High-affinity T cell receptor for recognizing SSX2 and application of high-affinity T cell receptor

Abstract

The invention provides a high-affinity T cell receptor for recognizing SSX2 and application of the high-affinity T cell receptor, and particularly relates to a T cell receptor (TCR), and the TCR has the characteristic of being combined with an AQIPEKIQK-HLA A1101 compound. The invention also provides a multivalent TCR compound, a nucleic acid molecule for coding the TCR, a carrier containing the nucleic acid molecule, a cell for expressing the TCR and a pharmaceutical composition containing the above substances, and applications thereof in preparation of drugs for diagnosis, treatment and prevention of SSX2 positive diseases. The invention also provides a preparation method of the TCR.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a high-affinity T cell receptor for recognizing SSX2 and its application; more specifically, it relates to a T cell receptor (T cell receptor, TCR) capable of recognizing a polypeptide derived from an SSX2 protein; the present invention also relates to a preparation method and application of the TCR. Background Art

[0002] There are only two types of molecules that can recognize antigens in a specific manner. One is the immunoglobulin or antibody; the other is the T cell receptor (TCR), which is a glycoprotein on the cell membrane surface that exists as a heterodimer of α chain / β chain or γ chain / δ chain. The composition of the immune system's TCR repertoire is generated in the thymus by V(D)J recombination followed by positive and negative selection. In the peripheral environment, TCR mediates the specific recognition of T cells to the major histocompatibility complex-peptide complex (pMHC), and is therefore crucial to the cellular immunity function of the immune system.

[0003] TCR is the only receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC). Such exogenous or endogenous peptides may be the only sign of abnormality in cells. In the immune system, the binding of antigen-specific TCR to pMHC complex triggers direct physical contact between T cells and antigen-presenting cells (APCs), and then other cell membrane surface molecules of both T cells and APCs interact, which causes a series of subsequent cell signaling and other physiological reactions, so that T cells with different antigen specificities can exert immune effects on their target cells.

[0004] The MHC class I and class II molecule ligands corresponding to TCR are also proteins of the immunoglobulin superfamily, but they are specific for antigen presentation. Different individuals have different MHCs, which can present different short peptides in a protein antigen to the surface of their respective APC cells. Human MHC is usually called HLA gene or HLA complex.

[0005] SSX2 is the synovial sarcoma X breakpoint, also known as HOM-MEL-40. SSX2 is one of the ten highly homologous nucleic acid proteins in the SSX family. SSX protein is a tumor testis antigen that is only expressed in tumor cells and testicular germ cells without MHC expression. SSX2 is expressed in a variety of human cancer cells, including but not limited to osteosarcoma, hepatocarcinoma, melanoma, synovial sarcoma, multiple myeloma, colon adenocarcinoma, breast cancer, etc., as described in the literature (Naka.N et al (2002) International journal of cancer 98 (4): 640-642; Chen et al (2001) Cancer letters 164 (2): 189-195; Tureci O et al (1996) Cancer research 56 (20): 4766-4772; Tureci O et al (1998) International journal of cancer 77 (1): 19-23; Zhang et al (2014) Molecular cancer 13: 25). SSX2 is degraded into small molecular peptides after being produced in cells, and binds to MHC (major histocompatibility complex) molecules to form a complex, which is presented on the cell surface. AQIPEKIQK is a short peptide derived from the SSX2 antigen. Chemotherapy and radiotherapy can be used to treat the above diseases, but they will cause damage to the body's normal cells.

[0006] Therefore, the AQIPEKIQK-HLA A1101 complex provides a marker for tumor cells that can be targeted by a TCR. TCRs that can bind to the AQIPEKIQK-HLA A1101 complex have high application value in the treatment of tumors. For example, TCRs that can target the tumor cell marker can be used to deliver cytotoxic agents or immunostimulants to target cells, or be transformed into T cells so that T cells expressing the TCR can destroy tumor cells so that they can be given to patients in a treatment process called adoptive immunotherapy. For the former purpose, the ideal TCR is one with high affinity so that the TCR can reside on the targeted cells for a long time. For the latter purpose, it is preferred to use TCRs with medium affinity. Therefore, those skilled in the art are committed to developing TCRs that can be used to target tumor cell markers to meet different purposes. Summary of the invention

[0007] In view of the shortcomings of the prior art, the object of the present invention is to provide a TCR with high affinity for the AQIPEKIQK-HLA A1101 complex. Another object of the present invention is to provide a method for preparing the TCR and the use of the TCR.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect of the present invention, a T cell receptor (TCR), the TCR comprising a TCR α chain variable domain and a TCR β chain variable domain, the TCR having an activity of binding to an AQIPEKIQK-HLA A1101 complex;

[0010] And the amino acid sequence of the TCR α chain variable domain has at least 90% (for example, it can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, etc.) sequence homology with the amino acid sequence shown in SEQ ID NO:1, and the amino acid sequence of the TCR β chain variable domain has at least 90% (for example, it can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, etc.) sequence homology with the amino acid sequence shown in SEQ ID NO:2.

[0011] SEQ ID NO: 1:

[0012] AQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQK ARKSANLVISASQLGDSAMYFCAMKPGGSYIPTFGRGTSLIVHP.

[0013] SEQ ID NO:2:

[0014] NAGVTQTPKFRILKIGQSMTLQCTQDMNHNYMYWYRQDPGMGLKLIYYSVGAGITDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVYFCASSYSVTEHRGKRNEQFFGPGTRLTVL.

[0015] In a preferred example, the amino acid sequence of the TCRα chain variable domain and the amino acid sequence of the TCRβ chain variable domain are not the amino acid sequence of the wild-type TCRα chain variable domain and the amino acid sequence of the wild-type TCRβ chain variable domain at the same time.

[0016] In another preferred embodiment, the α chain variable domain of the TCR comprises an amino acid sequence that has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the sequence shown in SEQ ID NO:1, or has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acid residue mutations relative to the sequence shown in SEQ ID NO:1.

[0017] In another preferred embodiment, the β chain variable domain of the TCR is an amino acid sequence that has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with the sequence shown in SEQ ID NO:2, or has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acid residue mutations relative to the sequence shown in SEQ ID NO:2.

[0018] In another preferred embodiment, the TCR undergoes mutations in CDR1α of the variable domain of the TCRα chain, and the number of mutations is 2-4.

[0019] In another preferred embodiment, the TCR undergoes mutations in CDR3β of the variable domain of the TCRβ chain, and the number of mutations is 3-5, preferably 3 or 4.

[0020] In another preferred embodiment, the amino acid sequence of the TCRα chain variable domain has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO:1, and the amino acid sequence of the TCRβ chain variable domain has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO:2.

[0021] In another preferred example, in the variable domain of the TCRα chain, CDR1α is TSDQSYG, CDR2α is QGSYDEQN, and CDR3α is AMKPGGSYIPT.

[0022] In another preferred example, CDR1β in the variable domain of the TCRβ chain is MNHNY, CDR2β is SVGAGI, and CDR3β is selected from ASSYSVTEHRGKRNEQF, ASSYSVTEHRGKRYPLK and ASSYSVTEHRGKRYPLF.

[0023] In another preferred example, the amino acid sequence of the TCR α chain variable domain is SEQ ID NO: 1.

[0024] In another preferred example, the amino acid sequence of the TCRβ chain variable domain is SEQ ID NO: 2.

[0025] In another preferred embodiment, the amino acid mutation sites of the TCRβ chain variable domain are positions 14, 15, 16 and 17 of CDR3β.

[0026] In another preferred embodiment, the amino acid mutation sites of the TCRβ chain variable domain are positions 14, 15 and 16 of CDR3β.

[0027] In another preferred embodiment, the TCR also has the activity of binding to the AQIPEKIQK-HLA A1101 complex.

[0028] In another preferred embodiment, the three CDRs of the TCRβ chain variable domain are:

[0029] CDR1β:MNHNY SEQ ID NO:68;

[0030] CDR2β: SVGAGI SEQ ID NO: 69;

[0031] CDR3β: ASSYSVTEHRGKRNEQF SEQ ID NO:70.

[0032] and contain at least one mutation listed in Table 1:

[0033] Table 1

[0034]

[0035]

[0036] In another preferred embodiment, the TCR mutation occurs in CDR3β, comprising:

[0037] Residue before mutation The mutated residue F at position 17 of CDR3β K Q at position 16 of CDR3β L E at position 15 of CDR3β P N at position 14 of CDR3β Y R at position 13 of CDR3β G or T K at position 12 of CDR3β G G at position 11 of CDR3β I or T R at position 10 of CDR3β W H at position 9 of CDR3β S or P E at position 8 of CDR3β T T at position 7 of CDR3β S

[0038] In another preferred embodiment, the TCR mutation comprises:

[0039] Residue before mutation The mutated residue Q at position 16 of CDR3β L E at position 15 of CDR3β P N at position 14 of CDR3β Y

[0040] In another preferred embodiment, the three CDRs of the TCRβ chain variable domain are:

[0041] CDR1α:TSDQSYG SEQ ID NO:41;

[0042] CDR2α: QGSYDEQN SEQ ID NO: 42;

[0043] CDR3α: AMKPGGSYIPT SEQ ID NO:43.

[0044] And contains at least one mutation in Table 2:

[0045] Table 2

[0046] Residue before mutation The mutated residue A at position 1 of CDR3α G The second M of CDR3α I The fourth P of CDR3α N S at position 7 of CDR3α A Position 9 of CDR3α W P at position 10 of CDR3α L G at position 2 of CDR2α S S at position 2 of CDR1α T Q at position 4 of CDR1α T or I or L S at position 5 of CDR1α Q or A Y at position 6 of CDR1α F G at position 7 of CDR1α S

[0047] In another preferred embodiment, the affinity of the TCR to the AQIPEKIQK-HLA A1101 complex is at least 4 times that of the wild-type TCR, for example, it can be 4, 4.5, 5, 5.5, 6, 6.5 or 7, etc.

[0048] In another preferred embodiment, the TCR undergoes a mutation in the α chain variable domain shown in SEQ ID NO: 1, and the mutation is selected from one or more groups of A93G, M94I, P96N, S99A, I101W, P102L, G52S, G33S, Y32F, S31Q / A, Q30T / I / L, and S28T, wherein the amino acid residues are numbered as shown in SEQ ID NO: 1.

[0049] In another preferred embodiment, the TCR undergoes a mutation in the β chain variable domain shown in SEQ ID NO: 2, and the mutation is selected from one or more groups of F108K, Q107L, E106P, N105Y, R104G / T, K103G, G102I / T, R101W, H100S / P, E99T, T98S, I54V, G53M / K / D / L, A52K / R / Q, N30R, N28S, and M27H, wherein the amino acid residues are numbered as shown in SEQ ID NO: 2.

[0050] In another preferred embodiment, the TCR has a CDR selected from Table 3:

[0051] Table 3

[0052]

[0053] The amino acid sequences of SEQ ID NO:44 to SEQ ID NO:67 are shown below:

[0054] SEQ ID NO:44: TSDTQFS;

[0055] SEQ ID NO:45: TSDISYS;

[0056] SEQ ID NO:46: TSDLAYS;

[0057] SEQ ID NO:47:TSDLSYS;

[0058] SEQ ID NO:48:TSDTSFS;

[0059] SEQ ID NO:49: TTDLSYG;

[0060] SEQ ID NO:50: QSSYDEQN;

[0061] SEQ ID NO:51:GIKNGGSYIPT;

[0062] SEQ ID NO:52:AMKPGGAYWLT;

[0063] SEQ ID NO:53: AIKNGGSYIPT;

[0064] SEQ ID NO:54: HSHRY;

[0065] SEQ ID NO:55: SVGKMV;

[0066] SEQ ID NO:56: SVGKKV;

[0067] SEQ ID NO:57:SVGRDV;

[0068] SEQ ID NO:58: SVGKLV;

[0069] SEQ ID NO:59: SVGQKV;

[0070] SEQ ID NO:60: ASSYSVTEHRGKRYPLK;

[0071] SEQ ID NO:61: ASSYSVTEHRGKRYPLF;

[0072] SEQ ID NO:62: ASSYSVSTSWGKRNEQF;

[0073] SEQ ID NO:63: ASSYSVTEHRGKGYPLF;

[0074] SEQ ID NO:64: ASSYSVTEHRGKGYPLK;

[0075] SEQ ID NO:65: ASSYSVTEHRGKTYPLF;

[0076] SEQ ID NO:66: ASSYSVTEPRIGRNEQF;

[0077] SEQ ID NO:67: ASSYSVTEPRTGRNEQF.

[0078] In another preferred embodiment, the TCR is soluble.

[0079] In another preferred embodiment, the TCR is an αβ heterodimeric TCR, and the αβ heterodimeric TCR comprises an α chain TRAC constant region sequence and a β chain TRBC1 or TRBC2 constant region sequence.

