TCR for identifying MAGE-A4 and coding sequence thereof

Abstract

The invention provides a T cell receptor (TCR) capable of being specifically combined with a short peptide GVYDGREHTV derived from an MAGE-A4 antigen. The antigen short peptide GVYDGREHTV and HLA A0201 can form a compound, and the compound and the HLA A0201 are presented to the surface of a cell together. The invention also provides a nucleic acid molecule for coding the TCR and a carrier containing the nucleic acid molecule. In addition, the invention also provides a cell transduced with the TCR provided by the invention.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and specifically relates to a T cell receptor that recognizes MAGE-A4 and its application, and more particularly to a TCR capable of recognizing a short peptide derived from a MAGE-A4 antigen and its coding sequence, MAGE-A4-specific T cells obtained by transducing the TCR, and its use in preventing and / or treating MAGE-A4-related diseases. Background Art

[0002] 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. The MAGE-A4 protein is expressed in a variety of 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.

[0003] T cell adoptive immunotherapy involves transferring reactive T cells specific for target cell antigens into a patient's body, allowing them to exert their effects against the target cells. The T cell receptor (TCR) is a membrane protein on the surface of T cells that can recognize short antigenic peptides on the surface of corresponding target cells. In the immune system, the binding of the antigenic peptide-specific TCR to the peptide-major histocompatibility complex (pMHC complex) triggers direct physical contact between T cells and antigen-presenting cells (APCs). Subsequently, other cell membrane surface molecules of both the T cell and the APC interact, triggering a series of subsequent cell signaling and other physiological reactions, thereby enabling T cells with different antigenic specificities to exert their immune effects on their target cells.

[0004] Therefore, those skilled in the art have devoted themselves to isolating TCRs specific to MAGE-A4 antigen short peptides and transducing the TCRs into T cells to obtain T cells specific to MAGE-A4 antigen short peptides, thereby enabling them to play a role in cellular immunotherapy. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a T cell receptor that recognizes MAGE-A4 and its application.

[0006] To achieve the purpose of this invention, the technical solution of the present invention includes:

[0007] In a first aspect, the present invention provides a T cell receptor (TCR), wherein the TCR comprises a TCR α chain variable domain and a TCR β chain variable domain, and the TCR is capable of binding to the GVYDGREHTV-HLAA0201 complex.

[0008] In another preferred embodiment, the three complementarity determining regions (CDRs) of the TCRα chain variable domain are:

[0009] αCDR1-VSGLRG SEQ ID NO: 10,

[0010] αCDR2-LYSAGEE SEQ ID NO:11, and,

[0011] αCDR3-AVHPLNYGGSQGNLI SEQ ID NO: 12; and / or

[0012] The three complementarity determining regions of the TCRβ chain variable domain are:

[0013] βCDR1-DFQATT SEQ ID NO: 13,

[0014] βCDR2-SNEGSKA SEQ ID NO: 14, and

[0015] βCDR3-SAREWRGTEAF SEQ ID NO:15.

[0016] In another preferred example, the TCR includes a TCRα chain variable domain and a TCRβ chain variable domain, and the sequence of the TCRα chain variable domain is an amino acid sequence that has at least 90% (for example, it can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, etc.) sequence identity with SEQ ID NO:1; and / or the sequence of the TCRβ chain variable domain is an amino acid sequence that has at least 90% (for example, it can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, etc.) sequence identity with SEQ ID NO:5.

[0017] In another preferred embodiment, the TCR sequence comprises the amino acid sequence of the α chain variable domain SEQ ID NO: 1.

[0018] In another preferred example, the sequence of the TCR comprises the amino acid sequence of the β chain variable domain SEQ ID NO: 5.

[0019] In another preferred embodiment, the TCR is an αβ heterodimer, and the αβ heterodimer comprises the TCRα chain constant region TRAC*01 and the TCRβ chain constant region TRBC1*01 or TRBC2*01.

[0020] In another preferred example, the amino acid sequence of the α chain of the TCR is SEQ ID NO: 3 and / or the amino acid sequence of the β chain of the TCR is SEQ ID NO: 7.

