High-affinity TCR targeting magea4

Abstract

Provided is a T cell receptor (TCR). The TCR has the property of binding to a GVYDGREHTV-HLA A0201 complex. Also provided are a multivalent TCR complex, a nucleic acid molecule encoding the TCR, a vector comprising the nucleic acid molecule, a cell expressing the TCR, a pharmaceutical composition comprising the aforementioned substances, and a use thereof in the preparation of a drug for diagnosing, treating and preventing MAGE-A4-positive diseases. Also provided is a preparation method for the TCR.

Description

High-affinity TCR targeting MAGEA4 Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a high-affinity T cell receptor that recognizes MAGE and its application; more specifically, to a T cell receptor (T cell receptor, TCR) that can recognize a polypeptide derived from the MAGE-A4 protein; the present invention also relates to a method for preparing the TCR and its application. 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), a glycoprotein on the cell membrane composed of either α / β or γ / δ chains in a heterodimer. The immune system's repertoire of TCRs is generated in the thymus by V(D)J recombination followed by positive and negative selection. In the peripheral environment, the TCR mediates the specific recognition of T cells against the major histocompatibility complex-peptide complex (pMHC) and is therefore crucial for the immune system's cellular immunity.

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

[0004] The MHC class I and class II ligands corresponding to TCRs are also proteins from the immunoglobulin superfamily, but they are specific for antigen presentation. Different individuals have different MHCs, enabling them to present different short peptides from a protein antigen to the surface of their respective APCs. The human MHC is often referred to as the HLA gene or HLA complex.

[0005] The MAGE family of proteins has homology regions that closely match the sequences of other MAGE proteins and contains peptides that are displayed as HLA / peptide complexes in immune recognition. Some MAGE gene family proteins (MAGE-A to MAGE-C families) are only expressed in germ cells and cancers; others (MAGE-D to MAGE-H) are widely expressed in normal tissues. MAGE-A4, as an endogenous tumor antigen, is produced in cells and degraded into small peptides. It then binds to MHC (major histocompatibility complex) molecules to form a complex that is presented on the cell surface. Studies have shown that GVYDGREHTV is a short peptide derived from MAGE-A4. MAGE-A4 protein is expressed in many tumor types, including melanoma, as well as other solid tumors such as gastric cancer, lung cancer, esophageal cancer, bladder cancer, and head and neck squamous cell carcinoma. For the treatment of the above diseases, chemotherapy and radiotherapy can be used, but both will cause damage to the body's normal cells.

[0006] Therefore, the GVYDGREHTV-HLA A0201 complex provides a TCR-targetable marker for tumor cells. TCRs capable of binding to the GVYDGREHTV-HLA A0201 complex have high therapeutic value in tumor treatment. For example, TCRs capable of targeting this tumor cell marker can be used to deliver cytotoxic or immunostimulatory agents to target cells, or can be transformed into T cells, enabling T cells expressing this TCR to destroy tumor cells, and then administered to patients in a treatment process known as adoptive immunotherapy. For the former purpose, an ideal TCR has high affinity, allowing the TCR to reside long-term on the targeted cells. For the latter purpose, TCRs with intermediate affinity are preferred. Therefore, those skilled in the art are committed to developing TCRs that can be used to target tumor cell markers for various purposes.

[0007] Summary of the Invention

[0008] In view of the shortcomings of the prior art, the present invention aims to provide a TCR with high affinity for the GVYDGREHTV-HLA A0201 complex. Another object of the present invention is to provide a method for preparing the TCR and the use of the TCR.

[0009] To achieve the purpose of this invention, the technical solution of the present invention includes:

[0010] In a first aspect of the present invention, a T cell receptor (TCR), comprising a TCR α chain variable domain and a TCR β chain variable domain, wherein the TCR has an activity of binding to a GVYDGREHTV-HLA A0201 complex;

[0011] The amino acid sequence of the TCRα chain variable domain has at least 90% (for example, 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 TCRβ chain variable domain has at least 90% (for example, 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.

[0012] SEQ ID NO: 1:

[0013] SEQ ID NO: 2:

[0014] 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.

[0015] 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.

[0016] 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.

[0017] In another preferred embodiment, in the variable domain of the TCRβ chain, CDR1β is DFQATT, CDR2β is SNEGSKA, and CDR3β is SAREWRGTEAF.

[0018] In another preferred embodiment, the TCR further has the activity of binding to the GVYDGREHTV-HLA A0201 complex.

[0019] In another preferred embodiment, the three CDRs of the TCRα chain variable domain are:

[0020] CDR1α: VSGLRG SEQ ID NO: 63;

[0021] CDR2α: LYSAGEE SEQ ID NO: 64; and,

[0022] CDR3α:AVHPLNYGGSQGNLI SEQ ID NO:65.

[0023] and contain at least one mutation listed in Table 1:

[0024] Table 1

[0025] In another preferred example, the amino acid sequence of the TCRβ chain variable domain is SEQ ID NO: 2.

[0026] In another preferred embodiment, the affinity of the TCR to the GVYDGREHTV-HLA A0201 complex is at least 2 times that of the wild-type TCR, for example, it can be 2, 2.5, 3, 3.5, 4, 4.5 or 5.

[0027] In another preferred embodiment, the TCR has 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 Y95A / N / H / S / T / D / K / R / W / M / V, G96Q / P / Y / K / T / D / A / N / L / M / R / V / H / S, G97T / P / Q / W / I / F / Y / K / N / L / M / R / H / V / E, S98P / T / I / K / M / R / L / Q / Y / F, Q99E / H / Y / D and G100S / N, wherein the amino acid residues are numbered according to the numbering shown in SEQ ID NO: 1.

[0028] In another preferred embodiment, the TCR has a CDR selected from Table 2:

[0029] Table 2

[0030] The amino acid sequences of SEQ ID NO:66 to SEQ ID NO:111 are shown below:

[0031] SEQ ID NO:66:AVHPLNYGTPQGNLI,

[0032] SEQ ID NO:67:AVHPLNAQPTQGNLI,

[0033] <h2 style=";text-align:left;direction:ltr">SEQ ID NO:68:AVHPLNYGQIESNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0034] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:69:AVHPLNNGWSQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0035] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:70:AVHPLNHPISQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0036] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:71:AVHPLNAYWTQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0037] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:72:AVHPLNSKFIQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0038] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:73:AVHPLNSTWKQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0039] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:74:AVHPLNSKYMQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0040] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:75:AVHPLNTDFMQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0041] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:76:AVHPLNTKKRQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0042] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:77:AVHPLNTAWRQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0043] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:78:AVHPLNTGWRQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0044] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:79:AVHPLNTTFLQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0045] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:80:AVHPLNDNPSQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0046] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:81:AVHPLNKGWLQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0047] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:82:AVHPLNKLFKQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0048] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:83:AVHPLNRMWRQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0049] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:84:AVHPLNRQPQQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0050] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:85:AVHPLNRAWRQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0051] <h2 style=";text-align:left;direction:ltr">SEQ ID NO:86:AVHPLNWKNMQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0052] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:87:AVHPLNMQFQQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0053] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:88:AVHPLNSRLIHGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0054] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:89:AVHPLNYGMPYGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0055] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:90:AVHPLNYGQPDGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0056] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:91:AVHPLNYGRPENNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0057] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:92:AVHPLNKGNSQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0058] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:93:AVHPLNVKNSQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0059] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:94:AVHPLNSQLSQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0060] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:95:AVHPLNYAQTQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0061] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:96:AVHPLNYVNSQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0062] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:97:AVHPLNYAKLQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0063] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:98:AVHPLNYGQSQNNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0064] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:99:AVHPLNNAGYQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0065] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:100:AVHPLNRMGPQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0066] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:101:AVHPLNYLPSQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0067] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:102:AVHPLNYTFSQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0068] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:103:AVHPLNYHHPQGNLI,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0069] SEQ ID NO:104:AVHPLNYKVPQGNLI,

[0070] SEQ ID NO:105:AVHPLNYMKFQGNLI,

[0071] SEQ ID NO:106:AVHPLNYSKRQGNLI,

[0072] SEQ ID NO:107:AVHPLNYDNPQGNLI,

[0073] SEQ ID NO:108:AVHPLNYGEPQGNLI,

[0074] SEQ ID NO:109:AVHPLNYGQPQGNLI,

[0075] SEQ ID NO:110:AVHPLNYGWPQGNLI,

[0076] SEQ ID NO:111:AVHPLNYGKYQGNLI.

[0077] In another preferred embodiment, the TCR is soluble.

[0078] In another preferred embodiment, the TCR is an αβ heterodimeric TCR, and the sequence of the αβ heterodimeric TCR comprises an α chain TRAC constant region sequence and a β chain TRBC1 or TRBC2 constant region sequence.

[0079] In another preferred embodiment, the TCR comprises (i) a TCRα chain variable domain and all or part of the TCRα chain constant region excluding the transmembrane domain; and (ii) a TCRβ chain variable domain and all or part of the TCRβ chain constant region excluding the transmembrane domain.