[0080] In another preferred embodiment, the TCR comprises (i) a TCR α chain variable domain and all or part of the TCR α chain constant region excluding the transmembrane domain; and (ii) a TCR β chain variable domain and all or part of the TCR β chain constant region excluding the transmembrane domain.

[0081] In another preferred embodiment, the TCR comprises an α chain constant region and a β chain constant region, and an artificial interchain disulfide bond is contained between the α chain constant region and the β chain constant region of the TCR.

[0082] In another preferred embodiment, the cysteine ​​residues that form the artificial interchain disulfide bond between the constant regions of the TCRα and TCRβ chains are substituted with one or more groups of sites selected from the following:

[0083] Thr48 of exon 1 of TRAC*01 and Ser57 of exon 1 of TRBC1*01 or TRBC2*01;

[0084] Thr45 of exon 1 of TRAC*01 and Ser77 of exon 1 of TRBC1*01 or TRBC2*01;

[0085] Tyr10 of exon 1 of TRAC*01 and Ser17 of exon 1 of TRBC1*01 or TRBC2*01;

[0086] Thr45 of exon 1 of TRAC*01 and Asp59 of exon 1 of TRBC1*01 or TRBC2*01;

[0087] Ser15 of exon 1 of TRAC*01 and Glu15 of exon 1 of TRBC1*01 or TRBC2*01;

[0088] Arg53 of exon 1 of TRAC*01 and Ser54 of exon 1 of TRBC1*01 or TRBC2*01;

[0089] Pro89 of exon 1 of TRAC*01 and Ala19 of exon 1 of TRBC1*01 or TRBC2*01;

[0090] or Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01.

[0091] In another preferred example, the amino acid sequence of the α chain variable domain of the TCR is one of SEQ ID NO:1, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:22-29; and / or the amino acid sequence of the β chain variable domain of the TCR is one of SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17-21, SEQ ID NO:30-36.

[0092] In another preferred embodiment, the TCR is selected from the following group:

[0093] (1) the α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 13;

[0094] (2) the α chain variable domain sequence is SEQ ID NO: 14, and the β chain variable domain sequence is SEQ ID NO: 2;

[0095] (3) the α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 15;

[0096] (4) the α chain variable domain sequence is SEQ ID NO: 16, and the β chain variable domain sequence is SEQ ID NO: 2;

[0097] (5) the α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 17;

[0098] (6) the α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 18;

[0099] (7) the α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 19;

[0100] (8) the α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 20;

[0101] (9) the α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 21;

[0102] (10) the α chain variable domain sequence is SEQ ID NO: 22, and the β chain variable domain sequence is SEQ ID NO: 2;

[0103] (11) the α chain variable domain sequence is SEQ ID NO: 23, and the β chain variable domain sequence is SEQ ID NO: 2;

[0104] (12) the α chain variable domain sequence is SEQ ID NO: 24, and the β chain variable domain sequence is SEQ ID NO: 2;

[0105] (13) the α chain variable domain sequence is SEQ ID NO: 25, and the β chain variable domain sequence is SEQ ID NO: 2;

[0106] (14) the α chain variable domain sequence is SEQ ID NO: 26, and the β chain variable domain sequence is SEQ ID NO: 2;

[0107] (15) the α chain variable domain sequence is SEQ ID NO: 27, and the β chain variable domain sequence is SEQ ID NO: 2;

[0108] (16) the α chain variable domain sequence is SEQ ID NO: 28, and the β chain variable domain sequence is SEQ ID NO: 2;

[0109] (17) the α chain variable domain sequence is SEQ ID NO: 29, and the β chain variable domain sequence is SEQ ID NO: 2;

[0110] (18) the α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 30;

[0111] (19) the α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 31;

[0112] (20) the α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 32;

[0113] (21) the α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 33;

[0114] (22) the α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 34;

[0115] (23) the α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 35;

[0116] (24) The α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 36.

[0117] In another preferred embodiment, the TCR is of human origin.

[0118] In another preferred embodiment, the TCR is isolated or purified.

[0119] In another preferred embodiment, the TCR is a single-chain TCR.

[0120] In another preferred embodiment, the TCR is a single-chain TCR composed of an α chain variable domain and a β chain variable domain, and the α chain variable domain and the β chain variable domain are connected by a flexible short peptide sequence (linker).

[0121] In another preferred example, the TCR comprises an α chain constant region and a β chain constant region, the α chain constant region is a mouse constant region and / or the β chain constant region is a mouse constant region.

[0122] In another preferred example, the TCR comprises an α chain constant region and a β chain constant region, the α chain constant region is a mouse constant region and / or the β chain constant region is a mouse constant region.

[0123] In another preferred embodiment, a conjugate is bound to the C- or N-terminus of the α chain of the TCR, and / or a conjugate is bound to the C- or N-terminus of the β chain of the TCR.

[0124] Preferably, the conjugate comprises any one or a combination of at least two of a detectable label, a therapeutic agent or a PK modifying moiety.

[0125] In another preferred embodiment, the therapeutic agent bound to the TCR is an anti-CD3 antibody linked to the C- or N-terminus of the α or β chain of the TCR.

[0126] A second aspect of the present invention provides a multivalent TCR complex, wherein the multivalent TCR complex comprises at least two TCRs, and at least one of the TCRs is the TCR described in the first aspect.

[0127] The third aspect of the present invention provides a nucleic acid molecule, which includes a nucleotide sequence encoding the TCR described in the first aspect of the present invention or the multivalent TCR complex described in the second aspect of the present invention, or a complementary sequence of the nucleotide sequence encoding the TCR described in the first aspect of the present invention or the multivalent TCR complex described in the second aspect of the present invention.

[0128] The fourth aspect of the present invention provides a vector, wherein the vector contains the nucleic acid molecule described in the third aspect of the present invention.

[0129] The fifth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the fourth aspect of the present invention, or the exogenous nucleic acid molecule described in the third aspect of the present invention is integrated into the chromosome of the host cell.

[0130] The sixth aspect of the present invention provides an isolated cell, wherein the isolated cell expresses the TCR described in the first aspect of the present invention.

[0131] Preferably, the isolated cells comprise T cells, NK cells or NKT cells.

[0132] Preferably, the isolated cells are T cells.

[0133] In another preferred embodiment, the cell expresses the TCR described in the first aspect of the present invention and also expresses exogenous CD8 receptor.

[0134] Preferably, the CD8 receptor is CD8α.

[0135] The seventh aspect of the present invention provides a pharmaceutical composition, which comprises any one or a combination of at least two of the TCR described in the first aspect of the present invention, the multivalent TCR complex described in the second aspect of the present invention, or the isolated cells described in the sixth aspect of the present invention.

[0136] The eighth aspect of the present invention provides a method for treating a disease, comprising administering an appropriate amount of the TCR described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, or the cell described in the sixth aspect of the present invention, or the pharmaceutical composition described in the seventh aspect of the present invention to a subject in need of treatment. Preferably, the disease is an SSX2-positive tumor, more preferably, malignant melanoma, liver cancer, human bile duct epithelial carcinoma cells, human thyroid duct carcinoma, osteosarcoma, synovial sarcoma, multiple myeloma, colon adenocarcinoma, breast cancer, etc.

[0137] The ninth aspect of the present invention provides the use of the TCR described in the first aspect of the present invention, the multivalent TCR complex described in the second aspect of the present invention, or the isolated cell described in the sixth aspect of the present invention in preparing a specific binding reagent for a target protein.

[0138] In another preferred embodiment, the specific binding agent for the target protein is a diagnostic agent and a therapeutic agent for tumors expressing the target protein. Preferably, the therapeutic agent is for SSX2-positive tumors.

[0139] The tenth aspect of the present invention provides the use of the TCR described in the first aspect of the present invention, the multivalent TCR complex described in the second aspect of the present invention, or the isolated cells described in the sixth aspect of the present invention, wherein the use includes use in preparing a drug for treating tumors.

[0140] Preferably, the tumor includes SSX2-positive tumors, more preferably, malignant melanoma, liver cancer, human bile duct epithelial carcinoma cells, human thyroid duct carcinoma, osteosarcoma, melanoma, synovial sarcoma, multiple myeloma, colon adenocarcinoma, breast cancer and the like.

[0141] In an eleventh aspect of the present invention, there is provided a method for preparing the TCR according to the first aspect of the present invention, the preparation method comprising the following steps:

[0142] (i) culturing the host cell according to the fifth aspect of the present invention, wherein the host cell expresses the TCR according to the first aspect of the present invention;

[0143] (ii) isolating or purifying the TCR.

[0144] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0145] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0146] Compared with the prior art, the present invention has the following beneficial effects:

[0147] (1) The affinity and / or binding half-life of the high-affinity TCR of the present invention for the AQIPEKIQK-HLA A1101 complex is at least 4 times that of the wild-type TCR.

[0148] (2) The high-affinity TCR of the present invention can specifically bind to the AQIPEKIQK-HLA A1101, and cells transfected with the high-affinity TCR of the present invention can be specifically activated.

[0149] (3) Effector cells transfected with the high-affinity TCR of the present invention have a strong specific killing effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0150] Figure 1 is the binding curve of the soluble reference TCR, i.e., wild-type TCR, and the AQIPEKIQK-HLA A1101 complex;

[0151] Figure 2a , Figure 2b They are respectively the activation function experimental results of T2 cells loaded with the short peptide AQIPEKIQK and effector cells transfected with the high-affinity TCR of the present invention;

[0152] Figure 3a , Figure 3b The results of the activation function experiments of effector cells transfected with the high-affinity TCR of the present invention against tumor cell lines;

[0153] Figure 4a , Figure 4b , Figure 4c and Figure 4d The results of the LDH experiment on the killing function of effector cells transfected with the high-affinity TCR of the present invention against tumor cell lines;

[0154] Figure 5 The results of IFN-γ release on positive tumor cell lines and human normal tissue cells were detected by ELISpot. DETAILED DESCRIPTION

[0155] Through extensive and in-depth research, the present invention has obtained a high-affinity T cell receptor (TCR) that recognizes the AQIPEKIQK short peptide (derived from the SSX2 protein), and the AQIPEKIQK short peptide is presented in the form of a peptide-HLA A1101 complex. The high-affinity TCR has three CDR regions in its α chain variable domain:

[0156] CDR1α:TSDQSYG SEQ ID NO:41;

[0157] CDR2α: QGSYDEQN SEQ ID NO: 42;

[0158] CDR3α: AMKPGGSYIPT mutated in SEQ ID NO:43;

[0159] and / or in the three CDR regions of the variable domain of its beta chain:

[0160] CDR1β:MNHNY SEQ ID NO:68;

[0161] CDR2β: SVGAGI SEQ ID NO: 69;

[0162] CDR3β: ASSYSVTEHRGKRNEQF mutated in SEQ ID NO:70;

[0163] Furthermore, after mutation, the affinity and / or binding half-life of the TCR of the present invention for the above-mentioned AQIPEKIQK-HLA A1101 complex is at least 4 times that of the wild-type TCR.

[0164] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terminology used herein is intended only to describe specific embodiments, and is not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0165] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0166] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0167] the term

[0168] T cell receptor (TCR)

[0169] The International Immunogenetics Information System (IMGT) can be used to describe TCRs. Natural αβ heterodimeric TCRs have α chains and β chains. Broadly speaking, each chain contains a variable region, a connecting region, and a constant region. The β chain usually also contains a short variable region between the variable region and the connecting region, but the variable region is often considered part of the connecting region. The connecting region of the TCR is determined by the unique IMGT TRAJ and TRBJ, and the constant region of the TCR is determined by IMGT TRAC and TRBC.

[0170] Each variable region contains three CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, embedded in the framework sequence. In the IMGT nomenclature, different numbers of TRAV and TRBV refer to different Vα types and Vβ types, respectively. In the IMGT system, the α chain constant domain has the following symbol: TRAC*01, where "TR" represents the T cell receptor gene; "A" represents the α chain gene; C represents the constant region; "*01" represents allele 1. The β chain constant domain has the following symbol: TRBC1*01 or TRBC2*01, where "TR" represents the T cell receptor gene; "B" represents the β chain gene; C represents the constant region; "*01" represents allele 1. The constant region of the α chain is uniquely determined, and in the form of the β chain, there are two possible constant region genes "C1" and "C2". Those skilled in the art can obtain the constant region gene sequences of TCRα and β chains through the public IMGT database.

[0171] The α and β chains of TCR are generally regarded as having two "domains" each, namely a variable domain and a constant domain. The variable domain is composed of a connected variable region and a connecting region. Therefore, in the specification and claims of the present application, "TCRα chain variable domain" refers to the connected TRAV and TRAJ regions, and similarly, "TCRβ chain variable domain" refers to the connected TRBV and TRBD / TRBJ regions. The three CDRs of the TCRα chain variable domain are CDR1α, CDR2α and CDR3α; the three CDRs of the TCRβ chain variable domain are CDR1β, CDR2β and CDR3β. The framework sequence of the TCR variable domain of the present invention can be of mouse or human origin, preferably of human origin. The constant domain of TCR comprises an intracellular portion, a transmembrane region and an extracellular portion.

