groups, i.e., A: a PBS injection group (with equal volume of PBS injected); B: a control TCR-T cell injection group (TCR-1G4 T cells); and C: an EBV TCR-T injection group (E141-TCRT cells), wherein the mice in group B/C were injected with 3×10.sup.6 T cells via the tail vein, and the mice in group A were injected with equal volume (200 μL) of PBS. The mice were monitored for tumor cell growth, T cell proliferation and mouse survival over the next few weeks. As shown in FIGS. 25, 26 and 27, compared with the control group, the EBV-specific E141-TCRT cells constructed in the example of the present invention were able to significantly kill tumor cells in mice (FIGS. 25 and 26), and the returned TCR T cells proliferated specifically in vivo (FIG. 27).

[0246] The preferred embodiments of the present invention are described in detail above, which, however, are not intended to limit the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, all of which will fall within the protection scope of the present invention.

[0247] In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, such combinations will not be illustrated separately.