[0021] In another preferred embodiment, the TCR is of human origin.

[0022] In another preferred embodiment, the TCR is soluble.

[0023] In another preferred embodiment, the TCR is isolated or purified.

[0024] In another preferred embodiment, the TCR is single chain.

[0025] In another preferred embodiment, the TCR is composed of an α chain variable domain and a β chain variable domain connected by a peptide linker.

[0026] In another preferred embodiment, the constant regions of the α and β chains of the TCR are respectively the constant regions of the α and β chains of mouse origin.

[0027] In another preferred embodiment, the TCR has one or more mutations at amino acid position 11, 13, 19, 21, 53, 76, 89, 91, or 94 of the α chain variable region, and / or the third to last, fifth to last, or seventh to last amino acid of the α chain J gene short peptide; and / or the TCR has one or more mutations at amino acid position 11, 13, 19, 21, 53, 76, 89, 91, or 94 of the β chain variable region, and / or the second to last, fourth to last, or sixth to last amino acid of the β chain J gene short peptide, wherein the amino acid positions are numbered according to the position numbers listed in IMGT (International Immunogenetics Information System).

[0028] In another preferred example, the amino acid sequence of the α chain variable domain of the TCR comprises SEQ ID NO: 30 and / or the amino acid sequence of the β chain variable domain of the TCR comprises SEQ ID NO: 32.

[0029] In another preferred embodiment, the amino acid sequence of the TCR is SEQ ID NO: 28.

[0030] In another preferred embodiment, the TCR comprises (a) all or part of the TCRα chain except the transmembrane domain; and (b) all or part of the TCRβ chain except the transmembrane domain;

[0031] And (a) and (b) each comprise a functional variable domain, or comprise a functional variable domain and at least a portion of the TCR chain constant domain.

[0032] In another preferred embodiment, the cysteine ​​residues form an artificial disulfide bond between the constant domains of the α and β chains of the TCR.

[0033] In another preferred embodiment, the cysteine ​​residues forming artificial disulfide bonds in the TCR are substituted with one or more sites selected from the following groups:

[0034] Thr48 of exon 1 of TRAC*01 and Ser57 of exon 1 of TRBC1*01 or TRBC2*01;

[0035] Thr45 of exon 1 of TRAC*01 and Ser77 of exon 1 of TRBC1*01 or TRBC2*01;

[0036] Tyr10 of exon 1 of TRAC*01 and Ser17 of exon 1 of TRBC1*01 or TRBC2*01;

[0037] Thr45 of exon 1 of TRAC*01 and Asp59 of exon 1 of TRBC1*01 or TRBC2*01;

[0038] Ser15 of exon 1 of TRAC*01 and Glu15 of exon 1 of TRBC1*01 or TRBC2*01;

[0039] Arg53 of exon 1 of TRAC*01 and Ser54 of exon 1 of TRBC1*01 or TRBC2*01;

[0040] Pro89 of exon 1 of TRAC*01 and Ala19 of exon 1 of TRBC1*01 or TRBC2*01; and,

[0041] or Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01.

[0042] In another preferred example, the amino acid sequence of the α chain of the TCR is SEQ ID NO: 26 and / or the amino acid sequence of the β chain of the TCR is SEQ ID NO: 27.

[0043] In another preferred embodiment, the TCR comprises an artificial interchain disulfide bond between the α chain variable region and the β chain constant region.

[0044] In another preferred embodiment, the cysteine ​​residues forming the artificial interchain disulfide bonds in the TCR are substituted with one or more sites selected from the following groups:

[0045] amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01;

[0046] amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01;

[0047] amino acid 46 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01;

[0048] and amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01.

[0049] In another preferred embodiment, the TCR comprises an α chain variable domain and a β chain variable domain and all or part of the β chain constant domain excluding the transmembrane domain, but does not comprise an α chain constant domain, and the α chain variable domain of the TCR forms a heterodimer with the β chain.