[0172] In the present invention, the amino acid sequences of the α and β chain variable domains of the wild-type TCR capable of binding to the AQIPEKIQK-HLA A1101 complex are SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The amino acid sequence of the α chain and the amino acid sequence of the β chain of the soluble "reference TCR" described in the present invention are SEQ ID NO: 11 and SEQ ID NO: 12, respectively. The extracellular amino acid sequence of the α chain and the extracellular amino acid sequence of the β chain of the "wild-type TCR" described in the present invention are SEQ ID NO: 37 and SEQ ID NO: 38, respectively. The TCR sequence used in the present invention is human. The amino acid sequence of the α chain and the amino acid sequence of the β chain of the "wild-type TCR" described in the present invention are SEQ ID NO: 39 and SEQ ID NO: 40, respectively. In the present invention, the terms "polypeptide of the present invention", "TCR of the present invention", and "T cell receptor of the present invention" are used interchangeably.

[0173] SEQ ID NO:37:

[0174] AQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMKPGGSYIPTF GRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS.

[0175] SEQ ID NO:38:

[0176] NAGVTQTPKFRILKIGQSMTLQCTQDMNHNYMYWYRQDPGMGLKLIYYSVGAGITDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVYFCASSYSVTEHRGKRNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD。

[0177] SEQ ID NO:39:

[0178] AQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMKPGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS。

[0179] SEQ ID NO:40:

[0180] NAGVTQTPKFRILKIGQSMTLQCTQDMNHNYMYWYRQDPGMGLKLIYYSVGAGITDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVYFCASSYSVTEHRGKRNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG。

[0181] Native interchain disulfide bonds and artificial interchain disulfide bonds

[0182] There is a set of disulfide bonds between the Cα and Cβ chains in the membrane-proximal region of the natural TCR, which are referred to as "natural interchain disulfide bonds" in the present invention. In the present invention, the artificially introduced interchain covalent disulfide bonds whose positions are different from those of the natural interchain disulfide bonds are referred to as "artificial interchain disulfide bonds".

[0183] For the convenience of description, the position numbers of the amino acid sequences of TRAC*01 and TRBC1*01 or TRBC2*01 in the present invention are numbered in order from the N-terminus to the C-terminus. For example, in TRBC1*01 or TRBC2*01, the 60th amino acid in the order from the N-terminus to the C-terminus is P (proline), then in the present invention it can be described as Pro60 of exon 1 of TRBC1*01 or TRBC2*01, or it can be expressed as the 60th amino acid in exon 1 of TRBC1*01 or TRBC2*01. For example, in TRBC1*01 or TRBC2*01, the 61st amino acid in the order from the N-terminus to the C-terminus is Q (glutamine), then in the present invention it can be described as Gln61 of exon 1 of TRBC1*01 or TRBC2*01, or it can be expressed as the 61st amino acid in exon 1 of TRBC1*01 or TRBC2*01, and so on. In the present invention, the position numbers of the amino acid sequences of variable regions TRAV and TRBV follow the position numbers listed in IMGT. For example, if a certain amino acid in TRAV is numbered 46 in IMGT, it will be described as the 46th amino acid in TRAV in the present invention, and so on. In the present invention, if there are special instructions for the sequence position numbers of other amino acids, they will be followed according to the special instructions.

[0184] Tumor

[0185] The term "tumor" refers to all types of cancer cell growth or carcinogenic processes, metastatic tissues or malignant transformed cells, tissues or organs, regardless of pathological type or stage of infection. Examples of tumors include, but are not limited to: solid tumors, soft tissue tumors, and metastatic lesions. Examples of solid tumors include: malignant tumors of different organ systems, such as sarcomas, squamous cell carcinomas of the lung and carcinomas. For example: infected prostate, lung, breast, lymph, gastrointestinal (e.g., colon), and genitourinary tract (e.g., kidney, epithelial cells), pharynx. DETAILED DESCRIPTION OF THE INVENTION

[0187] As is known to all, the α chain variable domain and β chain variable domain of TCR each contain 3 CDRs, similar to the complementary determining regions of antibodies. CDR3 interacts with the antigenic short peptide, and CDR1 and CDR2 interact with HLA. Therefore, the CDR of the TCR molecule determines its interaction with the antigenic short peptide-HLA complex. The amino acid sequence of the α chain variable domain and the amino acid sequence of the β chain variable domain of the wild-type TCR capable of binding the antigenic short peptide AQIPEKIQK-HLA A1101 complex (i.e., AQIPEKIQK-HLA A1101 complex) are SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and the sequences are first discovered by the inventors. The TCR has the following CDR regions:

[0188] α chain variable domain CDR:

[0189] CDR1α:TSDQSYG SEQ ID NO:41;

[0190] CDR2α: QGSYDEQN SEQ ID NO: 42;

[0191] CDR3α: AMKPGGSYIPT SEQ ID NO: 43;

[0192] and β chain variable domain CDRs:

[0193] CDR1β:MNHNY SEQ ID NO:68;

[0194] CDR2β: SVGAG SEQ ID NO: 69;

[0195] CDR3β: ASSYSVTEHRGKRNEQ SEQ ID NO:70.

[0196] The present invention obtains a high-affinity TCR whose affinity to the AQIPEKIQK-HLA A1101 complex is at least 4 times that of the wild-type TCR to the AQIPEKIQK-HLA A1101 complex by performing mutation screening on the above-mentioned CDR region.

[0197] Further, the TCR of the present invention is an αβ heterodimeric TCR, and the α chain variable domain of the TCR comprises an amino acid sequence having at least 85% (for example, at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, etc.) sequence homology with the amino acid sequence shown in SEQ ID NO: 1; preferably, an amino acid sequence having at least 90% sequence homology; more preferably, an amino acid sequence having at least 92% sequence homology; more preferably, an amino acid sequence having at least 94% sequence homology; and / or the β chain variable domain of the TCR comprises an amino acid sequence having at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO: 1. The amino acid sequence shown in NO:2 has an amino acid sequence with at least 90% (for example, it can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, etc.) sequence identity; preferably, an amino acid sequence with at least 92% sequence identity; more preferably, an amino acid sequence with at least 94% sequence identity.

[0198] Further, the TCR of the present invention is a single-chain TCR, and the α chain variable domain of the TCR comprises an amino acid sequence having at least 85% (e.g., at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, etc.) sequence homology with the amino acid sequence shown in SEQ ID NO: 3; preferably, an amino acid sequence having at least 90% sequence homology; more preferably, an amino acid sequence having at least 92% sequence homology; most preferably, an amino acid sequence having at least 94% sequence homology; and / or the β chain variable domain of the TCR comprises an amino acid sequence having at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO: 3. The amino acid sequence shown in NO:4 has an amino acid sequence with at least 85% (for example, it can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% etc.) sequence identity; preferably, an amino acid sequence with at least 90% sequence identity; more preferably, an amino acid sequence with at least 92% sequence identity; most preferably, an amino acid sequence with at least 94% sequence identity.

[0199] In the present invention, the three CDRs of the wild-type TCR α chain variable domain SEQ ID NO: 1, namely CDR1, CDR2 and CDR3, are located at positions 27-33, 51-58 and 93-103 of SEQ ID NO: 1, respectively. Accordingly, the amino acid residues are numbered as shown in SEQ ID NO: 1, 28S is the 2nd S of CDR1α, 30Q is the 4th Q of CDR1α, 31S is the 5th S of CDR1α, 32Y is the 6th Y of CDR1α, 33G is the 7th G of CDR1α, 52G is the 2nd G of CDR2α, 93A is the 1st A of CDR3α, 94M is the 2nd M of CDR3α, 96P is the 4th P of CDR3α, 99S is the 7th S of CDR3α, 101I is the 9th I of CDR3α, and 102P is the 10th P of CDR3α.

[0200] Specifically, the specific forms of the mutations in the α chain variable domain include one or more of A93G, M94I, P96N, S99A, I101W, P102L, G52S, G33S, Y32F, S31Q / A, Q30T / I / L, and S28T.

[0201] In the present invention, the three CDRs of the wild-type TCRβ chain variable domain SEQ ID NO: 2, namely CDR1, CDR2 and CDR3, are located at positions 27-31, 49-54 and 92-108 of SEQ ID NO: 2, respectively. Accordingly, the amino acid residue numbering adopts the numbering shown in SEQ ID NO: 2, 108F is the 17th F of CDR3β, 107Q is the 16th Q of CDR3β, 106E is the 15th E of CDR3β, 105N is the 14th N of CDR3β, 104R is the 13th R of CDR3β, 103K is the 12th K of CDR3β, 102G is the 11th G of CDR3β, 101R is the 15th E of CDR3β, 105N is the 14th N of CDR3β, 104R is the 13th R of CDR3β, 103K is the 12th K of CDR3β, 102G is the 11th G of CDR3β, and 101R is the 15th E of CDR3β. The 10th R, 100H is the 9th H of CDR3β, 99E is the 8th E of CDR3β, 98T is the 7th T of CDR3β, 54I is the 6th I of CDR2β, 53G is the 5th G of CDR2β, 52A is the 4th A of CDR2β, 30N is the 4th N of CDR1β, 28N is the 2nd N of CDR1β, and 27M is the 1st M of CDR3β.

[0202] Specifically, the specific forms of the mutations in the β chain variable domain include one or more groups of F108K, Q107L, E106P, N105Y, R104G / T, K103G, G102I / T, R101W, H100S / P, E99T, T98S, I54V, G53M / K / D / L, A52K / R / Q, N30R, N28S, and M27H.

[0203] It should be understood that the amino acid names herein are identified by internationally accepted single-letter English letters, and the corresponding three-letter abbreviations of the amino acid names are: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), and Val (V).

[0204] In the present invention, Pro60 or 60P both represent proline at position 60. In addition, the specific form of the mutation in the present invention is expressed as "S31Q / A", which means that S at position 31 is replaced by Q or A, and the same applies to the others.

[0205] According to the site-directed mutagenesis method well known to those skilled in the art, Thr48 of exon 1 of the wild-type TCR α chain constant region TRAC*01 is mutated to cysteine, and Ser57 of exon 1 of the β chain constant region TRBC1*01 or TRBC2*01 is mutated to cysteine, that is, a reference TCR is obtained, and the amino acid sequences of the reference TCR are SEQ ID NO: 11 and SEQ ID NO: 12, respectively, and the mutated cysteine ​​residues are represented by bold letters. The above-mentioned cysteine ​​substitution can form an artificial interchain disulfide bond between the constant regions of the α and β chains of the reference TCR to form a more stable soluble TCR, so that the binding affinity and / or binding half-life between the TCR and the AQIPEKIQK-HLA A1101 complex can be more conveniently evaluated. It should be understood that the CDR region of the TCR variable region determines its affinity with the pMHC complex, and therefore, the above-mentioned cysteine ​​substitution of the TCR constant region does not affect the binding affinity and / or binding half-life of the TCR. Therefore, in the present invention, the binding affinity between the reference TCR and the AQIPEKIQK-HLA A1101 complex is considered to be the binding affinity between the wild-type TCR and the AQIPEKIQK-HLA A1101 complex. Similarly, if the binding affinity between the TCR of the present invention and the AQIPEKIQK-HLA A1101 complex is at least 10 times the binding affinity between the reference TCR and the AQIPEKIQK-HLA A1101 complex, it is equivalent to the binding affinity between the TCR of the present invention and the AQIPEKIQK-HLA A1101 complex being at least 10 times the binding affinity between the wild-type TCR and the AQIPEKIQK-HLA A1101 complex.

[0206] Binding affinity (and dissociation equilibrium constant, K) can be determined by any suitable method. D inversely proportional to the binding half-life (expressed as T 1 / 2 ), such as by surface plasmon resonance. It should be understood that doubling the affinity of a TCR will result in a K D Halved. 1 / 2 Calculated as In2 ​​divided by the dissociation rate (K off ). Therefore, T 1 / 2 Doubling will result in K off The binding affinity or binding half-life of a given TCR is preferably measured several times, for example 3 times or more, using the same test protocol, and the results are averaged. In a preferred embodiment, the affinity of a soluble TCR is measured using the surface plasmon resonance (BIAcore) method described in the examples herein, under the conditions of a temperature of 25°C and a pH of 7.1-7.5. The method detects the dissociation equilibrium constant K of the reference TCR for the AQIPEKIQK-HLAA1101 complex.D is 6.81E-04M, i.e. 681μM. In the present invention, it is considered that the dissociation equilibrium constant K of the wild-type TCR for the AQIPEKIQK-HLA A1101 complex D is also 681 μM. Since doubling the affinity of the TCR will result in a K D So if the dissociation equilibrium constant K of the high affinity TCR for the AQIPEKIQK-HLA A1101 complex is detected D The affinity of the high-affinity TCR for the AQIPEKIQK-HLA A1101 complex is 10 times that of the wild-type TCR for the AQIPEKIQK-HLA A1101 complex. D Conversion relationship between value units, that is, 1M = 10 6 μM, 1 μM=1000 nM, 1 nM=1000 pM. In the present invention, the affinity of the TCR to the AQIPEKIQK-HLA A1101 complex is at least 4 times that of the wild-type TCR.