[0080] In another preferred embodiment, the TCR comprises an α chain constant region and a β chain constant region, and the α chain constant region and the β chain constant region of the TCR contain an artificial interchain disulfide bond; preferably, the cysteine ​​residues forming the artificial interchain disulfide bond between the constant regions of the TCR α and TCR β chains are substituted with one or more sites selected from the following groups:

[0081] Thr48 of exon 1 of TRAC*01 and Ser57 of exon 1 of TRBC1*01 or TRBC2*01;

[0082] Thr45 of exon 1 of TRAC*01 and Ser77 of exon 1 of TRBC1*01 or TRBC2*01;

[0083] Tyr10 of exon 1 of TRAC*01 and Ser17 of exon 1 of TRBC1*01 or TRBC2*01;

[0084] Thr45 of exon 1 of TRAC*01 and Asp59 of exon 1 of TRBC1*01 or TRBC2*01;

[0085] Ser15 of exon 1 of TRAC*01 and Glu15 of exon 1 of TRBC1*01 or TRBC2*01;

[0086] Arg53 of exon 1 of TRAC*01 and Ser54 of exon 1 of TRBC1*01 or TRBC2*01;

[0087] Pro89 of exon 1 of TRAC*01 and Ala19 of exon 1 of TRBC1*01 or TRBC2*01;

[0088] and Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01.

[0089] In another preferred example, the amino acid sequence of the α chain variable domain of the TCR is one of SEQ ID NOs: 13-58; and / or the amino acid sequence of the β chain variable domain of the TCR is SEQ ID NO: 2.

[0090] Preferably, the TCR is selected from the group consisting of:

[0091] (1) the α chain variable domain sequence is SEQ ID NO: 13, and the β chain variable domain sequence is SEQ ID NO: 2,

[0092] (2) the α chain variable domain sequence is SEQ ID NO: 14, and the β chain variable domain sequence is SEQ ID NO: 2,

[0093] (3) the α chain variable domain sequence is SEQ ID NO: 15, and the β chain variable domain sequence is SEQ ID NO: 2,

[0094] (4) the α chain variable domain sequence is SEQ ID NO: 16, and the β chain variable domain sequence is SEQ ID NO: 2,

[0095] (5) the α chain variable domain sequence is SEQ ID NO: 17, and the β chain variable domain sequence is SEQ ID NO: 2,

[0096] (6) the α chain variable domain sequence is SEQ ID NO: 18, and the β chain variable domain sequence is SEQ ID NO: 2,

[0097] (7) the α chain variable domain sequence is SEQ ID NO: 19, and the β chain variable domain sequence is SEQ ID NO: 2,

[0098] (8) the α chain variable domain sequence is SEQ ID NO: 20, and the β chain variable domain sequence is SEQ ID NO: 2,

[0099] (9) the α chain variable domain sequence is SEQ ID NO: 21, and the β chain variable domain sequence is SEQ ID NO: 2,

[0100] (10) the α chain variable domain sequence is SEQ ID NO: 22, and the β chain variable domain sequence is SEQ ID NO: 2,

[0101] (11) the α chain variable domain sequence is SEQ ID NO: 23, and the β chain variable domain sequence is SEQ ID NO: 2,

[0102] (12) the α chain variable domain sequence is SEQ ID NO: 24, and the β chain variable domain sequence is SEQ ID NO: 2,

[0103] (13) the α chain variable domain sequence is SEQ ID NO: 25, and the β chain variable domain sequence is SEQ ID NO: 2,

[0104] (14) the α chain variable domain sequence is SEQ ID NO: 26, and the β chain variable domain sequence is SEQ ID NO: 2,

[0105] (15) the α chain variable domain sequence is SEQ ID NO: 27, and the β chain variable domain sequence is SEQ ID NO: 2,

[0106] (16) The α chain variable domain sequence is SEQ ID NO: 28, and the β chain variable domain sequence is SEQ ID NO: 2,

[0107] (17) The α chain variable domain sequence is SEQ ID NO: 29, and the β chain variable domain sequence is SEQ ID NO: 2,

[0108] (18) The α chain variable domain sequence is SEQ ID NO: 30, and the β chain variable domain sequence is SEQ ID NO: 2,

[0109] (19) The α chain variable domain sequence is SEQ ID NO: 31, and the β chain variable domain sequence is SEQ ID NO: 2,

[0110] (20) The α chain variable domain sequence is SEQ ID NO: 32, and the β chain variable domain sequence is SEQ ID NO: 2,

[0111] (21) The α chain variable domain sequence is SEQ ID NO: 33, and the β chain variable domain sequence is SEQ ID NO: 2,

[0112] (22) The α chain variable domain sequence is SEQ ID NO: 34, and the β chain variable domain sequence is SEQ ID NO: 2,

[0113] (23) The α chain variable domain sequence is SEQ ID NO: 35, and the β chain variable domain sequence is SEQ ID NO: 2,

[0114] (24) The α chain variable domain sequence is SEQ ID NO: 36, and the β chain variable domain sequence is SEQ ID NO: 2,

[0115] (25) The α chain variable domain sequence is SEQ ID NO: 37, and the β chain variable domain sequence is SEQ ID NO: 2,

[0116] (26) The α chain variable domain sequence is SEQ ID NO: 38, and the β chain variable domain sequence is SEQ ID NO: 2,

[0117] (27) The α chain variable domain sequence is SEQ ID NO: 39, and the β chain variable domain sequence is SEQ ID NO: 2,

[0118] (28) The α chain variable domain sequence is SEQ ID NO: 40, and the β chain variable domain sequence is SEQ ID NO: 2,

[0119] (29) The α chain variable domain sequence is SEQ ID NO: 41, and the β chain variable domain sequence is SEQ ID NO: 2,

[0120] (30) The α chain variable domain sequence is SEQ ID NO: 42, and the β chain variable domain sequence is SEQ ID NO: 2,

[0121] (31) The α chain variable domain sequence is SEQ ID NO: 43, and the β chain variable domain sequence is SEQ ID NO: 2,

[0122] (32) The α chain variable domain sequence is SEQ ID NO: 44, and the β chain variable domain sequence is SEQ ID NO: 2,

[0123] (33) The α chain variable domain sequence is SEQ ID NO: 45, and the β chain variable domain sequence is SEQ ID NO: 2,

[0124] (34) The α chain variable domain sequence is SEQ ID NO: 46, and the β chain variable domain sequence is SEQ ID NO: 2,

[0125] (35) The α chain variable domain sequence is SEQ ID NO: 47, and the β chain variable domain sequence is SEQ ID NO: 2,

[0126] (36) The α chain variable domain sequence is SEQ ID NO: 48, and the β chain variable domain sequence is SEQ ID NO: 2,

[0127] (37) The α chain variable domain sequence is SEQ ID NO: 49, and the β chain variable domain sequence is SEQ ID NO: 2,

[0128] (38) The α chain variable domain sequence is SEQ ID NO: 50, and the β chain variable domain sequence is SEQ ID NO: 2,

[0129] (39) The α chain variable domain sequence is SEQ ID NO: 51, and the β chain variable domain sequence is SEQ ID NO: 2,

[0130] (40) The α chain variable domain sequence is SEQ ID NO: 52, and the β chain variable domain sequence is SEQ ID NO: 2,

[0131] (41) The α chain variable domain sequence is SEQ ID NO: 53, and the β chain variable domain sequence is SEQ ID NO: 2,

[0132] (42) The α chain variable domain sequence is SEQ ID NO: 54, and the β chain variable domain sequence is SEQ ID NO: 2,

[0133] (43) The α chain variable domain sequence is SEQ ID NO: 55, and the β chain variable domain sequence is SEQ ID NO: 2,

[0134] (44) The α chain variable domain sequence is SEQ ID NO: 56, and the β chain variable domain sequence is SEQ ID NO: 2,

[0135] (45) The α chain variable domain sequence is SEQ ID NO: 57, and the β chain variable domain sequence is SEQ ID NO: 2,

[0136] (46) The α chain variable domain sequence is SEQ ID NO: 58, and the β chain variable domain sequence is SEQ ID NO: 2.

[0137] In another preferred embodiment, the TCR is of human origin.

[0138] In another preferred embodiment, the TCR is isolated or purified.

[0139] In another preferred embodiment, the TCR is a single-chain TCR.

[0140] Preferably, the TCR is a single-chain TCR consisting 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 segment (linker).

[0141] In another preferred embodiment, 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.

[0142] In another preferred embodiment, 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.

[0143] In another preferred embodiment, a conjugate is bound to the C- or N-terminus of the α chain and / or β chain of the TCR.

[0144] Preferably, the conjugate is a detectable label or a therapeutic agent; more preferably, the therapeutic agent is an anti-CD3 antibody.

[0145] The 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.

[0146] In the third aspect of the present invention, a nucleic acid molecule is provided, the sequence of 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] Preferably, the isolated cells are T cells.

[0151] In another preferred embodiment, the cell expresses the TCR described in the first aspect of the present invention and also expresses an exogenous CD8 receptor.

[0152] Preferably, the CD8 receptor is CD8α; more preferably, the isolated cell is a T cell.

[0153] 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, and the isolated cells described in the sixth aspect of the present invention.

[0154] In an eighth aspect, 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 a MAGE-A4-positive tumor, including melanoma, and other solid tumors such as gastric cancer, lung cancer, esophageal cancer, bladder cancer, head and neck squamous cell carcinoma, etc.

[0155] The ninth aspect of the present invention provides 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.

[0156] 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 MAGE-A4 positive tumors.

[0157] 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 cell described in the sixth aspect of the present invention, wherein the use includes use in preparing a drug for treating tumors.

[0158] Preferably, the tumor includes MAGE-A4 positive tumors, including melanoma, and other solid tumors such as gastric cancer, lung cancer, esophageal cancer, bladder cancer, head and neck squamous cell carcinoma, etc.

[0159] 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 method comprising the following steps:

[0160] (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; and

[0161] (ii) isolating or purifying the TCR.

[0162] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one.

[0163] The numerical range described in the present invention includes not only the point values ​​listed above, but also 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.