[0050] In another preferred embodiment, a conjugate is bound to the C- or N-terminus of the α chain of the TCR, and / or a conjugate is bound to the C- or N-terminus of the β chain of the TCR.

[0051] In another preferred embodiment, the conjugate that binds to the T cell receptor comprises any one or a combination of at least two of a detectable label, a therapeutic agent, and a PK modifying moiety.

[0052] Preferably, the therapeutic agent is an anti-CD3 antibody.

[0053] The second aspect of the present invention provides a multivalent TCR complex, wherein the multivalent TCR complex comprises at least two TCR molecules, and at least one of the TCR molecules is the TCR described in the first aspect.

[0054] The third aspect of the present invention provides a nucleic acid molecule, the sequence of which includes a nucleotide sequence encoding the TCR described in the first aspect of the present invention, and / or a complementary sequence of the nucleotide sequence encoding the TCR described in the first aspect of the present invention.

[0055] In another preferred embodiment, the nucleic acid molecule is isolated or purified.

[0056] In another preferred example, the sequence of the nucleic acid molecule includes the nucleotide sequence encoding the TCR α chain variable domain SEQ ID NO: 2 or SEQ ID NO: 31.

[0057] In another preferred example, the sequence of the nucleic acid molecule includes the nucleotide sequence encoding the TCRβ chain variable domain SEQ ID NO: 6 or SEQ ID NO: 33.

[0058] In another preferred example, the sequence of the nucleic acid molecule includes the nucleotide sequence encoding the TCRα chain of SEQ ID NO: 4 and / or the nucleotide sequence encoding the TCRβ chain of SEQ ID NO: 8.

[0059] 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.

[0060] Preferably, the vector is a viral vector.

[0061] Preferably, the viral vector is a lentiviral vector.

[0062] The fifth aspect of the present invention provides an isolated host cell, wherein the isolated 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 genome of the isolated host cell.

[0063] The sixth aspect of the present invention provides an isolated cell, wherein the cell is transduced with the nucleic acid molecule according to the third aspect of the present invention or the vector according to the fourth aspect of the present invention.

[0064] Preferably, the cells include T cells, NK cells, NKT cells or stem cells.

[0065] 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, the nucleic acid molecule described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, and the cell described in the sixth aspect of the present invention.

[0066] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0067] The eighth aspect of the present invention provides the use of 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 cells described in the sixth aspect of the present invention, wherein the use includes the preparation of a drug for treating tumors or autoimmune diseases.

[0068] 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.

[0069] The ninth aspect of the present invention provides 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 cell described in the sixth aspect of the present invention for use as a drug for treating tumors or autoimmune diseases.

[0070] 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.

[0071] The tenth aspect of the present invention provides a method for treating a disease, comprising administering to a subject in need of treatment an appropriate amount of any one or a combination of at least two of the TCR described in the first aspect of the present invention, the TCR complex described in the second aspect of the present invention, the cell described in the sixth aspect of the present invention, and the pharmaceutical composition described in the seventh aspect of the present invention.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] Compared with the traditional technology, the present invention has the following beneficial effects:

[0076] (1) The TCR of the present invention can specifically bind to the MAGE-A4 mutant antigen short peptide complex GVYDGREHTV-HLA A0201, and the effector cells transduced with the TCR of the present invention can be specifically activated.

[0077] (2) The TCR of the present invention has a strong specific killing effect on MAGE-A4 positive tumor cell lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 This is the result of CD8-APC and tetramer-PE double positive staining of monoclonal cells;

[0079] Figure 2 is the SDS-PAGE electrophoresis gel image of the soluble TCR obtained after purification; wherein, Figure 2 a and Figure 2b The right lanes are reducing gel and non-reducing gel, respectively, and the left lanes are molecular weight markers;

[0080] Figure 3 The figure shows the SDS-PAGE electrophoresis gel of the purified soluble single-chain TCR. The right lane is a reducing gel, the left lane is a non-reducing gel, and the middle lane is a molecular weight marker.