[0207] Any suitable method may be used to perform mutations, including but not limited to those based on polymerase chain reaction (PCR), cloning based on restriction enzymes, or ligation-independent cloning (LIC) methods. Many standard molecular biology textbooks describe these methods in detail. More details on polymerase chain reaction (PCR) mutagenesis and cloning based on restriction enzymes can be found in Sambrook and Russell, (2001) Molecular Cloning-A Laboratory Manual (3rd Edition) CSHL Press. More information on the LIC method can be found in (Rashtchian, (1995) Curr Opin Biotechnol 6 (1): 30-6).

[0208] The method for producing the TCR of the present invention may be, but is not limited to, screening for a TCR with high affinity for the AQIPEKIQK-HLA A1101 complex from a diverse library of phage particles displaying such TCRs, as described in the literature (Li, et al (2005) Nature Biotech 23(3):349-354).

[0209] It should be understood that genes expressing wild-type TCR α and β chain variable domain amino acids or genes expressing slightly modified wild-type TCR α and β chain variable domain amino acids can be used to prepare template TCRs. The changes required to generate the high-affinity TCR of the present invention are then introduced into the DNA encoding the variable domain of the template TCR.

[0210] The high affinity TCR of the present invention comprises an α chain variable domain amino acid sequence of one of SEQ ID NO: 1, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 22-29; and / or the β chain variable domain amino acid sequence of the TCR is one of SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17-21, SEQ ID NO: 30-36. The amino acid sequences of the α chain variable domain and the β chain variable domain of the heterodimeric TCR molecule of the present invention are preferably selected from the following Table 4:

[0211] Table 4

[0212] TCR Number α chain variable domain sequence β chain variable domain sequence 1 SEQ ID NO:1 SEQ ID NO:13 2 SEQ ID NO:14 SEQ ID NO:2 3 SEQ ID NO:1 SEQ ID NO:15 4 SEQ ID NO:16 SEQ ID NO:2 5 SEQ ID NO:1 SEQ ID NO:17 6 SEQ ID NO:1 SEQ ID NO:18 7 SEQ ID NO:1 SEQ ID NO:19 8 SEQ ID NO:1 SEQ ID NO:20 9 SEQ ID NO:1 SEQ ID NO:21 10 SEQ ID NO:22 SEQ ID NO:2 11 SEQ ID NO:23 SEQ ID NO:2 12 SEQ ID NO:24 SEQ ID NO:2 13 SEQ ID NO:25 SEQ ID NO:2 14 SEQ ID NO:26 SEQ ID NO:2 15 SEQ ID NO:27 SEQ ID NO:2 16 SEQ ID NO:28 SEQ ID NO:2 17 SEQ ID NO:29 SEQ ID NO:2 18 SEQ ID NO:1 SEQ ID NO:30 19 SEQ ID NO:1 SEQ ID NO:31 20 SEQ ID NO:1 SEQ ID NO:32 21 SEQ ID NO:1 SEQ ID NO:33 22 SEQ ID NO:1 SEQ ID NO:34 23 SEQ ID NO:1 SEQ ID NO:35 24 SEQ ID NO:1 SEQ ID NO:36

[0213] Mutated residues in SEQ ID NO: 13-SEQ ID NO: 51 are indicated by single underlining.

[0214] SEQ ID NO:13:

[0215] NAGVTQTPKFRILKIGQSMTLQCTQDMNHNYMYWYRQDPGMGLKLIYYSVGAGITDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVYFCASSYSVTEHRGKR YPb FGPGTRLTVL.

[0216] SEQ ID NO:14:

[0217] AQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFC GI K N GGSYIPTFGRGTSLIVHP.

[0218] SEQ ID NO:15:

[0219] NAGVTQTPKFRILKIGQSMTLQCTQDMNHNYMYWYRQDPGMGLKLIYYSVGAGITDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVYFCASSYSVTEHRGKR YPL FFGPGTRLTVL.

[0220] SEQ ID NO:16:

[0221] AQKITQTQPGMFVQEKEAVTLDCTYDTSD QFS LFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMKPGGSYIPTFGRGTSLIVHP。

[0222] SEQ ID NO:17:

[0223] NAGVTQTPKFRILKIGQSMTLQCTQDMNHNYMYWYRQDPGMGLKLIYYSVG KMV TDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVYFCASSYSVTEHRGKRNEQFFGPGTRLTVL。

[0224] SEQ ID NO:18:

[0225] NAGVTQTPKFRILKIGQSMTLQCTQDMNHNYMYWYRQDPGMGLKLIYYSVG KV TDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVYFCASSYSVTEHRGKRNEQFFGPGTRLTVL。

[0226] SEQ ID NO:19:

[0227] NAGVTQTPKFRILKIGQSMTLQCTQDMNHNYMYWYRQDPGMGLKLIYYSVG RDV TDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVYFCASSYSVTEHRGKRNEQFFGPGTRLTVL。

[0228] SEQ ID NO:20:

[0229] NAGVTQTPKFRILKIGQSMTLQCTQDMNHNYMYWYRQDPGMGLKLIYYSVG KLV TDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVYFCASSYSVTEHRGKRNEQFFGPGTRLTVL。

[0230] SEQ ID NO:21:

[0231] <h2 style=";text-align:left;direction:ltr">NAGVTQTPKFRILKIGQSMTLQCTQDMNHNYMYWYRQDPGMGLKLIYYSVG<h2 style=";text-align:left;direction:ltr"> QUR <h2 style=";text-align:left;direction:ltr"> TDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVYFCASSYSVTEHRGKRNEQFFGPGTRLTVL。<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0232] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:22:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0233] <h2 style=";text-align:left;direction:ltr"> AQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMKPGG<h2 style=";text-align:left;direction:ltr"> A <h2 style=";text-align:left;direction:ltr"> Y<h2 style=";text-align:left;direction:ltr"> WL <h2 style=";text-align:left;direction:ltr"> TFGRGTSLIVHP.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0234] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:23:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0235] <h2 style=";text-align:left;direction:ltr"> AQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCA<h2 style=";text-align:left;direction:ltr"> I <h2 style=";text-align:left;direction:ltr"> K<h2 style=";text-align:left;direction:ltr"> N <h2 style=";text-align:left;direction:ltr"> GGSYIPTFGRGTSLIVHP.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0236] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:24:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0237] <h2 style=";text-align:left;direction:ltr"> AQKITQTQPGMFVQEKEAVTLDCTYDTSD<h2 style=";text-align:left;direction:ltr"> I <h2 style=";text-align:left;direction:ltr"> SY<h2 style=";text-align:left;direction:ltr"> S <h2 style=";text-align:left;direction:ltr"> LFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMKPGGSYIPTFGRGTSLIVHP。<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0238] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:25:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0239] <h2 style=";text-align:left;direction:ltr"> AQKITQTQPGMFVQEKEAVTLDCTYDTSD<h2 style=";text-align:left;direction:ltr"> LA <h2 style=";text-align:left;direction:ltr"> Y<h2 style=";text-align:left;direction:ltr"> S <h2 style=";text-align:left;direction:ltr"> LFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMKPGGSYIPTFGRGTSLIVHP。<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0240] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:26:<h2 style=";text-align:left;direction:ltr">

[0241] AQKITQTQPGMFVQEKEAVTLDCTYDTSD L SY S LFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMKPGGSYIPTFGRGTSLIVHP。

[0242] SEQ ID NO:27:

[0243] AQKITQTQPGMFVQEKEAVTLDCTYDTSD T S FS LFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMKPGGSYIPTFGRGTSLIVHP。

[0244] SEQ ID NO:28:

[0245] AQKITQTQPGMFVQEKEAVTLDCTYDT T D L SYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMKPGGSYIPTFGRGTSLIVHP。

[0246] SEQ ID NO:29:

[0247] AQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQ S SYDEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMKPGGSYIPTFGRGTSLIVHP。

[0248] SEQ ID NO:30:

[0249] NAGVTQTPKFRILKIGQSMTLQCTQDMNHNYMYWYRQDPGMGLKLIYYSVGAGITDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVYFCASSYSV STSW GKRNEQFFGPGTRLTVL。

[0250] SEQ ID NO:31:

[0251] NAGVTQTPKFRILKIGQSMTLQCTQDMNHNYMYWYRQDPGMGLKLIYYSVGAGITDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVYFCASSYSVTEHRGK GYPL FFGPGTRLTVL。

[0252] SEQ ID NO:32:

[0253] NAGVTQTPKFRILKIGQSMTLQCTQDMNHNYMYWYRQDPGMGLKLIYYSVGAGITDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVYFCASSYSVTEHRGK G JZ FGPGTRLTVL。

[0254] SEQ ID NO:33:

[0255] NAGVTQTPKFRILKIGQSMTLQCTQDMNHNYMYWYRQDPGMGLKLIYYSVGAGITDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVYFCASSYSVTEHRGK TYPL FFGPGTRLTVL。

[0256] SEQ ID NO:34:

[0257] NAGVTQTPKFRILKIGQSMTLQCTQDMNHNYMYWYRQDPGMGLKLIYYSVGAGITDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVYFCASSYSVTE P R IG RNEQFFGPGTRLTVL。

[0258] SEQ ID NO:35:

[0259] NAGVTQTPKFRILKIGQSMTLQCTQDMNHNYMYWYRQDPGMGLKLIYYSVGAGITDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVYFCASSYSVTE P R TG RNEQFFGPGTRLTVL。

[0260] SEQ ID NO:36:

[0261] NAGVTQTPKFRILKIGQSMTLQCTQD HS H R YMYWYRQDPGMGLKLIYYSVGAGITDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVYFCASSYSVTEHRGKRNEQFFGPGTRLTVL.

[0262] For the purposes of the present invention, the TCR of the present invention is a part having at least one TCRα and / or TCRβ chain variable domain. They generally contain both TCRα chain variable domain and TCRβ chain variable domain. They can be αβ heterodimers or single chain forms or any other form that can be stably present. In adoptive immunotherapy, the full-length chain of αβ heterodimeric TCR (including cytoplasmic and transmembrane domains) can be transfected. The TCR of the present invention can be used as a targeting agent for delivering therapeutic agents to antigen presenting cells or in combination with other molecules to prepare bifunctional polypeptides to direct effector cells, and at this time, the TCR is preferably in a soluble form.

[0263] For stability, the prior art discloses that the introduction of an artificial interchain disulfide bond between the α and β chain constant domains of the TCR can obtain a soluble and stable TCR molecule, as described in patent document PCT / CN2015 / 093806. Therefore, the TCR of the present invention may be a TCR that introduces an artificial interchain disulfide bond between the residues of its α and β chain constant domains. Cysteine ​​residues form an artificial interchain disulfide bond between the α and β chain constant domains of the TCR. Cysteine ​​residues can replace other amino acid residues at suitable sites in natural TCRs to form artificial interchain disulfide bonds. For example, replacing Thr48 of TRAC*01 exon 1 and replacing Ser57 of TRBC1*01 or TRBC2*01 exon 1 to form a disulfide bond. Other sites for introducing cysteine ​​residues to form disulfide bonds may also be:

[0264] Thr45 of exon 1 of TRAC*01 and Ser77 of exon 1 of TRBC1*01 or TRBC2*01;

[0265] Tyr10 of exon 1 of TRAC*01 and Ser17 of exon 1 of TRBC1*01 or TRBC2*01;

[0266] Thr45 of exon 1 of TRAC*01 and Asp59 of exon 1 of TRBC1*01 or TRBC2*01;

[0267] Ser15 of exon 1 of TRAC*01 and Glu15 of exon 1 of TRBC1*01 or TRBC2*01;

[0268] Arg53 of exon 1 of TRAC*01 and Ser54 of exon 1 of TRBC1*01 or TRBC2*01;

[0269] Pro89 of exon 1 of TRAC*01 and Ala19 of exon 1 of TRBC1*01 or TRBC2*01;

[0270] or Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01.

[0271] That is, cysteine ​​residues replace any of the above-mentioned α and β chain constant domains. Up to 15, or up to 10, or up to 8 or fewer amino acids can be truncated at one or more C-termini of the TCR constant domain of the present invention so that it does not include cysteine ​​residues to achieve the purpose of missing the natural interchain disulfide bond, and the above purpose can also be achieved by mutating the cysteine ​​residues that form the natural interchain disulfide bond to another amino acid.

[0272] As described above, the TCR of the present invention may contain artificial interchain disulfide bonds introduced between residues of the constant domains of its α and β chains. It should be noted that the TCR of the present invention may contain a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, with or without the artificial disulfide bonds introduced as described above between the constant domains. The TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the TCR may be connected by a natural interchain disulfide bond present in the TCR.