[0164] Compared with the traditional technology, the present invention has the following beneficial effects:

[0165] (1) The affinity and / or binding half-life of the high-affinity TCR of the present invention for the GVYDGREHTV-HLA A0201 complex is at least twice that of the wild-type TCR.

[0166] (2) The high-affinity TCR of the present invention can specifically bind to the GVYDGREHTV-HLA A0201, and cells transfected with the high-affinity TCR of the present invention can be specifically activated.

[0167] (3) Effector cells transfected with the high-affinity TCR of the present invention have a strong specific killing effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0168] FIG1 is a binding curve of a soluble reference TCR, i.e., wild-type TCR, and the GVYDGREHTV-HLA A0201 complex;

[0169] Figures 2a and 2b are respectively the results of activation function experiments on T2 cells loaded with the short peptide GVYDGREHTV and effector cells transfected with the high-affinity TCR of the present invention;

[0170] Figures 3a, 3b and 3c are respectively the results of activation function experiments on the number of IFN-γ spots released by effector cells transfected with the high-affinity TCR of the present invention for tumor cell lines;

[0171] FIG4 shows the activation function experimental results of the number of granzyme B spots released by effector cells transfected with the high-affinity TCR of the present invention against tumor cell lines;

[0172] FIG5a and FIG5b 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. DETAILED DESCRIPTION

[0173] Through extensive and in-depth research, the present invention has developed a high-affinity T cell receptor (TCR) that recognizes the GVYDGREHTV short peptide (derived from the MAGE-A4 protein), which is presented as a peptide-HLA A0201 complex. The high-affinity TCR has three CDR regions in its α chain variable domain:

[0174] CDR1α:VSGLRG SEQ ID NO:63,

[0175] CDR2α: LYSAGEE SEQ ID NO: 64, and

[0176] CDR3α: AVHPLNYGGSQGNLI SEQ ID NO: 65 is mutated;

[0177] and / or in the three CDR regions of its beta chain variable domain:

[0178] CDR1β:DFQATT SEQ ID NO:112,

[0179] CDR2β: SNEGSKA SEQ ID NO:113, and,

[0180] CDR3β: SAREWRGTEAF mutated in SEQ ID NO: 114;

[0181] Moreover, after mutation, the affinity and / or binding half-life of the TCR of the present invention for the GVYDGREHTV-HLA A0201 complex is at least twice that of the wild-type TCR.

[0182] 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 may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, and the scope of the present invention will be limited only by the appended claims.

[0183] 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.

[0184] 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.

[0185] the term

[0186] T cell receptor (TCR)

[0187] The International Immunogenetics Information System (IMGT) can be used to describe TCRs. Natural αβ heterodimeric TCRs have α and β chains. Broadly speaking, each chain contains a variable region, a joining region, and a constant region. The β chain typically also contains a short multivariable region between the variable and joining regions, but this multivariable region is often considered part of the joining region. The joining 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.

[0188] Each variable region contains three CDRs (complementarity determining regions), CDR1, CDR2, and CDR3, embedded in the framework sequence. In the IMGT nomenclature, the different numbers TRAV and TRBV refer to different Vα 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; and "*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; and "*01" represents allele 1. The constant region of the α chain is uniquely defined, 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 the TCRα and β chains through the public IMGT database.

[0189] The α and β chains of a TCR are generally considered to each have two "domains," 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 this application, "TCR α chain variable domain" refers to the connected TRAV and TRAJ regions, and similarly, "TCR β chain variable domain" refers to the connected TRBV and TRBD / TRBJ regions. The three CDRs of the TCR α chain variable domain are CDR1α, CDR2α, and CDR3α; the three CDRs of the TCR β chain variable domain are CDR1β, CDR2β, and CDR3β. The framework sequences of the TCR variable domains of the present invention can be murine or human, preferably human. The constant domain of a TCR comprises an intracellular portion, a transmembrane region, and an extracellular portion.

[0190] In the present invention, the amino acid sequences of the α and β chain variable domains of a wild-type TCR capable of binding to the GVYDGREHTV-HLA A0201 complex are SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The amino acid sequences of the α and β chains 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 sequences of the α and β chains of the "wild-type TCR" described in the present invention are SEQ ID NO: 59 and SEQ ID NO: 60, respectively. The TCR sequences used in the present invention are human. The amino acid sequences of the α and β chains of the "wild-type TCR" described in the present invention are SEQ ID NO: 61 and SEQ ID NO: 62, 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.

[0191] SEQ ID NO: 59:

[0192] SEQ ID NO: 60:

[0193] SEQ ID NO: 61:

[0194] SEQ ID NO: 62:

[0195] Natural interchain disulfide bonds and artificial interchain disulfide bonds

[0196] A set of disulfide bonds exists between the Cα and Cβ chains in the juxtamembrane region of the native TCR, referred to herein as "native interchain disulfide bonds." In the present invention, artificially introduced interchain covalent disulfide bonds that are located at a different position than the native interchain disulfide bonds are referred to herein as "artificial interchain disulfide bonds."

[0197] 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, if 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, if 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 numbering of the amino acid sequences of the variable regions TRAV and TRBV follows the position numbering listed in IMGT. For example, if an amino acid in TRAV is numbered 46 in IMGT, it will be described in the present invention as amino acid position 46 of TRAV, and the same applies to other amino acids. In the present invention, if the sequence position numbering of other amino acids is specifically indicated, it will be used in accordance with the specific instructions.

[0198] tumor

[0199] The term "tumor" is intended to include all types of cancerous growths or oncogenic processes, metastatic tissues, or malignantly 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 malignancies of various organ systems, such as sarcomas, squamous cell carcinomas of the lung, and carcinomas of the prostate, lung, breast, lymphoid, gastrointestinal (e.g., colon), and genitourinary tract (e.g., kidney, epithelial cells), and pharynx.

[0200] Detailed Description of the Invention

[0201] As is well known, the α chain variable domain and β chain variable domain of TCR each contain three CDRs, similar to the complementary determining regions of antibodies. CDR3 interacts with the antigen short peptide, and CDR1 and CDR2 interact with HLA. Therefore, the CDRs of the TCR molecule determine its interaction with the antigen 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 to the antigen short peptide GVYDGREHTV-HLA A0201 complex (i.e., GVYDGREHTV-HLA A0201 complex) are SEQ ID NO: 1 and SEQ ID NO: 2, respectively, which are sequences discovered for the first time by the inventors. The TCR has the following CDR regions:

[0202] α chain variable domain CDRs:

[0203] CDR1α: VSGLRG SEQ ID NO: 63;

[0204] CDR2α: LYSAGEE SEQ ID NO: 64; and,

[0205] CDR3α:AVHPLNYGGSQGNLI SEQ ID NO:65;

[0206] and β chain variable domain CDRs:

[0207] CDR1β: DFQATT SEQ ID NO: 112;

[0208] CDR2β: SNEGSKA SEQ ID NO: 113; and,

[0209] CDR3β: SAREWRGTEAF SEQ ID NO:114.

[0210] The present invention obtains a high-affinity TCR whose affinity to the GVYDGREHTV-HLA A0201 complex is at least twice that of the wild-type TCR to the GVYDGREHTV-HLA A0201 complex by performing mutation screening on the above-mentioned CDR region.

[0211] Furthermore, the TCR of the present invention is an αβ heterodimeric TCR, and the sequence of the α chain variable domain of the TCR comprises an amino acid sequence that has at least 85% (for example, at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, etc.) sequence homology to the amino acid sequence shown in SEQ ID NO: 1; preferably, an amino acid sequence with at least 90% sequence homology; more preferably, an amino acid sequence with at least 92% sequence homology; more preferably, an amino acid sequence with at least 94% sequence homology; and / or the sequence of the β chain variable domain of the TCR comprises an amino acid sequence with at least 95% sequence homology to 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.

[0212] Furthermore, the TCR of the present invention is a single-chain TCR, and the sequence of the α chain variable domain of the TCR comprises an amino acid sequence that has at least 85% (e.g., at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence shown in SEQ ID NO: 3; preferably, an amino acid sequence that has at least 90% sequence homology; more preferably, an amino acid sequence that has at least 92% sequence homology; most preferably, an amino acid sequence that has at least 94% sequence homology; and / or the sequence of the β chain variable domain of the TCR comprises an amino acid sequence that has at least 85% (e.g., at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology to 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.

[0213] 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 27-32, 50-56 and 89-103 of SEQ ID NO: 1, respectively. Accordingly, the amino acid residue numbering adopts the numbering shown in SEQ ID NO: 1, 95Y is the 7th Y of CDR3α, 96G is the 8th G of CDR3α, 97G is the 9th G of CDR3α, 98S is the 10th S of CDR3α, 99Q is the 11th Q of CDR3α, and 100G is the 12th G of CDR3α.

[0214] Specifically, the specific forms of the mutations in the α chain variable domain include one or more groups of Y95A / N / H / S / T / D / K / R / W / M / V, G96Q / P / Y / K / T / D / A / N / L / M / R / V / H / S, G97T / P / Q / W / I / F / Y / K / N / L / M / R / H / V / E, S98P / T / I / K / M / R / L / Q / Y / F, Q99E / H / Y / D and G100S / N.

[0215] 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).

[0216] In the present invention, Pro60 or 60P both represent proline at position 60. In addition, the specific expression of the mutation in the present invention, such as "G100S / N", represents that G at position 100 is substituted by S or by N, and the same applies to other expressions.