[0081] Figure 4 BIAcore kinetic profile of the binding of soluble TCR to the GVYDGREHTV-HLA A0201 complex;

[0082] Figure 5 This is the BIAcore kinetic profile of the binding of soluble single-chain TCR to the GVYDGREHTV-HLA A0201 complex;

[0083] Figure 6 The results of ELISPOT activation function verification of the obtained T cell clones;

[0084] Figure 7 This is the ELISPOT activation function verification result of effector cells transfected with the TCR of the present invention against T2 cells loaded with short peptides;

[0085] Figure 8 The results of ELISPOT activation function verification of effector cells transfected with the TCR of the present invention for T2 cells loaded with short peptides at gradient concentrations;

[0086] Figure 9 The results are experimental results of activation function of IFN-γ released by effector cells transfected with the TCR of the present invention against tumor cell lines. DETAILED DESCRIPTION

[0087] After extensive and in-depth research, the inventors have discovered a TCR that specifically binds to the MAGE-A4 antigen peptide GVYDGREHTV (SEQ ID NO: 9). This peptide can form a complex with HLA A0201 and be presented on the cell surface. The present invention also provides a nucleic acid molecule encoding this TCR and a vector containing this nucleic acid molecule. Furthermore, the present invention provides cells transduced with this TCR.

[0088] the term

[0089] MHC molecules are proteins from the immunoglobulin superfamily and can be either class I or class II. Therefore, they are specific for antigen presentation. Different individuals have different MHCs, capable of presenting different short peptides from a protein antigen on their APC cell surfaces. The human MHC is often referred to as the HLA gene or HLA complex.

[0090] The T cell receptor (TCR) is the only receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC). In the immune system, the binding of the antigen-specific TCR to the pMHC complex triggers direct physical contact between the T cell and the antigen-presenting cell (APC). Subsequently, other cell membrane surface molecules of both the T cell and the APC 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.

[0091] TCRs are glycoproteins found on the cell membrane, consisting of heterodimers of either α / β or γ / δ chains. In 95% of T cells, TCR heterodimers are composed of α and β chains, while 5% have TCRs composed of γ and δ chains. Natural αβ heterodimeric TCRs have α and β chains, which together constitute the subunits of the αβ heterodimeric TCR. Broadly speaking, each α and β 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. Each variable region consists of three complementarity-determining regions (CDRs): CDR1, CDR2, and CDR3, embedded within a framework structure. The CDR regions determine TCR binding to the pMHC complex. CDR3, formed by the recombination of the variable and joining regions, is known as the hypervariable region. The α and β chains of TCRs are generally considered to each have two "domains": a variable domain and a constant domain. The variable domain consists of a variable region and a connecting region. Sequences of TCR constant domains can be found in the public database of the International Immunogenetics Information System (IMGT). For example, the constant domain sequence of the TCR α chain is "TRAC*01," and the constant domain sequence of the TCR β chain is "TRBC1*01" or "TRBC2*01." In addition, the TCR α and β chains also contain transmembrane regions and cytoplasmic regions, the latter of which are very short.

[0092] 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.

[0093] Natural interchain disulfide bonds and artificial interchain disulfide bonds

[0094] 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."

[0095] To facilitate the description of the position of the disulfide bond, 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, if the 60th amino acid in the order from the N-terminus to the C-terminus in TRBC1*01 or TRBC2*01 is P (proline), then it can be described in the present invention 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, if the 61st amino acid in the order from the N-terminus to the C-terminus in TRBC1*01 or TRBC2*01 is Q (glutamine), then it can be described in the present invention 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. Detailed Description of the Invention

[0097] TCR molecules

[0098] During antigen processing, antigens are degraded within the cell and then carried to the cell surface by MHC molecules. T cell receptors are able to recognize peptide-MHC complexes on the surface of antigen-presenting cells. Therefore, a first aspect of the present invention provides a TCR molecule capable of binding to the GVYDGREHTV-HLAA0201 complex.

[0099] Preferably, the TCR molecule is isolated or purified. The α and β chains of the TCR each have three complementarity determining regions (CDRs).