[0273] In addition, regarding stability, patent document PCT / CN2016 / 077680 also discloses that the introduction of an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR can significantly improve the stability of the TCR. Therefore, the α chain variable region and the β chain constant region of the high-affinity TCR of the present invention may also contain an artificial interchain disulfide bond. Specifically, the cysteine ​​residue that forms the artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR is replaced by:

[0274] amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01;

[0275] amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01;

[0276] amino acid 46 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01;

[0277] or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01.

[0278] Preferably, the TCR may comprise (i) all or part of the TCR α chain excluding its transmembrane domain, and (ii) all or part of the TCR β chain excluding its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least a portion of the constant domain of the TCR chain, and the α chain and the β chain form a heterodimer.

[0279] More preferably, the TCR may comprise an α chain variable domain and a β chain variable domain and all or part of the β chain constant domain excluding the transmembrane domain, but does not comprise an α chain constant domain, and the α chain variable domain of the TCR forms a heterodimer with the β chain.

[0280] For stability, on the other hand, the TCR of the present invention also includes a TCR that mutates in its hydrophobic core region, and the mutations in these hydrophobic core regions are preferably mutations that can improve the stability of the TCR of the present invention, as described in the patent document with publication number WO2014 / 206304. The TCR may mutate at the following variable domain hydrophobic core positions: (α and / or β chain) variable region amino acids 11, 13, 19, 21, 53, 76, 89, 91, 94, and / or α chain J gene (TRAJ) short peptide amino acid position penultimate 3, 5, 7, and / or β chain J gene (TRBJ) short peptide amino acid position penultimate 2, 4, 6, wherein the position numbering of the amino acid sequence is according to the position numbering listed in the International Immunogenetics Information System (IMGT). Those skilled in the art are aware of the above-mentioned International Immunogenetics Information System, and can obtain the position numbering of the amino acid residues of different TCRs in IMGT according to the database.

[0281] More specifically, the TCR in which the hydrophobic core region is mutated in the present invention can be a highly stable single-chain TCR composed of a flexible peptide chain connecting the variable domains of the α chain and the β chain of the TCR. The CDR region of the TCR variable region determines its affinity with the short peptide-HLA complex. The mutation of the hydrophobic core can make the TCR more stable, but it does not affect its affinity with the short peptide-HLA complex. It should be noted that the flexible peptide chain in the present invention can be any peptide chain suitable for connecting the variable domains of the TCR α and β chains. The template chain for screening high-affinity TCRs constructed in Example 1 of the present invention is the above-mentioned highly stable single-chain TCR containing a hydrophobic core mutation. Using a TCR with higher stability, it is more convenient to evaluate the affinity between the TCR and the AQIPEKIQK-HLA A1101 complex.

[0282] The CDR regions of the α chain variable domain and the β chain variable domain of the single-chain template TCR are exactly the same as the CDR regions of the wild-type TCR. That is, the three CDRs of the α chain variable domain are CDR1α: TSDQSYG, CDR2α: QGSYDEQN, CDR3α: AMKPGGSYIPT, and the three CDRs of the β chain variable domain are CDR1β: MNHNY, CDR2β: SVGAGI, CDR3β: ASSYSVTEHRGKRNEQF. The amino acid sequence of the single-chain template TCR is shown in SEQ ID NO: 9, and the nucleotide sequence of the single-chain template TCR is shown in SEQ ID NO: 10. In this way, a single-chain TCR composed of an α chain variable domain and a β chain variable domain with high affinity for the AQIPEKIQK-HLAA1101 complex is screened out.

[0283] The αβ heterodimer with high affinity for the AQIPEKIQK-HLA A1101 complex of the present invention is obtained by transferring the CDR regions of the α and β chain variable domains of the screened high-affinity single-chain TCR to the corresponding positions of the wild-type TCR α chain variable domain (SEQ ID NO: 1) and β chain variable domain (SEQ ID NO: 2).

[0284] The TCR of the present invention can also be provided in the form of a multivalent complex. The multivalent TCR complex of the present invention comprises a polymer formed by combining two, three, four or more TCRs of the present invention, such as a tetramerization domain of p53 can be used to produce a tetramer, or a complex formed by combining multiple TCRs of the present invention with another molecule. The TCR complex of the present invention can be used to track or target cells presenting specific antigens in vitro or in vivo, and can also be used to produce intermediates of other multivalent TCR complexes with such applications.

[0285] The TCR of the present invention can be used alone or in combination with a conjugate, preferably covalently, wherein the conjugate includes a detectable marker (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the AQIPEKIQK-HLA A1101 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these substances.

[0286] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0287] Therapeutic agents that can be combined or coupled to the TCR of the present invention include, but are not limited to:

[0288] 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539);

[0289] 2. Biological toxins (Chaudhary et al., 1989, Nature 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy 51, 565);

[0290] 3. Cytokines such as IL-2 (Gillies et al., 1992, Proceedings of the National Academy of Sciences (PNAS) 89, 1428; Card et al., 2004, Cancer Immunology and Immunotherapy 53, 345; Halin et al., 2003, Cancer Research 63, 3202);

[0291] 4. Antibody Fc fragment (Mosquera et al., 2005, The Journal of Immunology 174, 4381);

[0292] 5. Antibody scFv fragment (Zhu et al., 1995, International Journal of Cancer 62, 319);

[0293] 6. Gold nanoparticles / nanorods (Lapotko et al., 2005, Cancer letters 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115);

[0294] 7. Virus particles (Peng et al., 2004, Gene therapy 11, 1234);

[0295] 8. Liposomes (Mamot et al., 2005, Cancer Research 65, 11631);

[0296] 9.Nanomagnetic particles;

[0297] 10. Prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL));

[0298] 11. Chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, etc.

[0299] The antibodies or fragments thereof bound to the TCR of the present invention include anti-T cell or NK-cell determining antibodies, such as anti-CD3 or anti-CD28 or anti-CD16 antibodies, and the binding of the above antibodies or fragments thereof to the TCR can direct effector cells to better target target cells. A preferred embodiment is that the TCR of the present invention is bound to an anti-CD3 antibody or a functional fragment or variant of the anti-CD3 antibody. Specifically, the fusion molecule of the TCR of the present invention and the anti-CD3 single-chain antibody includes a TCR α chain variable domain amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 22-29; and / or the amino acid sequence of the β chain variable domain of the TCR is SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17-21, SEQ ID NO: 30-36.

[0300] The present invention also relates to nucleic acid molecules encoding the TCR of the present invention. The nucleic acid molecules of the present invention can be in the form of DNA or RNA. The DNA can be a coding strand or a non-coding strand. For example, the nucleic acid sequence encoding the TCR of the present invention can be the same as the nucleic acid sequence shown in the present invention or a degenerate variant. To illustrate the meaning of "degenerate variant", as used herein, "degenerate variant" in the present invention refers to a nucleotide sequence that encodes a protein sequence having SEQ ID NO: 3, but is different from the sequence of SEQ ID NO: 5.

[0301] The full-length sequence of the nucleic acid molecule of the present invention or its fragment can usually be obtained by, but not limited to, PCR amplification, recombination or artificial synthesis. At present, the DNA sequence encoding the TCR of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The obtained DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0302] The present invention also relates to vectors comprising the nucleic acid molecules of the present invention, and host cells produced by genetic engineering using the vectors or coding sequences of the present invention.

[0303] The present invention also includes isolated cells, particularly T cells, expressing the TCR of the present invention. There are many methods suitable for transfecting T cells with DNA or RNA encoding the high-affinity TCR of the present invention (e.g., Robbins et al., (2008) J. Immunol. 180: 6116-6131). T cells expressing the high-affinity TCR of the present invention can be used for adoptive immunotherapy. Those skilled in the art will be aware of many suitable methods for adoptive therapy (e.g., Rosenberg et al., (2008) Nat Rev Cancer 8 (4): 299-308).

[0304] The present invention also provides a pharmaceutical composition, which contains a pharmaceutically acceptable carrier and the TCR of the present invention, or the TCR complex of the present invention, or a cell presenting the TCR of the present invention.

[0305] The present invention also provides a method for treating a disease, comprising administering an appropriate amount of the TCR of the present invention, or the TCR complex of the present invention, or a cell presenting the TCR of the present invention, or the pharmaceutical composition of the present invention to a subject in need of treatment.

[0306] In the art, when amino acids with similar or similar properties are substituted, the function of the protein is generally not changed. Adding one or more amino acids at the C-terminus and / or N-terminus generally does not change the structure and function of the protein. Therefore, the TCR of the present invention also includes at most 5, preferably at most 3, more preferably at most 2, and most preferably 1 amino acid (especially amino acids outside the CDR region) of the TCR of the present invention, which is replaced by amino acids with similar or similar properties and can still maintain its functionality.

[0307] The present invention also includes TCRs that are slightly modified from the TCRs of the present invention. Modifications (usually without changing the primary structure) include: chemical derivatization forms of the TCRs of the present invention such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of the TCRs of the present invention or in further processing steps. This modification can be accomplished by exposing the TCR to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylation enzyme). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, or phosphothreonine). It also includes TCRs that have been modified to improve their anti-proteolytic properties or optimize their solubility properties.

[0308] The TCR, TCR complex of the present invention or T cells transfected with the TCR of the present invention may be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier. The TCR, multivalent TCR complex or cell of the present invention is typically provided as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier. The pharmaceutical composition may be in any suitable form (depending on the desired method of administration to the patient). The pharmaceutical composition may be provided in unit dosage form, typically in a sealed container, and may be provided as part of a kit. Such a kit (but not necessarily) includes instructions for use. It may include a plurality of such unit dosage forms.

[0309] In addition, the TCR of the present invention can be used alone or in combination or coupled with other therapeutic agents (such as formulated in the same pharmaceutical composition).

[0310] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent. The term refers to such pharmaceutical carriers: they do not induce the production of antibodies harmful to the individual receiving the composition, and there is no excessive toxicity after administration. These carriers are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991). Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, adjuvants and combinations thereof.

[0311] Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffer substances, etc. may also be present in these carriers.

[0312] Typically, therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid vehicles prior to injection can also be prepared.

[0313] Once formulated, the composition of the present invention can be administered by conventional routes, including (but not limited to): intraocular, intramuscular, intravenous, subcutaneous, intradermal or topical administration, preferably parenteral including subcutaneous, intramuscular or intravenous. The subject to be prevented or treated can be an animal; especially a human.

[0314] When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition can be used according to the usage conditions, preferably, injections, oral preparations, etc. can be cited.

[0315] These pharmaceutical compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and appropriate pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers are occasionally added, and the formulation process can be carried out in a conventional manner according to the dosage form.

[0316] The pharmaceutical composition of the present invention can also be administered in the form of a sustained release agent. For example, the TCR of the present invention can be incorporated into a pill or microcapsule with a sustained release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated. As examples of sustained release polymers, ethylene-vinyl acetate copolymers, polyhydroxymethylacrylate (polyhydrometaacrylate), polyacrylamide, polyvinyl pyrrolidone, methylcellulose, lactic acid polymers or lactic acid-glycolic acid copolymers, etc., preferably biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers can be cited.

[0317] When the pharmaceutical composition of the present invention is used for actual treatment, the TCR or TCR complex of the present invention or the cell presenting the TCR of the present invention as the active ingredient can be reasonably determined based on the weight, age, gender, and symptom severity of each patient to be treated, and the reasonable dosage is ultimately determined by the physician.

[0318] The main advantages of the present invention are:

[0319] (1) The affinity and / or binding half-life of the high-affinity TCR of the present invention for the AQIPEKIQK-HLA A1101 complex is at least 4 times that of the wild-type TCR.

[0320] (2) The high-affinity TCR of the present invention can specifically bind to the AQIPEKIQK-HLA A1101, and cells transfected with the high-affinity TCR of the present invention can be specifically activated.

[0321] (3) Effector cells transfected with the high-affinity TCR of the present invention have a strong specific killing effect.

[0322] The following specific examples further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in (Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (Molecular Cloning-A Laboratory Manual) (3rd Edition) (2001) CSHL Press), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0323] Materials and methods

[0324] Unless otherwise specified, the experimental materials used in the examples of the present invention can be obtained from commercial channels, among which E. coli DH5α was purchased from Tiangen, E. coli BL21 (DE3) was purchased from Tiangen, E. coli Tuner (DE3) was purchased from Novagen, and plasmid pET28a was purchased from Novagen.