[0217] Using site-directed mutagenesis methods well known to those skilled in the art, Thr48 in exon 1 of the wild-type TCR α chain constant region TRAC*01 was mutated to cysteine, and Ser57 in exon 1 of the β chain constant region TRBC1*01 or TRBC2*01 was mutated to cysteine, resulting in a reference TCR. The amino acid sequences of the reference TCRs are SEQ ID NO:11 and SEQ ID NO:12, respectively, with the mutated cysteine ​​residues indicated in bold. These cysteine ​​substitutions enable the formation of artificial interchain disulfide bonds between the constant regions of the α and β chains of the reference TCR, resulting in a more stable soluble TCR. This facilitates the assessment of the binding affinity and / or half-life of the TCR to the GVYDGREHTV-HLA A0201 complex. It should be understood that the CDR regions of the TCR variable region determine its affinity for the pMHC complex; therefore, these cysteine ​​substitutions in the TCR constant region do not affect the binding affinity and / or half-life of the TCR. Therefore, in the present invention, the measured binding affinity between a reference TCR and the GVYDGREHTV-HLA A0201 complex is considered to be the binding affinity between the wild-type TCR and the GVYDGREHTV-HLA A0201 complex. Similarly, if the measured binding affinity between the TCR of the present invention and the GVYDGREHTV-HLA A0201 complex is at least 10 times greater than the binding affinity between the reference TCR and the GVYDGREHTV-HLA A0201 complex, it is equivalent to the binding affinity between the TCR of the present invention and the GVYDGREHTV-HLA A0201 complex being at least 10 times greater than the binding affinity between the wild-type TCR and the GVYDGREHTV-HLA A0201 complex.

[0218] Binding affinity (and the dissociation equilibrium constant, K) can be determined by any suitable method. D inversely proportional to) and 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 offThe binding affinity or binding half-life of a given TCR is preferably measured several times, for example three times or more, using the same assay 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 following conditions: temperature 25°C, pH 7.1-7.5. This method measures the dissociation equilibrium constant K of the reference TCR for the GVYDGREHTV-HLA A0201 complex. D is 1.55E-05M, i.e. 15.49 μM. In the present invention, it is considered that the dissociation equilibrium constant K of the wild-type TCR for the GVYDGREHTV-HLA A0201 complex is D is also 15.49 μM. Since doubling the affinity of TCR will result in K D So if the dissociation equilibrium constant K of high affinity TCR for GVYDGREHTV-HLA A0201 complex is detected, D The affinity of the high-affinity TCR for the GVYDGREHTV-HLA A0201 complex is 10 times that of the wild-type TCR for the GVYDGREHTV-HLA A0201 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 GVYDGREHTV-HLA A0201 complex is at least 2 times that of the wild-type TCR.

[0219] Mutagenesis can be performed using any suitable method, including but not limited to those based on polymerase chain reaction (PCR), restriction enzyme-based cloning, or ligation-independent cloning (LIC) methods. Many standard molecular biology textbooks describe these methods in detail. Further details of polymerase chain reaction (PCR) mutagenesis and restriction enzyme-based cloning 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).

[0220] The method for producing the TCR of the present invention can be, but is not limited to, screening TCRs with high affinity for the GVYDGREHTV-HLA A0201 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).

[0221] It should be understood that genes expressing the amino acids of the wild-type TCR α and β chain variable domains 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 domains of the template TCR.

[0222] The high-affinity TCR of the present invention comprises an α chain variable domain amino acid sequence of one of SEQ ID NOs: 13-58; and / or the β chain variable domain amino acid sequence of the TCR is SEQ ID NO: 2. The amino acid sequences of the α chain variable domain and the β chain variable domain of the high-affinity TCR molecule of the present invention are preferably selected from the following Table 3:

[0223] Table 3

[0224] Mutated residues in SEQ ID NO: 13-SEQ ID NO: 58 are single underlined.

[0225] SEQ ID NO: 13:

[0226] SEQ ID NO: 14:

[0227] SEQ ID NO: 15:

[0228] SEQ ID NO: 16:

[0229] SEQ ID NO: 17:

[0230] SEQ ID NO: 18:

[0231] SEQ ID NO: 19:

[0232] SEQ ID NO: 20:

[0233] SEQ ID NO: 21:

[0234] SEQ ID NO:22:

[0235] SEQ ID NO:23:

[0236] SEQ ID NO:24:

[0237] SEQ ID NO:25:

[0238] SEQ ID NO:26:

[0239] SEQ ID NO:27:

[0240] SEQ ID NO:28:

[0241] SEQ ID NO:29:

[0242] SEQ ID NO:30:

[0243] SEQ ID NO:31:

[0244] SEQ ID NO:32:

[0245] SEQ ID NO:33:

[0246] SEQ ID NO:34:

[0247] SEQ ID NO:35:

[0248] SEQ ID NO:36:

[0249] SEQ ID NO:37:

[0250] SEQ ID NO:38:

[0251] SEQ ID NO:39:

[0252] SEQ ID NO:40:

[0253] SEQ ID NO:41:

[0254] SEQ ID NO:42:

[0255] SEQ ID NO:43:

[0256] SEQ ID NO:44:

[0257] SEQ ID NO:45:

[0258] SEQ ID NO:46:

[0259] SEQ ID NO:47:

[0260] SEQ ID NO:48:

[0261] SEQ ID NO:49:

[0262] SEQ ID NO:50:

[0263] SEQ ID NO:51:

[0264] SEQ ID NO:52:

[0265] SEQ ID NO:53:

[0266] SEQ ID NO:54:

[0267] SEQ ID NO:55:

[0268] SEQ ID NO:56:

[0269] SEQ ID NO:57:

[0270] SEQ ID NO:58:

[0271] For the purposes of the present invention, the TCR of the present invention is a portion having at least one TCRα and / or TCRβ chain variable domain. They typically contain both TCRα chain variable domains and TCRβ chain variable domains. They can be αβ heterodimers or single chain forms or any other form that can stably exist. In adoptive immunotherapy, the full-length chain of the 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, in which case the TCR is preferably in a soluble form.

[0272] As for stability, it is disclosed in the prior art that the introduction of an artificial interchain disulfide bond between the α and β chain constant domains of TCR can obtain a soluble and stable TCR molecule, as described in patent document PCT / CN2015 / 093806. Therefore, the TCR of the present invention can be a TCR in which an artificial interchain disulfide bond is introduced 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 TCR to form artificial interchain disulfide bonds. For example, Thr48 of TRAC*01 exon 1 is replaced and Ser57 of TRBC1*01 or TRBC2*01 exon 1 is replaced to form a disulfide bond. Other sites for introducing cysteine ​​residues to form disulfide bonds can also be:

[0273] Thr45 of exon 1 of TRAC*01 and Ser77 of exon 1 of TRBC1*01 or TRBC2*01;

[0274] Tyr10 of exon 1 of TRAC*01 and Ser17 of exon 1 of TRBC1*01 or TRBC2*01;

[0275] Thr45 of exon 1 of TRAC*01 and Asp59 of exon 1 of TRBC1*01 or TRBC2*01;

[0276] Ser15 of exon 1 of TRAC*01 and Glu15 of exon 1 of TRBC1*01 or TRBC2*01;

[0277] Arg53 of exon 1 of TRAC*01 and Ser54 of exon 1 of TRBC1*01 or TRBC2*01;

[0278] Pro89 of exon 1 of TRAC*01 and Ala19 of exon 1 of TRBC1*01 or TRBC2*01;

[0279] or Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01.

[0280] That is, cysteine ​​residues are substituted for any of the aforementioned groups of sites in the α and β chain constant domains. The C-terminus of one or more TCR constant domains of the present invention can be truncated by up to 15, or up to 10, or up to 8 or fewer amino acids to exclude cysteine ​​residues, thereby eliminating native interchain disulfide bonds. This can also be achieved by mutating cysteine ​​residues that form native interchain disulfide bonds to another amino acid.

[0281] As described above, the TCRs of the present invention may include artificial interchain disulfide bonds introduced between residues in their α and β chain constant domains. It should be noted that the TCRs 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 between the constant domains as described above. The TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the TCR may be linked by a native interchain disulfide bond present in the TCR.

[0282] 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:

[0283] amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01;

[0284] amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01;

[0285] amino acid 46 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01;

[0286] or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01.

[0287] 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.

[0288] 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 it does not comprise an α chain constant domain, and the α chain variable domain of the TCR forms a heterodimer with the β chain.

[0289] For stability, on the other hand, the TCR of the present invention also includes a TCR that is mutated 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 acid positions 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 based on the database.

[0290] More specifically, the TCR with a mutation in the hydrophobic core region of 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 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, the affinity between the TCR and the GVYDGREHTV-HLA A0201 complex can be more conveniently evaluated.

[0291] The CDR regions of the α and β chain variable domains of the single-chain template TCR are identical to those of a wild-type TCR. Specifically, the three CDRs of the α chain variable domain are CDR1α: VSGLRG, CDR2α: LYSAGEE, and CDR3α: AVHPLNYGGSQGNLI, and the three CDRs of the β chain variable domain are CDR1β: DFQATT, CDR2β: SNEGSKA, and CDR3β: SAREWRGTEAF. 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. This method screened for single-chain TCRs composed of α and β chain variable domains that have high affinity for the GVYDGREHTV-HLA A0201 complex.

[0292] The αβ heterodimeric TCR (also referred to as a high-affinity TCR) of the present invention with high affinity for the GVYDGREHTV-HLA A0201 complex 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).

[0293] The TCRs of the present invention can also be provided as multivalent complexes. The multivalent TCR complexes of the present invention comprise multimers formed by the association of two, three, four, or more TCRs of the present invention, such as tetramers produced using the tetramerization domain of p53, or complexes formed by the association of multiple TCRs of the present invention with another molecule. The TCR complexes 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 generate intermediates for other multivalent TCR complexes with similar applications.