[0325] Example 1 Stability of hydrophobic core mutations Generation of single-stranded TCR template chains

[0326] The present invention utilizes the method of site-directed mutagenesis, according to the patent document WO2014 / 206304, to construct a stable single-chain TCR molecule consisting of a flexible short peptide (linker) connecting the TCRα and β chain variable domains, and the amino acid and DNA sequences of the single-chain TCR molecule are shown in SEQ ID NO:9 and SEQ ID NO:10, respectively. And the single-chain TCR molecule is used as a template for screening high-affinity TCR molecules. The amino acid sequence of the α chain variable domain of the template chain is shown in SEQ ID NO:3, and the amino acid sequence of the β chain variable domain is shown in SEQ ID NO:4; the corresponding nucleotide sequences are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively; the amino acid sequence and nucleotide sequence of the flexible short peptide (linker) are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0327] The target gene carrying the template strand was double-digested with NcoⅠ and NotⅠ, and then connected to the pET28a vector double-digested with NcoⅠ and NotⅠ. The ligation product was transformed into E.coli DH5α, coated with LB plates containing kanamycin, and inverted and cultured at 37℃ overnight. Positive clones were selected for PCR screening, and positive recombinants were sequenced. After confirming that the sequence was correct, the recombinant plasmid was extracted and transformed into E.coli BL21 (DE3) for expression.

[0328] SEQ ID NO:3:

[0329] AQKITQTQPGLNVQEGENVTIDCTYDTSDQSYGLFWYKQPSSGEPIFLIYQGSYDEQNATEGRYSLNFQK ARKSANLEISDVQPGDSAMYFCAMKPGGSYIPTFGRGTSLSVHP。

[0330] SEQ ID NO:4:

[0331] NAGVTQTPKYLSVKTGQSVTLQCTQDMNHNYMYWYRQDPGQGLKLIYYSVGAGITDKGEVPNRYNVSRSTTEDFPLRIESVTPSDTAVYFCASSYSVTEHRGKRNEQFFGPGTRLTVD。

[0332] SEQ ID NO:5:

[0333] gctcaaaaaattactcaaacccaaccgggtctgaacgtgcaagagggcgaaaacgttaccattgactgcacctatgacaccagcgatcagagctatggtctgttctgg

[0334] tacaaacaaccgagcagcggcgagccgatctttctgatttatcagggcagctacgatgagcaaaacgcgaccgaaggtcgttacagcctgaacttccagaaggcgc

[0335] gtaaaagcgcgaacctggaaatcagcgacgtgcaaccgggcgatagcgcgatgtatttctgcgcgatgaagccgggtggcagctacattccgacctttggtcgtggtaccagcctgagcgttcatccg。

[0336] SEQ ID NO:6:

[0337] aacgcgggcgtgacccagaccccgaagtatctgagcgtgaaaaccggtcaaagcgttaccctgcagtgcacccaagacatgaaccacaactacatgtattggtacc

[0338] gtcaggacccgggtcaaggcctgaagctgatctactatagcgttggtgcgggcattaccgacaaaggcgaggtgccgaaccgttacaacgttagccgtagcaccac

[0339] cgaggacttcccgctgcgtatcgaaagcgtgaccccgagcgataccgcggtttatttttgcgcgagcagctacagcgtgaccgagcaccgtggtaaacgtaacgaacagttctttggtccgggcacccgtctgaccgttgat。

[0340] SEQ ID NO:7:

[0341] GGGSEGGGSEGGGSEGGGSEGGTG。

[0342] SEQ ID NO:8:

[0343] ggtggcggtagcgagggcggtggcagcgaaggtggcggtagcgagggcggtggcagcgaaggtggcaccggt。

[0344] SEQ ID NO:9:

[0345] AQKITQTQPGLNVQEGENVTIDCTYDTSDQSYGLFWYKQPSSGEPIFLIYQGSYDEQNATEGRYSLNFQKARKSANLEISDVQPGDSAMYFCAMKPGGSYIPTFGRGTSLSVHPGGGSEGGGSEGGGSEGGGSEGGTGNAGVTQTPKYLSVKTGQSVTLQCTQDMNHNYMYWYRQDPGQGLKLIYYSVGAGITDKGEVPNRYNVSRSTTEDFPLRIESVTPSDTAVYFCASSYSVTEHRGKRNEQFFGPGTRLTVD。

[0346] SEQ ID NO:10:

[0347] .

[0348] Example 2 Expression, renaturation and purification of the stable single-chain TCR constructed in Example 1

[0349] All BL21 (DE3) colonies containing the recombinant plasmid pET28a-template chain prepared in Example 1 were inoculated into LB medium containing kanamycin and cultured at 37°C until OD 600The concentration of IPTG was 0.6-0.8, and IPTG was added to a final concentration of 0.5mM. The culture was continued at 37°C for 4h. The cell precipitate was harvested by centrifugation at 5000rpm for 15min, and the cell precipitate was lysed with Bugbuster Master Mix (Merck). The inclusion bodies were recovered by centrifugation at 6000rpm for 15min, and then washed with Bugbuster (Merck) to remove cell debris and membrane components. The inclusion bodies were collected by centrifugation at 6000rpm for 15min. The inclusion bodies were dissolved in a buffer (20mM Tris-HCl pH8.0, 8M urea), and the insoluble matter was removed by high-speed centrifugation. The supernatant was quantified by BCA method and then packaged and stored at -80°C for later use.

[0350] To 5 mg of dissolved single-chain TCR inclusion body protein, add 2.5 mL of buffer (6M Gua-HCl, 50 mM Tris-HCl pH 8.1, 100 mM NaCl, 10 mM EDTA), then add DTT to a final concentration of 10 mM, and treat at 37°C for 30 min. Use a syringe to add the above-treated single-chain TCR to 125 mL of refolding buffer (100 mM Tris-HCl pH 8.1, 0.4 M L-arginine, 5 M urea, 2 mM EDTA, 6.5 mM β-mercapthoethylamine, 1.87 mM Cystamine), stir at 4°C for 10 min, and then load the refolding solution into a cellulose membrane dialysis bag with a cutoff of 4 kDa, place the dialysis bag in 1 L of pre-cooled water, and stir slowly at 4°C overnight. After 17 hours, the dialysate was replaced with 1L pre-cooled buffer (20mM Tris-HCl pH 8.0), and the dialysis was continued for 8 hours at 4°C, and then the dialysate was replaced with the same fresh buffer and the dialysis was continued overnight. After 17 hours, the sample was filtered through a 0.45μm filter membrane, vacuum degassed, and passed through an anion exchange column (HiTrap Q HP, GE Healthcare). The protein was purified using a 0-1M NaCl linear gradient eluent prepared with 20mM Tris-HCl pH 8.0. The collected elution fractions were analyzed by SDS-PAGE, and the fractions containing the single-chain TCR were concentrated and further purified using a gel filtration column (Superdex 75 10 / 300, GE Healthcare). The target fraction was also analyzed by SDS-PAGE.

[0351] The eluted fractions used for BIAcore analysis were further tested for purity by gel filtration. The conditions were: chromatographic column Agilent Bio SEC-3 (300A, ), the mobile phase was 150 mM phosphate buffer, the flow rate was 0.5 mL / min, the column temperature was 25 °C, and the UV detection wavelength was 214 nm.

[0352] Example 3 Binding Characterization

[0353] BIAcore analysis

[0354] The binding activity of TCR molecules to the AQIPEKIQK-HLA A1101 complex was detected using the BIAcore T200 real-time analysis system. Anti-streptavidin antibodies (GenScript) were added to the coupling buffer (10mM sodium acetate buffer, pH4.77), and then the antibodies were passed through the CM5 chip pre-activated with EDC and NHS to fix the antibodies on the chip surface. Finally, the unreacted activated surface was blocked with ethanolamine hydrochloric acid solution to complete the coupling process. The coupling level was about 15000RU. The conditions were: temperature 25°C, pH 7.1-7.5.

[0355] A low concentration of streptavidin was passed through the antibody-coated chip surface, and then the AQIPEKIQK-HLAA1101 complex was passed through the detection channel. The other channel was used as the reference channel, and 0.05mM biotin was passed through the chip at a flow rate of 10μL / min for 2min to block the remaining binding sites of streptavidin. The affinity was determined by single-cycle kinetic analysis. TCR was diluted to several different concentrations with HEPES-EP buffer (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.005% P20 pH 7.4) and passed through the chip surface in sequence at a flow rate of 30μL / min. The binding time for each injection was 120s, and the dissociation was allowed for 600s after the last injection. After each round of measurement, the chip was regenerated with 10mM Gly-HCl at pH 1.75. The kinetic parameters were calculated using BIAcore Evaluation software.

[0356] The preparation process of the above AQIPEKIQK-HLAA1101 complex is as follows:

[0357] a. Purification: Collect 100 mL of E. coli culture induced to express heavy chain or light chain, centrifuge at 4°C and 8000 g for 10 min, wash the cells once with 10 mL PBS, then resuspend the cells with 5 mL BugBuster Master Mix Extraction Reagents (Merck) by vigorous shaking, incubate with rotation at room temperature for 20 min, then centrifuge at 4°C and 6000 g for 15 min, discard the supernatant, and collect the inclusion bodies.

[0358] The inclusion bodies were resuspended in 5 mL BugBuster Master Mix and incubated with rotation at room temperature for 5 min; 30 mL of 10-fold diluted BugBuster was added, mixed, and centrifuged at 4°C and 6000 g for 15 min; the supernatant was discarded, 30 mL of 10-fold diluted BugBuster was added to resuspend the inclusion bodies, mixed, and centrifuged at 4°C and 6000 g for 15 min, repeated twice, 30 mL of 20 mM Tris-HCl pH 8.0 was added to resuspend the inclusion bodies, mixed, and centrifuged at 4°C and 6000 g for 15 min, and finally 20 mM Tris-HCl and 8 M urea were used to dissolve the inclusion bodies, and the purity of the inclusion bodies was detected by SDS-PAGE and the concentration was measured by BCA kit.

[0359] b. Renaturation: The synthetic short peptide AQIPEKIQK (synthesized by Jiangsu GenScript Biotechnology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg / mL. The inclusion bodies of the light chain and heavy chain were dissolved with a solution containing 8M urea, 20mM Tris pH 8.0, and 10mM DTT, and 3M guanidine hydrochloride, 10mM sodium acetate, and 10mM EDTA were added for further denaturation before renaturation. The AQIPEKIQK peptide was added to renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4°C) at 25mg / L (final concentration), and then 20mg / L of light chain and 90mg / L of heavy chain were added in sequence (final concentration, heavy chain was added three times, 8h / time), and renaturation was carried out at 4°C for at least 3 days until completion, and SDS-PAGE was used to detect whether renaturation was successful.

[0360] c. Purification after renaturation: Use 10 volumes of 20mM Tris pH 8.0 for dialysis to replace the renaturation buffer, and replace the buffer at least twice to fully reduce the ionic strength of the solution. After dialysis, filter the protein solution with a 0.45μm cellulose acetate filter and then load it onto a HiTrap Q HP (GE General Electric) anion exchange column (5mL bed volume). Use an Akta purifier (GE General Electric) to elute the protein with a 0-400mM NaCl linear gradient solution prepared with 20mM Tris pH 8.0. pMHC is eluted at about 250mM NaCl. Collect the peak components and detect the purity by SDS-PAGE.

[0361] d. Biotinylation: The purified pMHC molecules were concentrated using Millipore ultrafiltration tubes, and the buffer was replaced with 20 mM Tris pH 8.0. Then, biotinylation reagents 0.05 M Bicine pH 8.3, 10 mM ATP, 10 mM MgOAc, 50 μM D-Biotin, and 100 μg / mL BirA enzyme (GST-BirA) were added. The mixture was incubated at room temperature overnight, and SDS-PAGE was used to detect whether the biotinylation was complete.

[0362] e. Purification of biotinylated complex: The biotinylated pMHC molecules were concentrated to 1 mL using Millipore ultrafiltration tubes, and the biotinylated pMHC was purified by gel filtration chromatography. The Akta purifier (GE General Electric) was used to pre-equilibrate HiPrep with filtered PBS. TM 16 / 60S200 HR column (GE General Electric Company) was loaded with 1 mL of concentrated biotinylated pMHC molecules, and then eluted with PBS at a flow rate of 1 mL / min. The biotinylated pMHC molecules appeared as a single peak eluted at about 55 mL. The protein-containing fractions were combined and concentrated using Millipore ultrafiltration tubes. The protein concentration was determined by the BCA method (Thermo). Protease inhibitor cocktail (Roche) was added and the biotinylated pMHC molecules were aliquoted and stored at -80°C.

[0363] Example 4 Generation of high affinity TCR

[0364] Phage display technology is a means of generating a library of TCR high-affinity variants to screen for high-affinity variants. The TCR phage display and screening method described by Li et al. ((2005) Nature Biotech 23(3):349-354) was applied to the single-chain TCR template in Example 1. A library of high-affinity single-chain TCRs was established by mutating the CDR region of the template chain and panning was performed. After several rounds of panning, the phage libraries all had specific binding to the corresponding antigens, from which monoclones were picked and analyzed. The CDR region combinations of the α and β chains of the screened high-affinity single-chain TCRs are shown in Table 3 below.

[0365] Table 3

[0366]

[0367] The CDR region combination of the α and β chains of the screened high-affinity single-chain TCR, i.e., CDR numbers 1-23 in the above table, are introduced one by one into the corresponding sites of the variable domain of the wild-type TCR, and the affinity with the AQIPEKIQK-HLAA1101 complex is detected by BIAcore. Since the wild-type TCR is an αβ heterodimer, the wild-type TCR introduced with the above CDR combination can also be called a high-affinity αβ heterodimer. The introduction of the high-affinity mutation point in the above CDR region adopts the method of site-directed mutagenesis well known to those skilled in the art. The amino acid sequences of the α chain and β chain variable domains of the above wild-type TCR are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0368] It should be noted that in order to obtain a more stable soluble TCR so as to more conveniently evaluate the binding affinity and / or binding half-life between the TCR and the AQIPEKIQK-HLAA1101 complex, the αβ heterodimeric TCR can be a TCR in which a cysteine ​​residue is introduced into the constant region of each of the α and β chains to form an artificial interchain disulfide bond. In this example, the amino acid sequences of the TCR α and β chains after the introduction of the cysteine ​​residues are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively, and the introduced cysteine ​​residues are represented by bold and double underlined letters.

[0369] SEQ ID NO:11:

[0370] AQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMKPGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFF C SPESS.

[0371] SEQ ID NO:12:

[0372] NAGVTQTPKFRILKIGQSMTLQCTQDMNHNYMYWYRQDPGMGLKLIYYSVGAGITDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVYFCASSYSVTEHRGKRNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSE CEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD.

[0373] The extracellular sequence genes of the TCRα chain and TCRβ chain to be expressed were synthesized and inserted into the expression vector pET28a+ (Novagene) by the standard method described in Molecular Cloning a Laboratory Manual (3rd edition, Sambrook and Russell), and the upstream and downstream cloning sites were Nco I and Not I, respectively. Mutations in the CDR region were introduced by overlap PCR, which is well known to those skilled in the art. The inserted fragments were confirmed to be correct by sequencing.

[0374] Example 5 Expression, renaturation and purification of high affinity TCR

[0375] The expression vectors of TCRα chain and TCRβ chain were transformed into expression bacteria BL21(DE3) by chemical transformation, and the bacteria were grown in LB medium. 600 =0.6, induced with a final concentration of 0.5 mM IPTG, the α and β chains of TCR expressed, the formed inclusion bodies were extracted with BugBuster Mix (Novagene), and repeatedly washed with BugBuster solution. The inclusion bodies were finally dissolved in a solution containing 6 M guanidine hydrochloride, 10 mM dithiothreitol (DTT), 10 mM ethylenediaminetetraacetic acid (EDTA), and 20 mM Tris (pH 8.1).

[0376] The dissolved TCRα chain and TCRβ chain were quickly mixed in a solution containing 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine, and 6.6mMβ-mercapoethylamine (4℃) at a mass ratio of 1:1, with a final concentration of 60mg / mL. After mixing, the solution was dialyzed in 10 volumes of deionized water (4℃). After 12h, the deionized water was replaced with buffer (20mM Tris, pH 8.0) and continued to be dialyzed at 4℃ for 12h. After the dialysis was completed, the solution was filtered through a 0.45μm filter membrane and purified by an anion exchange column (HiTrap Q HP, 5mL, GE Healthcare). The elution peak contained TCRs with successfully refolded α and β dimers, which were confirmed by SDS-PAGE gel. TCR was then further purified by gel filtration chromatography (HiPrep 16 / 60, Sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was greater than 90% as determined by SDS-PAGE, and the concentration was determined by the BCA method.

[0377] Example 6 BIAcore analysis results

[0378] The method described in Example 3 was used to detect the affinity of the wild-type TCR introduced with the screened high-affinity single-chain CDR region to the AQIPEKIQK-HLA A1101 complex.

[0379] The amino acid sequences of the novel TCR α chain and TCR β chain variable domains obtained by the present invention are shown in SEQ ID NO: 13-SEQ ID NO: 36. Since the CDR region of the TCR molecule determines its affinity with the corresponding pMHC complex, those skilled in the art can expect that the wild-type TCR introduced with a high-affinity mutation point also has a high affinity for the AQIPEKIQK-HLA A1101 complex. The expression vector was constructed using the method described in Example 4, and the wild-type TCR introduced with a high-affinity mutation was expressed, renatured and purified using the method described in Example 5, and then its affinity with the AQIPEKIQK-HLA A1101 complex was determined using BIAcore T200. Figure 1 is the binding curve of the soluble reference TCR, i.e., the wild-type TCR, and the AQIPEKIQK-HLA A1101 complex. The affinity test results of the high-affinity αβ heterodimeric TCR (wild-type TCR with CDR mutation sites introduced) are shown in Table 5 below:

[0380] Table 5

[0381]

[0382]

[0383] From the results in Table 5, it can be seen that the affinity of the high-affinity TCR obtained after introducing CDR mutations is at least 4 times that of the wild-type TCR for the AQIPEKIQK-HLA A1101 complex.

[0384] Example 7 Expression, refolding and purification of anti-CD3 antibody fusion with high affinity αβ heterodimeric TCR

[0385] The anti-CD3 single-chain antibody (scFv) is fused with a high-affinity αβ heterodimeric TCR to prepare a fusion molecule. The anti-CD3 scFv is fused with the β chain of TCR, and the TCR β chain can include any of the above-mentioned high-affinity αβ heterodimeric TCR β chain variable domains, and the TCR α chain of the fusion molecule can include any of the above-mentioned high-affinity αβ heterodimeric TCR α chain variable domains. The construction of the fusion molecule expression vector includes the following steps:

[0386] (1) Construction of α chain expression vector: The target gene carrying the α chain of high-affinity αβ heterodimeric TCR was double-digested with NcoⅠ and NotⅠ, and ligated to the pET28a vector double-digested with NcoⅠ and NotⅠ. The ligation product was transformed into E.coli DH5α, spread on an LB plate containing kanamycin, and incubated inverted at 37℃ overnight. Positive clones were selected for PCR screening, and positive recombinants were sequenced. After confirming that the sequence was correct, the recombinant plasmid was extracted and transformed into E.coli Tuner (DE3) for expression.

[0387] (2) Construction of anti-CD3 (scFv)-β chain expression vector: By overlapping PCR, primers were designed to connect the anti-CD3 scFv and high-affinity heterodimeric TCRβ chain genes. The intermediate linker was GGGGS, and the gene fragment of the fusion protein of anti-CD3 scFv and high-affinity heterodimeric TCRβ chain was endowed with restriction endonuclease sites NcoⅠ (CCATGG) and NotⅠ (GCGGCCGC). The PCR amplification product was double-digested with NcoⅠ and NotⅠ, and ligated with the pET28a vector double-digested with NcoⅠ and NotⅠ. The ligation product was transformed into E.coli DH5α competent cells, coated with LB plates containing kanamycin, and inverted and cultured at 37℃ overnight. Positive clones were selected for PCR screening, and positive recombinants were sequenced. After confirming that the sequence was correct, the recombinant plasmid was extracted and transformed into E.coli Tuner (DE3) competent cells for expression.

[0388] (3) Expression, renaturation and purification of fusion protein: The expression plasmids were transformed into E. coli Tuner (DE3) competent cells, coated with LB plates (kanamycin 50 μg / mL) and cultured at 37°C overnight. The next day, the clones were inoculated into 10 mL LB liquid culture medium (kanamycin 50 μg / mL) and cultured for 2-3 h, and then inoculated into 1 L LB culture medium at a volume ratio of 1:100 and continued to culture until OD 600 The pH value was 0.5-0.8, and the final concentration of 1mM IPTG was added to induce the expression of the target protein. After 4 hours of induction, the cells were harvested by centrifugation at 6000rpm for 10min. The cells were washed once with PBS buffer and aliquoted. The cells equivalent to 200mL of bacterial culture were lysed with 5mL BugBuster Master Mix (Merck) and the inclusion bodies were collected by centrifugation at 6000g for 15min. Then 4 detergent washes were performed to remove cell debris and membrane components. Then, the inclusion bodies were washed with a buffer such as PBS to remove detergents and salts. Finally, the inclusion bodies were dissolved with a buffer solution containing 6M guanidine hydrochloride, 10mM dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA), and 20mM Tris pH 8.1, and the inclusion body concentration was determined. After aliquoting, they were placed in a -80°C freezer for storage.

[0389] The dissolved TCR α chain and anti-CD3 (scFv) -β chain were quickly mixed in a solution containing 5 M urea, 0.4 M L-arginine, 20 mM Tris pH 8.1, 3.7 mM cystamine, and 6.6 mM β-mercapoethylamine (4°C) at a mass ratio of 2:5. The final concentrations of the α chain and anti-CD3 (scFv) -β chain were 0.1 mg / mL and 0.25 mg / mL, respectively.

[0390] After mixing, the solution was dialyzed in 10 volumes of deionized water (4°C), and after 12 hours, the deionized water was replaced with a buffer solution (10mM Tris, pH 8.0) and continued to be dialyzed at 4°C for 12 hours. After the dialysis was completed, the solution was filtered through a 0.45μm filter membrane and purified by an anion exchange column (HiTrap QHP 5ml, GE healthcare). The elution peak contained TCRs with successfully renatured TCRα chains and anti-CD3 (scFv)-β chain dimers, which were confirmed by SDS-PAGE gel. The TCR fusion molecule was then further purified by size exclusion chromatography (S-100 16 / 60, GE healthcare) and purified again by an anion exchange column (HiTrap Q HP 5ml, GE healthcare). The purity of the purified TCR fusion molecule was greater than 90% by SDS-PAGE, and the concentration was determined by the BCA method.

[0391] Example 8 Activation function experiment of effector cells transfected with high affinity TCR of the present invention on T2 cells loaded with short peptides

[0392] IFN-γ is a powerful immunomodulatory factor produced by activated T lymphocytes. Therefore, in this example, the ELISPOT assay known to those skilled in the art was used to detect the amount of IFN-γ to verify the activation function and antigen specificity of cells transfected with the high-affinity TCR of the present invention. The high-affinity TCR of the present invention was transfected into CD3 T cells isolated from the blood of healthy volunteers. + T cells were used as effector cells, and CD3 T cells transfected with other TCRs (A6) or wild-type TCRs (WT-TCR) from the same volunteers were used. + T cells were used as controls. The target cells used were T2 cells loaded with the SSX2 antigen peptide AQIPEKIQK, T2 cells loaded with other antigen peptides, or empty T2 cells. The following experiments were conducted in two batches (I) and (II):

[0393] (I) The high-affinity TCRs are TCR1, TCR2, TCR3, TCR10, TCR11, TCR19, TCR20, and TCR22.

[0394] (II) The high-affinity TCRs are TCR4, TCR5, TCR6, TCR7, TCR8, TCR9, and TCR24.

[0395] Perform the following steps: First, prepare the ELISPOT plate and add 2 × 10 target cells. 4 cells / well, effector cells 2×10 3Cells / well (calculated according to the transfection positive rate) were added to the corresponding wells, and then the SSX2 antigen short peptide AQIPEKIQK solution was added to the experimental group, and other antigen short peptide solutions were added to the control group, and the final concentration of the short peptide was 10 -6 M, the blank group was added with an equal volume of culture medium, and two replicate wells were set up. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, wash the plate and perform secondary detection and color development, dry the plate, and then use an immunospot plate reader (ELISPOT READER system, AID20 company) to count the spots formed on the membrane.

[0396] Figure 2a and Figure 2b The results of the activation function experiment of the effector cells transfected with the high-affinity TCR of the present invention against T2 cells loaded with the short peptide AQIPEKIQK.

[0397] Figure 2a and Figure 2b The results showed that for target cells loaded with the SSX2 antigen short peptide AQIPEKIQK, the effector cells transfected with the high-affinity TCR of the present invention exhibited a very obvious activation effect compared with the effector cells transfected with the wild type, while the effector cells transfected with other TCRs were inactive; at the same time, the effector cells transfected with the TCR of the present invention were basically inactive to target cells loaded with other antigen short peptides or empty.

[0398] Example 9 Activation function experiment of effector cells transfected with high affinity TCR of the present invention for tumor cell lines

[0399] In this example, tumor cell lines were used to verify the activation function and specificity of effector cells transfected with the high-affinity TCR of the present invention. The detection was also performed by the ELISPOT assay well known to those skilled in the art. The high-affinity TCR of the present invention was transfected into CD3 T cells isolated from the blood of healthy volunteers. + T cells were used as effector cells, and CD3 T cells transfected with other TCRs (A6) or wild-type TCRs (WT-TCR) from the same volunteers were used. + T cells were used as controls. The tumor cell lines used in the examples were SK-MEL-28, Huh-1, 8305C, SNU423, SK-MEL-1, SNU449, HCC827, COV504, SK-MEL-5, and HUCCT1. Among them, SK-MEL-28, SK-MEL-1, HUCC-T1, SNU449, 8305C, COV504, and SNU423 were purchased from Guangzhou Saiku Biotechnology Co., Ltd., Huh-1 was purchased from Nanjing Kebai Biotechnology Co., Ltd., SK-MEL-5 was purchased from ATCC, and HCC827 was purchased from the Cell Bank of the Chinese Academy of Sciences. The following experiments were carried out in two batches (I) and (II):

[0400] (I) The high affinity TCRs are TCR1, TCR2, TCR3, TCR19, and TCR20. The SSX2 positive tumor cell lines used in this batch are SK-MEL-28-SSX2 (SSX2 overexpression) and Huh-1-A11 (HLA-A1101 overexpression), and the negative cell lines are 8305C, SNU423, SK-MEL-1, SNU449, HCC827, and COV504.