[0294] The TCRs of the present invention can be used alone or covalently or otherwise, preferably covalently, bound to a conjugate comprising a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the GVYDGREHTV-HLA A0201 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination thereof.

[0295] 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.

[0296] Therapeutic agents that can be combined or coupled to the TCR of the present invention include, but are not limited to:

[0297] 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539);

[0298] 2. Biological toxicity (Chaudhary et al., 1989, Nature 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy 51, 565);

[0299] 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);

[0300] 4. Antibody Fc fragment (Mosquera et al., 2005, The Journal of Immunology 174, 4381);

[0301] 5. Antibody scFv fragment (Zhu et al., 1995, International Journal of Cancer 62, 319);

[0302] 6. Gold nanoparticles / nanorods (Lapotko et al., 2005, Cancer letters 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115);

[0303] 7. Viral particles (Peng et al., 2004, Gene therapy 11, 1234);

[0304] 8. Liposomes (Mamot et al., 2005, Cancer Research 65, 11631);

[0305] 9. Nanomagnetic particles;

[0306] 10. Prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL));

[0307] 11. Chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, etc.

[0308] The antibodies or fragments thereof that bind to the TCR of the present invention include anti-T cell or NK cell determinants, such as anti-CD3, anti-CD28, or anti-CD16 antibodies. The binding of the above antibodies or fragments to the TCR can direct effector cells to better target target cells. A preferred embodiment is that the TCR of the present invention is combined with 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 one of the amino acid sequences of the TCR α chain variable domain selected from SEQ ID NO: 13-58; and / or the amino acid sequence of the β chain variable domain of the TCR is SEQ ID NO: 2.

[0309] The present invention also relates to nucleic acid molecules encoding the TCRs 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 identical to the nucleic acid sequence set forth herein or a degenerate variant. To illustrate the meaning of "degenerate variant," as used herein, a "degenerate variant" in the present invention refers to a nucleotide sequence that encodes a protein sequence having SEQ ID NO:3 but differs from the sequence of SEQ ID NO:5.

[0310] The full-length sequence of the nucleic acid molecule of the present invention or its fragments can generally be obtained by, but not limited to, PCR amplification, recombinant methods, or artificial synthesis. Currently, DNA sequences encoding the TCR of the present invention (or its fragments, or derivatives thereof) can be obtained entirely by chemical synthesis. The resulting DNA sequence can then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.

[0311] The present invention also relates to vectors comprising the nucleic acid molecules of the present invention, and host cells genetically engineered with the vectors or coding sequences of the present invention.

[0312] The present invention also includes isolated cells, particularly T cells, expressing the TCRs of the present invention. Many methods are suitable for transfecting T cells with DNA or RNA encoding the high-affinity TCRs of the present invention (e.g., Robbins et al., (2008) J. Immunol. 180:6116-6131). T cells expressing the high-affinity TCRs of the present invention can be used for adoptive immunotherapy. Those skilled in the art will recognize many suitable methods for adoptive therapy (e.g., Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).

[0313] The present invention also provides a pharmaceutical composition comprising 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.

[0314] 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 the cell presenting the TCR of the present invention, or the pharmaceutical composition of the present invention to a subject in need of treatment.

[0315] In the art, substitution with amino acids of similar or similar properties generally does not alter protein function. Adding one or more amino acids to the C-terminus and / or N-terminus generally does not alter protein structure and function. Therefore, the TCRs of the present invention also include TCRs in which up to 5, preferably up to 3, more preferably up to 2, and most preferably 1 amino acid (especially amino acids outside the CDR regions) of the TCRs of the present invention are replaced with amino acids of similar or similar properties, while still maintaining their functionality.

[0316] The present invention also includes TCRs that have been slightly modified from the TCRs of the present invention. Modifications (generally without altering the primary structure) include chemical derivatization of the TCRs of the present invention, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation during the synthesis and processing of the TCRs of the present invention or in further processing steps. Such modifications can be accomplished by exposing the TCR to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylation enzyme). Modified forms also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, or phosphothreonine). Also included are TCRs that have been modified to increase their resistance to proteolysis or optimize their solubility.

[0317] The TCR, TCR complex or T cell transfected with the TCR of the present invention can 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 can be in any suitable form (depending on the desired method of administration to the patient). The pharmaceutical composition can be provided in unit dosage form, typically in a sealed container, which can 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.

[0318] In addition, the TCR of the present invention can be used alone or in combination or coupled with other therapeutic agents (eg, formulated in the same pharmaceutical composition).

[0319] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier used to administer a therapeutic agent. The term refers to pharmaceutical carriers that do not themselves induce the production of antibodies harmful to the individual receiving the composition and are not unduly toxic upon administration. Such carriers are well known to those of ordinary skill in the art. A comprehensive 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, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.

[0320] Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances such as wetting agents or emulsifiers, pH buffering substances, etc. may also be present in these carriers.

[0321] 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.

[0322] 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 administration. The subject to be prevented or treated can be an animal; in particular, a human.

[0323] When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition can be adopted according to the usage conditions, preferably injections, oral preparations, etc.

[0324] These pharmaceutical compositions can be formulated according to conventional methods by mixing, diluting or dissolving, and occasionally suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers are added, and the formulation process can be carried out in a conventional manner according to the dosage form.

[0325] The pharmaceutical compositions of the present invention can also be administered in the form of a sustained-release formulation. For example, the TCR of the present invention can be incorporated into a pill or microcapsule containing a sustained-release polymer as a carrier, and then the pill or microcapsule can be surgically implanted into the tissue to be treated. Examples of sustained-release polymers include ethylene-vinyl acetate copolymer, polyhydroxymethylacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, or lactic acid-glycolic acid copolymers. Preferred examples include biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.

[0326] When the pharmaceutical composition of the present invention is used for actual treatment, the TCR or TCR complex of the present invention or the cells 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.

[0327] The main advantages of the present invention are:

[0328] (1) The affinity and / or binding half-life of the high-affinity TCR of the present invention for the GVYDGREHTV-HLA A0201 complex is at least twice that of the wild-type TCR.

[0329] (2) The high-affinity TCR of the present invention can specifically bind to the GVYDGREHTV-HLA A0201, and cells transfected with the high-affinity TCR of the present invention can be specifically activated.

[0330] (3) Effector cells transfected with the high-affinity TCR of the present invention have a strong specific killing effect.

[0331] The following specific examples further illustrate the present invention. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, were generally performed under conventional conditions, such as those described in (Sambrook and Russell et al., 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.

[0332] Materials and methods

[0333] Unless otherwise specified, the experimental materials used in the examples of the present invention were 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.

[0334] Example 1 Stability of Hydrophobic Core Mutations Generation of Single-Chain TCR Template Chains

[0335] 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 TCR α 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.

[0336] The target gene carrying the template strand was double-digested with Nco I and Not I and ligated into the pET28a vector, which had also been double-digested with Nco I and Not I. The ligation product was transformed into E. coli DH5α, plated on LB plates containing kanamycin, and incubated inverted at 37°C overnight. Positive clones were selected for PCR screening and sequenced. After confirming the correct sequence, the recombinant plasmid was extracted and transformed into E. coli BL21(DE3) for expression.

[0337] SEQ ID NO: 3:

[0338] SEQ ID NO: 4:

[0339] SEQ ID NO: 5:

[0340] SEQ ID NO: 6:

[0341] SEQ ID NO: 7:

[0342] SEQ ID NO:8:

[0343] SEQ ID NO:9:

[0344] SEQ ID NO: 10:

[0345] Example 2 Expression, renaturation and purification of the stable single-chain TCR constructed in Example 1

[0346] 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 600 The pH value was 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and culture was continued at 37°C for 4 hours. The cell pellet was harvested by centrifugation at 5000 rpm for 15 minutes, lysed with Bugbuster Master Mix (Merck), and centrifuged at 6000 rpm for 15 minutes to recover the inclusion bodies. The inclusion bodies were then washed with Bugbuster (Merck) to remove cell debris and membrane components, and collected by centrifugation at 6000 rpm for 15 minutes. The inclusion bodies were dissolved in buffer (20 mM Tris-HCl pH 8.0, 8 M urea), and insoluble matter was removed by high-speed centrifugation. The supernatant was quantified by BCA assay, aliquoted, and stored at -80°C until further use.

[0347] To 5 mg of dissolved single-chain TCR inclusion body protein, add 2.5 mL of buffer (6 M 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 treated single-chain TCR dropwise 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. The dialysis bag is placed in 1 L of pre-chilled water and slowly stirred at 4°C overnight. After 17 hours, the dialysate was exchanged for 1 L of pre-chilled buffer (20 mM Tris-HCl pH 8.0), and dialysis was continued at 4°C for 8 hours. The dialysate was then exchanged for the same fresh buffer and dialysis continued overnight. After 17 hours, the sample was filtered through a 0.45 μm filter, degassed under vacuum, and passed through an anion exchange column (HiTrap Q HP, GE Healthcare). The protein was purified using a linear gradient of 0-1 M NaCl in 20 mM Tris-HCl pH 8.0. The collected elution fractions were analyzed by SDS-PAGE. 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.

[0348] 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.

[0349] Example 3 Binding Characterization

[0350] BIAcore analysis

[0351] The binding activity of TCR molecules to the GVYDGREHTV-HLA A0201 complex was measured using a BIAcore T200 real-time analysis system. Anti-streptavidin antibodies (GenScript) were added to the coupling buffer (10 mM sodium acetate buffer, pH 4.77). The antibodies were then passed over a CM5 chip pre-activated with EDC and NHS to immobilize the antibodies on the chip surface. The unreacted activated surface was blocked with ethanolamine hydrochloride solution to complete the coupling process, achieving a coupling level of approximately 15,000 RU. The conditions were: temperature 25°C, pH 7.1-7.5.

[0352] A low concentration of streptavidin was passed over the antibody-coated chip surface, followed by the GVYDGREHTV-HLA A0201 complex through the detection channel. Another channel served as a reference channel. 0.05 mM biotin was then passed through the chip at a flow rate of 10 μL / min for 2 minutes to block the remaining streptavidin binding sites. Affinity was determined using a single-cycle kinetic analysis method. TCRs were diluted to several concentrations in HEPES-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, pH 7.4) and sequentially passed over the chip surface at a flow rate of 30 μL / min. Each injection allowed 120 seconds of binding time, followed by a 600-second dissociation period after the final injection. After each round of measurement, the chip was regenerated with 10 mM Gly-HCl, pH 1.75. Kinetic parameters were calculated using BIAcore Evaluation software.

[0353] The preparation process of the above-mentioned GVYDGREHTV-HLA A0201 complex is as follows:

[0354] a. Purification: Collect 100 mL of E. coli culture induced to express heavy or light chain, centrifuge at 8000 g for 10 min at 4°C, wash the cells once with 10 mL of PBS, and resuspend the cells in 5 mL of BugBuster Master Mix Extraction Reagents (Merck) by vigorous vortexing. Incubate with rotation at room temperature for 20 min, and centrifuge at 6000 g for 15 min at 4°C. Discard the supernatant and collect the inclusion bodies.

[0355] The inclusion bodies were resuspended in 5 mL of 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, and the inclusion bodies were resuspended in 30 mL of 10-fold diluted BugBuster, mixed, and centrifuged at 4°C and 6000 g for 15 min. Repeat twice, and 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. Finally, the inclusion bodies were solubilized with 20 mM Tris-HCl and 8 M urea. The purity of the inclusion bodies was detected by SDS-PAGE, and the concentration was measured by BCA kit.

[0356] b. Renaturation: The synthetic short peptide GVYDGREHTV (synthesized by Jiangsu GenScript Biotechnology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg / mL. Light and heavy chain inclusion bodies were solubilized in a solution containing 8 M urea, 20 mM Tris pH 8.0, and 10 mM DTT. Before renaturation, 3 M guanidine hydrochloride, 10 mM sodium acetate, and 10 mM EDTA were added for further denaturation. The GVYDGREHTV peptide was added to renaturation buffer (0.4 M L-arginine, 100 mM Tris pH 8.3, 2 mM EDTA, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, 0.2 mM PMSF, cooled to 4°C) at a final concentration of 25 mg / L. Then, 20 mg / L of the light chain and 90 mg / L of the heavy chain were added sequentially (final concentration, the heavy chain was added in three additions, 8 h / time). Renaturation was carried out at 4°C for at least 3 days until completion. SDS-PAGE was used to detect successful renaturation.

[0357] c. Purification after renaturation: The renaturation buffer was exchanged by dialysis with 10 volumes of 20 mM Tris pH 8.0. The buffer was exchanged at least twice to fully reduce the ionic strength of the solution. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and loaded onto a HiTrap Q HP (GE) anion exchange column (5 mL bed volume). The protein was eluted using an Akta purifier (GE) with a linear gradient of 0-400 mM NaCl in 20 mM Tris pH 8.0. pMHC eluted at approximately 250 mM NaCl. Fractions were collected and analyzed for purity by SDS-PAGE.

[0358] d. Biotinylation: Purified pMHC molecules were concentrated using Millipore ultrafiltration tubes, and the buffer was replaced with 20 mM Tris pH 8.0. 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 then added. The mixture was incubated at room temperature overnight, and complete biotinylation was detected by SDS-PAGE.

[0359] e. Purification of the 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 using an Akta purifier (GE General Electric) and pre-equilibrated with filtered PBS. TM A 16 / 60S200 HR column (GE) was loaded with 1 mL of concentrated biotinylated pMHC molecules and eluted with PBS at a flow rate of 1 mL / min. The biotinylated pMHC molecules eluted as a single peak at approximately 55 mL. Protein fractions were pooled and concentrated using Millipore ultrafiltration tubes. Protein concentration was determined by the BCA assay (Thermo Fisher Scientific). A protease inhibitor cocktail (Roche) was added and the biotinylated pMHC molecules were aliquoted and stored at -80°C.

[0360] Example 4 Generation of high-affinity TCR

[0361] Phage display technology is a means of generating a library of high-affinity TCR variants for screening 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 then panning was performed. After several rounds of panning, the phage library showed specific binding to the corresponding antigen, and single clones were selected from it and analyzed. The CDR regions of the α chain and β chain of the high-affinity single-chain TCR screened were as follows:

[0362] Table 5

[0363] The CDR region mutations of the screened high-affinity single-chain TCRs, i.e., CDR numbers 1-46, were introduced one by one into the corresponding sites of the α and β chain variable domains of the wild-type TCR, respectively, to obtain high-affinity TCRs, with TCR numbers 1-46. Their affinity for the GVYDGREHTV-HLA A0201 complex was tested using BIAcore. The introduction of these high-affinity CDR region mutations was performed using site-directed mutagenesis methods well known to those skilled in the art. The amino acid sequences of the α and β chain variable domains of the wild-type TCR are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0364] It should be noted that a cysteine ​​residue can be introduced into the constant region of each of the wild-type α and β chains to form an artificial interchain disulfide bond to obtain a more stable soluble TCR, thereby facilitating the assessment of the binding affinity and / or half-life of the TCR to the GVYDGREHTV-HLA A0201 complex. In this example, the amino acid sequences of the TCR α and β chains after the introduction of the cysteine ​​residue are shown in SEQ ID NO:11 and SEQ ID NO:12, respectively, with the introduced cysteine ​​residue indicated by a bold, double-underlined letter.

[0365] SEQ ID NO: 11:

[0366] SEQ ID NO: 12:

[0367] The extracellular sequences of the TCRα and TCRβ chains to be expressed were synthesized using standard methods described in the Molecular Cloning a Laboratory Manual (3rd edition, Sambrook and Russell) and inserted into the expression vector pET28a+ (Novagene), with the upstream and downstream cloning sites Nco I and Not I, respectively. Mutations in the CDR regions were introduced using overlap PCR, a technique well known to those skilled in the art. The inserts were confirmed by sequencing.

[0368] Example 5 Expression, renaturation and purification of high-affinity TCR

[0369] 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, and the inclusion bodies formed after the expression of the α and β chains of the TCR were extracted using 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).

[0370] The solubilized TCR α and β chains were rapidly mixed in a 1:1 mass ratio in a solution containing 5 M urea, 0.4 M arginine, 20 mM Tris (pH 8.1), 3.7 mM cystamine, and 6.6 mM β-mercapoethylamine (4°C) to a final concentration of 60 mg / mL. After mixing, the solution was dialyzed against 10 volumes of deionized water (4°C). After 12 hours, the deionized water was exchanged for 20 mM Tris (pH 8.0) buffer, and dialysis was continued at 4°C for another 12 hours. The dialyzed solution was filtered through a 0.45 μm filter and purified by anion exchange column (HiTrap Q HP, 5 mL, GE Healthcare). The elution peak containing successfully refolded α and β dimers of the TCR was confirmed by SDS-PAGE. The 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.

[0371] Example 6 BIAcore analysis results

[0372] The method described in Example 3 was used to detect the affinity of the wild-type TCR introduced with the high-affinity CDR region to the GVYDGREHTV-HLA A0201 complex.

[0373] The novel TCR α chain variable domain amino acid sequences obtained by the present invention are shown in SEQ ID NO:13-SEQ ID NO:58. Since the CDR region of a TCR molecule determines its affinity for the corresponding pMHC complex, those skilled in the art would anticipate that a wild-type TCR with introduced high-affinity mutations would also have high affinity for the GVYDGREHTV-HLA A0201 complex. An expression vector was constructed using the method described in Example 4, and the wild-type TCR with introduced high-affinity mutations was expressed, renatured, and purified using the method described in Example 5. The affinity of the wild-type TCR for the GVYDGREHTV-HLA A0201 complex was then determined using a BIAcore T200. Figure 1 shows the binding curve of a soluble reference TCR, i.e., a wild-type TCR, to the GVYDGREHTV-HLA A0201 complex. The results of the high-affinity TCR affinity assay are shown in Table 6 below:

[0374] Table 6

[0375] From the results in Table 5, it can be seen that the high-affinity TCR obtained in the present invention has an affinity for the GVYDGREHTV-HLA A0201 complex of at least 2 times.

[0376] Example 7 Expression, Renaturation and Purification of Anti-CD3 Antibody Fusion with High Affinity TCR

[0377] A fusion molecule is prepared by fusing an anti-CD3 single-chain antibody (scFv) to a high-affinity TCR. The anti-CD3 scFv is fused to the β chain of a TCR. The TCR β chain can comprise any of the aforementioned high-affinity TCR β chain variable domains, and the TCR α chain of the fusion molecule can comprise any of the aforementioned high-affinity TCR α chain variable domains. Construction of the fusion molecule expression vector includes the following steps:

[0378] (1) Construction of α chain expression vector: The target gene carrying the α chain of the high-affinity TCR was double-digested with Nco I and Not I and ligated to the pET28a vector that had been double-digested with Nco I and Not I. The ligation product was transformed into E. coli DH5α, plated on an LB plate containing kanamycin, and incubated inverted at 37°C overnight. Positive clones were selected for PCR screening, and positive recombinants were sequenced. After confirming the correct sequence, the recombinant plasmid was extracted and transformed into E. coli Tuner (DE3) for expression.