[0401] (II) The high affinity TCRs are TCR4, TCR5, TCR6, TCR7, TCR8, and TCR9. The SSX2 positive tumor cell lines used in this batch are SK-MEL-28-SSX2 (SSX2 overexpression) and Huh-1-A11 (HLA-A1101 overexpression), and the negative cell lines are SK-MEL-1, SK-MEL-5, SNU423, and HUCCT1.

[0402] The following steps were performed: First, prepare the ELISPOT plate. The ELISPOT plate was activated and coated with ethanol at 4°C overnight. On the first day of the experiment, the coating solution was removed, the plate was washed and blocked, and the plate was incubated at room temperature for two hours. The blocking solution was removed and the components of the test were added to the ELISPOT plate: the target cells of batch (I) were 2*10 4 / well, effector cells: 4*10 3 / well (calculated according to the positive rate of transfection), and set up two duplicate wells; the target cells of batch (II) are 2*10 4 / well, effector cells are 2*10 3 100 / well (calculated according to the positive rate of transfection), and set up two duplicate wells. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, wash the plate and perform secondary detection and color development. Dry the plate and count the spots formed on the membrane using an ELISPOT READER system (AID20 company).

[0403] Figure 3a and Figure 3b The results of the activation function experiment of the effector cells transfected with the high-affinity TCR of the present invention against tumor cell lines.

[0404] Figure 3a and Figure 3b The results showed that, for SSX2-positive tumor cell lines, the wild type was inactive, and the effector cells transfected with the high-affinity TCR of the present invention showed a very obvious activation effect compared with the effector cells transfected with the wild type, while the effector cells transfected with other TCRs were inactive; at the same time, the effector cells transfected with the high-affinity TCR of the present invention were basically inactive against the SSX2-negative cell line.

[0405] Example 10 Killing function experiment of effector cells transfected with high affinity TCR of the present invention against tumor cell lines

[0406] Lactate dehydrogenase (LDH) is abundant in the cytoplasm and cannot pass through the cell membrane under normal circumstances. When the cell is damaged or dead, it can be released outside the cell. At this time, the LDH activity in the cell culture medium is proportional to the number of cell deaths. In this example, the release of LDH is also measured by a non-radioactive cytotoxicity experiment well known to those skilled in the art, thereby verifying the killing function of the cells transfected with the TCR of the present invention. In this example, the LDH experiment uses CD3 isolated from the blood of healthy volunteers. + T cells were transfected with the high affinity TCR of the present invention as effector cells, and the CD3 + T cells were used as controls. The tumor cell lines used in the examples were SK-MEL-28, A375, 8305C, SNU423, COV504, HUCC-T1, Huh-1, SK-MEL-1, SK-MEL-5, SKM-1, and TT_THYROID. Among them, SK-MEL-28, SNU423, HUCC-T1, SK-MEL-1, 8305C, COV504, and TT_THYROID were purchased from Guangzhou Saiku Biotechnology Co., Ltd., SK-MEL-5 and A375 were purchased from ATCC, Huh-1 was purchased from Nanjing Kebai Biotechnology Co., Ltd., and SKM-1 was purchased from Guangzhou Weijia Biotechnology Co., Ltd. The following experiments were carried out in three batches (Ⅰ), (Ⅱ), (Ⅲ), and (Ⅳ):

[0407] (I) The high affinity TCRs are TCR1, TCR3, and TCR11. The SSX2 positive tumor cell line used in this batch is SK-MEL-28-SSX2 (SSX2 overexpression), and the negative tumor cell lines are A375, 8305C, SNU423, COV504, and HUCC-T1.

[0408] (II) The high affinity TCRs are TCR4, TCR5, TCR6, TCR7, TCR8, and TCR9. The SSX2 positive tumor cell lines used in this batch are SK-MEL-28-SSX2 (SSX2 overexpression) and Huh-1-A11 (HLA-A1101 overexpression), and the negative tumor cell lines are SK-MEL-1, SK-MEL-5, and SKM-1.

[0409] (III) The high affinity TCRs are TCR2 and TCR20. The SSX2 positive tumor cell lines used in this batch are SK-MEL-28-SSX2 (SSX2 overexpression), HUCC-T1-SSX2 (SSX2 overexpression), Huh-1-A11 (HLA-A1101 overexpression), and the negative tumor cell lines are SNU423 and HUCC-T1.

[0410] (IV) The high affinity TCRs are TCR1, TCR3 T, and CR19. The SSX2 positive tumor cell lines used in this batch are SK-MEL-28-SSX2 (SSX2 overexpression) and TT_THYROID-A1101 (HLA-A1101 overexpression), and the negative tumor cell lines are TT_THYROID, SNU423, and HUCC-T1.

[0411] The following experimental steps were performed: First, prepare the LDH plate and add the components of the test to the plate in the following order: batches (I), (III) and (IV): target cells 3×10 4 cells / well, effector cells 3×10 4 cells / well (calculated according to the transfection positive rate); Batch (II): target cells 3×10 4 cells / well, effector cells 6×10 4 Cells / well (calculated by transfection positive rate) were added to the corresponding wells, and three replicate wells were set. At the same time, effector cell spontaneous wells, target cell spontaneous wells, target cell maximum wells, volume correction control wells and culture medium background control wells were set. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, the color was detected, and the absorbance value was recorded at 490nm using a microplate reader (Bioteck) after the reaction was terminated.

[0412] Figure 4a , Figure 4b , Figure 4c Figure d and Figure d are the results of LDH experiments on the killing function of effector cells transfected with the high-affinity TCR of the present invention against tumor cell lines.

[0413] Figure 4a , Figure 4b , Figure 4c The results in Figure d show that, for SSX2-positive tumor cell lines, effector cells transfected with the high-affinity TCR of the present invention exhibit very obvious strong killing efficacy compared to effector cells transfected with the wild type, while T cells transfected with other TCRs and T cells not transfected with TCR basically do not react. At the same time, T cells transfected with the high-affinity TCR of the present invention have almost no killing effect on negative tumor cell lines.

[0414] Example 11 Verification of the specificity of effector cells transduced with the high affinity TCR of the present invention on normal human tissue cells

[0415] This example uses normal human tissue cells to verify the specificity of effector cells transduced with the high-affinity TCR of the present invention. The detection is also performed by the ELISPOT assay well known to those skilled in the art. The high-affinity TCR (TCR1, TCR2, TCR9) of the present invention is transduced into CD3 T cells isolated from the blood of healthy volunteers via lentivirus. + T cells were used as effector cells and transduced with other TCRs (A6) or empty transduced (NC) or with only target cell CD3 + T cells served as control.

[0416] The positive tumor cell line used in the experiment was Huh-1-A1101, the negative tumor cell line was SNU423, and the human normal tissue cells used in the experiment were: human CD34+ progenitor cells (hCD34+_CB1-45A), human aortic endothelial cells (HAoEC-46A), human aortic endothelial cells (HAoEC-46B), human aortic adventitial fibroblasts (HAoAF-47), human coronary artery endothelial cells (HCAEC-49A), human coronary artery endothelial cells (HCAEC-49B), human coronary artery endothelial cells (HCAEC-49A), human coronary artery endothelial cells (HCAEC-49A), human coronary artery endothelial cells (HCAEC-49B), human aortic adventitial fibroblasts (HAoAF-47), human coronary artery endothelial cells (HCAEC-49A), human coronary artery endothelial cells (HCAEC-49B ...B), human coronary artery endothelial cells (HCAEC-49A), human coronary artery endothelial cells (HCAEC-49B), human coronary artery endothelial cells (HCAEC-49B), human coronary artery endo Human cardiac artery endothelial cells (HCAEC-49B), human cardiac fibroblasts (HCF-51), human cardiac microvascular endothelial cells (HCMEC-52C), human cardiac microvascular endothelial cells (HCMEC-52A), human uterine microvascular endothelial cells (HUtMEC-55A), human uterine microvascular endothelial cells (HUtMEC-55A-A1101), human uterine microvascular endothelial cells (HUtMEC-55B), human uterine fibroblasts (HUF- 56), human uterine smooth muscle cells (HUtSMC-57), human renal cortical epithelial cells (HRCEpiC-58), human renal epithelial cells (HREpC-59), human chondrocytes (HCH-61B-A1101), human uterine smooth muscle cells (HUtSMC-57-A1101), human bronchial smooth muscle cells (HBSMC-71), human bronchial epithelial cells (HBEpC-70A), human bronchial epithelial cells (HBEpC-7 0B), human nasal epithelial cells (HNEPC-74), human nasal epithelial cells (HNEPC-73-A1101), human dermal microvascular endothelial cells (HDMEC-74), human dermal microvascular endothelial cells (HDMEC-74-A1101), human dermal microvascular endothelial cells (HDMEC-75), human dermal fibroblasts (NHDF-76B), human cardiomyocytes (HCM-DonorB), human hepatocytes (HUM183231). The human normal tissue cells used were purchased from ScienCell. The following experiments were carried out in two batches (I) and (II):

[0417] First, prepare the ELISPOT plate. Activate the ELISPOT plate with ethanol and incubate overnight at 4°C. On the first day of the experiment, remove the coating solution, wash and block, incubate at room temperature for two hours, remove the blocking solution, and add the various components of the test to the ELISPOT plate: 2×10 target cells 4 / well, effector cells: 2×10 3 100 / well (calculated according to the positive rate of transduction), and set up two duplicate wells. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, wash the plate and perform secondary detection and color development. Dry the plate and count the spots formed on the membrane using an ELISPOT READER system (AID20 company).

[0418] Figure 5 The results of IFN-γ release on positive tumor cell lines and human normal tissue cells were detected by ELISPOT by effector cells transfected with the high-affinity TCR of the present invention. Figure 5 The results showed that the effector cells transduced with the high-affinity TCR of the present invention significantly activated positive tumor cell lines and had little activity against normal human tissue cells, further verifying the high specificity of the TCR of the present invention.

[0419] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0420] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A T cell receptor (TCR), comprising a TCR α chain variable domain and a TCR β chain variable domain, characterized in that: It has the activity of binding to the AQIPEKIQK-HLA A1101 complex; And the amino acid sequence of the TCRα chain variable domain has at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO:1, and the amino acid sequence of the TCRβ chain variable domain has at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO:

2.

2. A multivalent TCR complex, characterized in that The multivalent TCR complex comprises at least two TCRs, and at least one of the TCRs is the TCR of claim 1.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleic acid sequence encoding the TCR of claim 1 or a complementary sequence thereof.

4. A carrier, characterized in that The vector contains the nucleic acid molecule described in claim 3.

5. A host cell, characterized in that The host cell contains the vector described in claim 4 or the chromosome in which the exogenous nucleic acid molecule described in claim 3 is integrated.

6. An isolated cell, characterized in that The cell expresses the TCR of claim 1, preferably, the cell also expresses an exogenous CD8 receptor; more preferably, the CD8 receptor is CD8α; preferably, the isolated cell is a T cell.

7. A pharmaceutical composition, characterized in that The composition contains a pharmaceutically acceptable carrier and the TCR of claim 1 , or the TCR complex of claim 2 , or the cell of claim 6 .

8. A method for treating a disease, characterized in that: The method comprises administering the TCR of claim 1, the TCR complex of claim 2, the cell of claim 6, or the pharmaceutical composition of claim 7 to a subject in need of treatment, wherein the disease is an SSX2-positive tumor, and more preferably, malignant melanoma, liver cancer, human bile duct epithelial carcinoma cell, human thyroid duct carcinoma, osteosarcoma, melanoma, synovial sarcoma, multiple myeloma, colon adenocarcinoma or breast cancer.

9. Use of the T cell receptor according to claim 1, the TCR complex according to claim 2 or the cell according to claim 6, characterized in that: For preparing a drug for treating tumors, preferably, the disease is an SSX2-positive tumor, more preferably, malignant melanoma, liver cancer, human bile duct epithelial carcinoma cells, human thyroid duct carcinoma, osteosarcoma, melanoma, synovial sarcoma, multiple myeloma, colon adenocarcinoma or breast cancer.

10. A method for preparing the T cell receptor according to claim 1, characterized in that: Includes steps: (i) culturing the cell of claim 5 so as to express the T cell receptor of claim 1; and, (ii) isolating or purifying the T cell receptor.

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