[0379] (2) Construction of anti-CD3 (scFv)-β chain expression vector: Using the overlap PCR method, primers were designed to link the anti-CD3 scFv and high-affinity TCRβ chain genes. The intermediate linker was GGGGS, and the gene fragment of the fusion protein of the anti-CD3 scFv and high-affinity TCRβ chain was endowed with restriction endonuclease sites Nco Ⅰ (CCATGG) and Not Ⅰ (GCGGCCGC). The PCR amplified product was double-digested with Nco Ⅰ and Not Ⅰ and ligated to the pET28a vector double-digested with Nco Ⅰ and Not Ⅰ. The ligated product was transformed into E. coli DH5α competent cells, coated with LB plates containing kanamycin, and inverted and cultured at 37°C overnight. Positive clones were picked for PCR screening. The positive recombinants were sequenced. After confirming the correct sequence, the recombinant plasmid was extracted and transformed into E. coli Tuner (DE3) competent cells for expression.

[0380] (3) Expression, renaturation and purification of fusion proteins: The expression plasmids were transformed into E. coli Tuner (DE3) competent cells, coated on LB plates (kanamycin 50 μg / mL) and cultured at 37°C overnight. The next day, clones were inoculated into 10 mL LB liquid medium (kanamycin 50 μg / mL) and cultured for 2-3 hours. Then, the clones were inoculated into 1 L LB medium at a volume ratio of 1:100 and cultured until the OD 600 The pH value was 0.5-0.8, and IPTG was added at a final concentration of 1 mM to induce the expression of the target protein. After 4 hours of induction, the cells were harvested by centrifugation at 6000 rpm for 10 minutes. The cells were washed once with PBS buffer and aliquoted. The cells equivalent to 200 mL of bacterial culture were lysed with 5 mL of BugBuster Master Mix (Merck) and the inclusion bodies were collected by centrifugation at 6000 g for 15 minutes. Then, 4 detergent washes were performed to remove cell debris and membrane components. Then, the inclusion bodies were washed with a buffer solution such as PBS to remove detergents and salts. Finally, the inclusion bodies were dissolved with a buffer solution containing 6 M guanidine hydrochloride, 10 mM dithiothreitol (DTT), 10 mM ethylenediaminetetraacetic acid (EDTA), and 20 mM Tris pH 8.1, and the inclusion body concentration was determined. After aliquoting, the inclusion bodies were stored frozen at -80°C.

[0381] The dissolved TCR α chain and anti-CD3 (scFv)-β chain were rapidly mixed at a mass ratio of 2:5 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). The final concentrations of the α chain and anti-CD3 (scFv)-β chain were 0.1 mg / mL and 0.25 mg / mL, respectively.

[0382] After mixing, the solution was dialyzed against 10 volumes of deionized water (4°C). After 12 hours, the deionized water was replaced with a buffer solution (10 mM Tris, pH 8.0) and the dialysis was continued at 4°C for another 12 hours. The dialysis solution was filtered through a 0.45 μm filter membrane and purified by an anion exchange column (HiTrap QHP 5 ml, GE healthcare). The elution peak containing the TCR with successfully renatured TCRα chain and anti-CD3 (scFv)-β chain dimer was 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 5 ml, GE healthcare). The purity of the purified TCR fusion molecule was greater than 90% as determined by SDS-PAGE, and the concentration was determined by BCA assay.

[0383] Example 8 Activation Function Experiment of Effector Cells Transfected with the High-Affinity TCR of the Present Invention on T2 Cells Loaded with Short Peptides

[0384] IFN-γ is a powerful immunoregulatory factor produced by activated T lymphocytes. Therefore, in this example, the IFN-γ count was detected by an ELISPOT experiment familiar to those skilled in the art 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 as effector cells, and CD3+T cells transfected with other TCRs (A6) or wild-type TCRs (WT-TCR) from the same volunteers were used as controls. The target cells used were T2 loaded with the MAGE-A4 antigen short peptide GVYDGREHTV, T2 loaded with other antigen short peptides, or empty T2. The following experiments were carried out in two batches (I) and (II):

[0385] (I) The high-affinity TCRs are TCR5, TCR11, TCR27, TCR28, TCR29, TCR30, TCR31, TCR32, and TCR33.

[0386] (II) The high-affinity TCRs are TCR1, TCR2, TCR3, TCR4, TCR15, TCR19, TCR23, TCR25, TCR34, TCR35, TCR36, TCR38, TCR39, TCR40, TCR41, TCR42, and TCR46.

[0387] The following steps were performed: First, prepare the ELISPOT plate. In batch (I), the target cells were 1×10 4 cells / well, effector cells 2×10 3 cells / well (calculated according to the positive transfection rate), batch (II) first calculated the target cells as 1×10 4 cells / well, effector cells 4×10 3 Cells / well (calculated by transfection positive rate) were added to the corresponding wells, and then MAGE-A4 antigen short peptide GVYDGREHTV 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: For the blank group, add an equal volume of culture medium and set up two replicate wells. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, wash the plates and perform secondary detection and color development. Dry the plates, and count the spots formed on the membrane using an ELISPOT READER system (AID20).

[0388] FIG2a and FIG2b are respectively the activation function experimental results of T2 cells loaded with the short peptide GVYDGREHTV and effector cells transfected with the high-affinity TCR of the present invention.

[0389] The results in Figures 2a and 2b show that, against target cells loaded with the MAGE-A4 antigen short peptide GVYDGREHTV, effector cells transfected with the high-affinity TCR of the present invention have a more significant activation effect than effector cells transfected with the wild-type TCR, while effector cells transfected with other TCRs are inactive; at the same time, effector cells transfected with the TCR of the present invention are essentially inactive against target cells loaded with other antigen short peptides or empty cells.

[0390] Example 8 Activation Function Experiment of IFN-γ Spot Release by Effector Cells Transfected with High-Affinity TCR of the Present Invention for Tumor Cell Lines

[0391] This example uses tumor cell lines to verify the activation function and specificity of effector cells transfected with the high-affinity TCR of the present invention. ELISPOT assays, which are well known to those skilled in the art, are also used for testing. The high-affinity TCR of the present invention is transfected into CD3 T cells isolated from the blood of healthy volunteers. + T cells were used as effector cells and CD3 T cells were transfected with other TCRs (A6) from the same volunteers.+ T cells were used as controls. The tumor cell lines used in the examples were U2OS, A375, NUGC4, U251, COLO205, SK-HEP-1, Caski, and MCF-7. Among them, A375, COLO205, and MCF-7 were purchased from ATCC, NUGC4 and U251 were purchased from Nanjing Kebai Biotechnology Co., Ltd., SK-HEP-1 was purchased from the Cell Bank of the Chinese Academy of Sciences, Caski was purchased from Wuhan Punosai Life Science Co., Ltd., and U2OS was purchased from Guangzhou Saiku Biotechnology Co., Ltd. The following experiments were carried out in three batches (I), (II), and (III):

[0392] (I) The high-affinity TCRs are TCR1, TCR2, TCR3, TCR4, and TCR5. The MAGE-A4-positive tumor cell lines used are U2OS and A375, and the negative cell lines are NUGC4, U251, COLO205, and effector cells only.

[0393] (II) The high-affinity TCRs are TCR1, TCR2, TCR3, TCR4, TCR15, TCR19, TCR23, TCR25, TCR34, TCR35, TCR36, TCR38, TCR39, TCR40, TCR41, TCR42, and TCR46. The MAGE-A4-positive tumor cell line used was NUGC4-MAGE-A4 (MAGE-A4 overexpression), and the negative cell lines were NUGC4 and SK-HEP-1.

[0394] (III) The high-affinity TCRs are TCR5, TCR11, TCR27, TCR28, TCR29, TCR30, TCR31, TCR32, and TCR33. The MAGE-A4-positive tumor cell lines used are U2OS and NUGC4-MAGE-A4 (MAGE-A4 overexpression), and the negative cell lines are NUGC4, SK-HEP-1, Caski, and MCF-7.

[0395] The following steps were performed: First, prepare the ELISPOT plate. Activate the ELISPOT plate with ethanol and coat it overnight at 4°C. On the first day of the experiment, remove the coating solution, wash and block the plate, incubate at room temperature for two hours, remove the blocking solution, and add the test components to the ELISPOT plate: Batch (I) target cells were 2 × 10 4 cells / well, effector cells: 2×10 3 / well (calculated according to the positive rate of transfection), the target cells in batch (II) were 2×10 4 Effector cells: 4×10 3 / well (calculated according to the positive rate of transfection), the target cells in batch (III) were 2×104 cells / well, effector cells: 2×10 3 Count the number of transfections / well (calculated based on the positive transfection rate), and set up duplicate wells. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, wash the plates and perform secondary detection and color development. Dry the plates, and count the spots formed on the membrane using an ELISPOT READER system (AID20).

[0396] Figures 3a, 3b, and 3c show the activation function experimental results of effector cells transfected with the high-affinity TCR of the present invention against tumor cell lines. The results of Figures 3a, 3b, and 3c show that the effector cells transfected with the high-affinity TCR of the present invention exhibit a very obvious activation effect against the MAGE-A4-positive tumor cell line, while the effector cells transfected with other TCRs (A6) are basically inactive; at the same time, the effector cells transfected with the high-affinity TCR of the present invention are basically inactive against the MAGE-A4-negative cell line.

[0397] Example 9 Activation Function Experiment of Granzyme Spot Number Released by Effector Cells Transfected with High-Affinity TCR of the Present Invention for Tumor Cell Lines

[0398] This example uses tumor cell lines to verify the activation function and specificity of effector cells transfected with the high-affinity TCR of the present invention. The ELISPOT assay, which is well known to those skilled in the art, is also used for detection. The high-affinity TCR of the present invention is transfected into CD3 T cells isolated from the blood of healthy volunteers. + T cells were used as effector cells and CD3 T cells were transfected with other TCRs (A6) from the same volunteers. + T cells were used as controls. The tumor cell lines used in the examples were U2OS, A375, NUGC4, U251, and T84. U2OS was purchased from Guangzhou Saiku Biotechnology Co., Ltd., A375 and T84 were purchased from ATCC, and NUGC4 and U251 were purchased from Nanjing Kebai Biotechnology Co., Ltd. The experiments were performed as follows:

[0399] The high-affinity TCRs are TCR1, TCR2, TCR3, TCR4, and TCR5. The MAGE-A4 positive tumor cell lines used are U2OS and A375, and the negative cell lines are NUGC4, U251, T84, and effector cells only.

[0400] The following steps were performed: First, prepare the ELISPOT plate. Activate the ELISPOT plate with ethanol and coat it overnight at 4°C. On the first day of the experiment, remove the coating solution, wash and block the plate, incubate at room temperature for two hours, remove the blocking solution, and add the test components to the ELISPOT plate: target cells were 1×10 4 cells / well, effector cells: 1×103 Count the number of transfections per well (calculated based on the positive transfection rate), and set up duplicate wells. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, wash the plates and perform secondary detection and color development. Dry the plates, and count the spots formed on the membrane using an ELISPOT READER system (AID20).

[0401] Figure 4 shows the activation function experimental results of effector cells transfected with the high-affinity TCR of the present invention against tumor cell lines. The results in Figure 4 show that the effector cells transfected with the high-affinity TCR of the present invention exhibit a very obvious activation effect against MAGE-A4-positive tumor cell lines, while the effector cells transfected with other TCRs (A6) are basically inactive; at the same time, the effector cells transfected with the high-affinity TCR of the present invention are basically inactive against MAGE-A4-negative cell lines.

[0402] Example 10 Killing function experiment of effector cells transfected with high affinity TCR of the present invention against tumor cell lines

[0403] Lactate dehydrogenase (LDH) is abundant in the cytoplasm and cannot pass through the cell membrane under normal circumstances. When cells are damaged or die, they can be released outside the cells. 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 non-radioactive cytotoxicity experiments well known to those skilled in the art, thereby verifying the killing function of 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 of the same volunteer was transfected with other TCR (A6) + T cells were used as controls. The tumor cell lines used in the examples were U-2OS, NUGC4, SK-HEP-1, and A375. U2OS was purchased from Guangzhou Saiku Biotechnology Co., Ltd., NUGC4 was purchased from Nanjing Kebai Biotechnology Co., Ltd., SK-HEP-1 was purchased from the Cell Bank of the Chinese Academy of Sciences, and A375 was purchased from ATCC. The following experiments were conducted in two batches (I) and (II):

[0404] The high-affinity TCRs in batch (I) are TCR1, TCR2, TCR3, TCR4, TCR5, TCR15, TCR19, TCR31, TCR32, TCR36, TCR41, and TCR46. The MAGE-A4-positive tumor cell lines used are U-2OS and NUGC4-MAGE-A4 (MAGE-A4 overexpression), and the negative tumor cell lines are SK-HEP-1 and NUGC4.

[0405] The high affinity TCRs in batch (II) are TCR1, TCR2, TCR3, TCR4, and TCR5. The MAGE-A4 positive tumor cell lines used are U-2OS and A375, and the negative tumor cell line is NUGC4.

[0406] Experimental steps: First, prepare the LDH plate and add the test components to the plate in the following order: Batch (I) 3×10 4 cells / well, effector cells 9×10 4 cells / well (calculated based on the positive transfection rate), batch (II) target cells 3×10 4 cells / well, effector cells 2×10 4 Cells / well (calculated based on the transfection positive rate) were added to the corresponding wells, and triplicate wells were set up. Also, wells for effector cells, target cells, a maximum target cell well, a volume correction control well, and a culture medium background control well were set up. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, color development was detected. After stopping the reaction, absorbance was recorded at 490 nm using a microplate reader (Bioteck).

[0407] Figures 5a and 5b show the results of LDH assays of the killing function of effector cells transfected with the high-affinity TCR of the present invention against tumor cell lines. The results in Figures 5a and 5b demonstrate that effector cells transfected with the high-affinity TCR of the present invention exhibit significant and potent killing efficacy against MAGE-A4-positive tumor cell lines, while T cells transfected with another TCR (A6) exhibit virtually no response. Furthermore, T cells transfected with the high-affinity TCR of the present invention exhibit almost no killing efficacy against negative tumor cell lines.

[0408] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

[0409] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection 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 fall within the scope of protection and disclosure 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 GVYDGREHTV-HLA A0201 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. The TCR according to claim 1, wherein In the TCR β chain variable domain, CDR1 β is DFQATT, CDR2 β is SNEGSKA, and CDR3 β is SAREWRGTEAF.

3. The TCR according to claim 1, wherein: The amino acid sequence of the TCRβ chain variable domain is SEQ ID NO:

2.

4. [Corrected 11.11.2024 in accordance with Rule 26] A TCR as claimed in claim 1, characterized in that The reference sequences of the three CDR regions (complementarity determining regions) of the TCRα chain variable domain are as follows: CDR1α:VSGLRG CDR2α: LYSAGEE CDR3α: AVHPLNYGGSQGNLI and contains at least one of the following mutations:

5. [Corrected 11.11.2024 in accordance with Rule 26] A TCR as claimed in claim 1, characterized in that The TCR has CDRs selected from the group consisting of:

6. The TCR according to claim 1, wherein: The TCR is soluble.

7. The TCR according to claim 1, wherein: The TCR is an αβ heterodimeric TCR, comprising an α chain TRAC constant region sequence and a β chain TRBC1 or TRBC2 constant region sequence.

8. The TCR according to claim 1, wherein: 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.

9. The TCR according to claim 1, wherein: 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; preferably, the cysteine ​​residues forming the artificial interchain disulfide bond between the constant regions of the TCR α and β chains are substituted with one or more groups of sites selected from the following: Thr48 of exon 1 of TRAC*01 and Ser57 of exon 1 of TRBC1*01 or TRBC2*01; Thr45 of exon 1 of TRAC*01 and Ser77 of exon 1 of TRBC1*01 or TRBC2*01; Tyr10 of exon 1 of TRAC*01 and Ser17 of exon 1 of TRBC1*01 or TRBC2*01; Thr45 of exon 1 of TRAC*01 and Asp59 of exon 1 of TRBC1*01 or TRBC2*01; Ser15 of exon 1 of TRAC*01 and Glu15 of exon 1 of TRBC1*01 or TRBC2*01; Arg53 of exon 1 of TRAC*01 and Ser54 of exon 1 of TRBC1*01 or TRBC2*01; Pro89 of exon 1 of TRAC*01 and Ala19 of exon 1 of TRBC1*01 or TRBC2*01; and Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01.

10. The TCR according to claim 1, wherein: The amino acid sequence of the α chain variable domain of the TCR is one of SEQ ID NOs: 13-58; and / or the amino acid sequence of the β chain variable domain of the TCR is SEQ ID NO:

2.

11. The TCR according to claim 1, wherein: The TCR is a single-chain TCR; preferably, 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).

12. A TCR as claimed in any preceding claim, characterised in that The C- or N-terminus of the α chain and / or β chain of the TCR is bound to a conjugate; preferably, The conjugate is a detectable marker or a therapeutic agent; more preferably, the therapeutic agent is an anti-CD3 antibody.

13. 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 described in claim 1.

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

15. A carrier, characterized in that The vector contains the nucleic acid molecule described in claim 14.

16. A host cell, characterized in that The host cell contains the vector described in claim 15 or the chromosome in which the exogenous nucleic acid molecule described in claim 14 is integrated.

17. An isolated cell, characterized in that The cell expresses the TCR described in claim 1, and preferably, the isolated cell is a T cell.

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

19. A pharmaceutical composition, characterized in that The composition contains the TCR described in claim 1, or the TCR complex described in claim 13, or the isolated cell described in claim 17 or 18.

20. A method for treating a disease, characterized in that: The method comprises administering the TCR described in claim 1, or the TCR complex described in claim 13, or the cell described in claim 17 or 18, or the pharmaceutical composition described in claim 19 to a subject in need of treatment. Preferably, the disease is a MAGE-A4 positive tumor, including melanoma, and other solid tumors such as gastric cancer, lung cancer, esophageal cancer, bladder cancer, head and neck squamous cell carcinoma, etc.

21. Use of the T cell receptor of claim 1, the TCR complex of claim 13, or the cell of claim 17 or 18 in preparing a specific binding agent for a target protein.

22. The use according to claim 21, characterized in that 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 MAGE-A4 positive tumors.

23. Use of the T cell receptor according to claim 1, the TCR complex according to claim 13 or the cell according to claim 17 or 18, characterized in that: Used for preparing a drug for treating tumors, preferably, the tumor is a MAGE-A4 positive tumor, including melanoma, and other solid tumors such as gastric cancer, lung cancer, esophageal cancer, bladder cancer, head and neck squamous cell carcinoma, etc.

24. A method for preparing the T cell receptor according to claim 1, characterized in that: Includes steps: (i) culturing the cell of claim 16 so as to express the T cell receptor of claim 1; (ii) isolating or purifying the T cell receptor.

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