[0100] In a preferred embodiment of the present invention, the α chain of the TCR comprises a CDR having the following amino acid sequence:

[0101] αCDR1-VSGLRG SEQ ID NO: 10,

[0102] αCDR2-LYSAGEE SEQ ID NO:11, and,

[0103] αCDR3-AVHPLNYGGSQGNLI SEQ ID NO: 12; and / or

[0104] The three complementarity determining regions of the TCRβ chain variable domain are:

[0105] βCDR1-DFQATT SEQ ID NO: 13,

[0106] βCDR2-SNEGSKA SEQ ID NO: 14, and

[0107] βCDR3-SAREWRGTEAF SEQ ID NO:15.

[0108] The CDR region amino acid fragments of the present invention can be embedded into any suitable framework structure to prepare a chimeric TCR. As long as the framework structure is compatible with the CDR region of the TCR of the present invention, those skilled in the art can design or synthesize TCR molecules with corresponding functions based on the CDR regions disclosed in the present invention. Therefore, the TCR molecule of the present invention refers to a TCR molecule comprising the above-mentioned α and / or β chain CDR region sequences and any suitable framework structure.

[0109] The sequence of the TCR α chain variable domain of the present invention is an amino acid sequence having at least 90%, preferably 95%, and more preferably 98% sequence identity with SEQ ID NO: 1;

[0110] And / or the sequence of the TCR β chain variable domain of the present invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity with SEQ ID NO: 5.

[0111] In a preferred embodiment of the present invention, the TCR molecule of the present invention is a heterodimer composed of α and β chains. Specifically, on one hand, the α chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the amino acid sequence of the α chain variable domain comprises CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11), and CDR3 (SEQ ID NO: 12) of the above-mentioned α chain.

[0112] Preferably, the sequence of the TCR molecule comprises the amino acid sequence of the α chain variable domain SEQ ID NO: 1.

[0113] More preferably, the amino acid sequence of the α chain variable domain of the TCR molecule is SEQ ID NO: 1.

[0114] On the other hand, the β chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the amino acid sequence of the β chain variable domain comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 14) and CDR3 (SEQ ID NO: 15) of the above-mentioned β chain.

[0115] Preferably, the sequence of the TCR molecule comprises the amino acid sequence of the β chain variable domain SEQ ID NO: 5.

[0116] More preferably, the amino acid sequence of the β chain variable domain of the TCR molecule is SEQ ID NO: 5.

[0117] In a preferred embodiment of the present invention, the TCR molecule of the present invention is a single-chain TCR molecule composed of part or all of the α chain and / or part or all of the β chain. For a description of single-chain TCR molecules, reference can be made to the document Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-12658. According to the description in the literature, those skilled in the art can easily construct a single-chain TCR molecule comprising the CDRs region of the present invention. Specifically, the single-chain TCR molecule comprises Vα, Vβ and Cβ, preferably connected in the order from N-terminus to C-terminus.

[0118] The amino acid sequence of the α chain variable domain of the single-chain TCR molecule comprises CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the above-mentioned α chain.

[0119] Preferably, the sequence of the single-chain TCR molecule comprises the amino acid sequence of the α chain variable domain SEQ ID NO: 1.

[0120] More preferably, the amino acid sequence of the α chain variable domain of the single-chain TCR molecule is SEQ ID NO: 1.

[0121] The amino acid sequence of the β chain variable domain of the single-chain TCR molecule comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 14) and CDR3 (SEQ ID NO: 15) of the above-mentioned β chain.

[0122] Preferably, the sequence of the single-chain TCR molecule comprises the amino acid sequence of the β chain variable domain SEQ ID NO: 5.

[0123] More preferably, the amino acid sequence of the β chain variable domain of the single-chain TCR molecule is SEQ ID NO: 5.

[0124] In a preferred embodiment of the present invention, the constant domain of the TCR molecule of the present invention is a human constant domain. Those skilled in the art know or can obtain the human constant domain amino acid sequence by consulting relevant books or the public database of IMGT (International Immunogenetics Information System). For example, the constant domain sequence of the α chain of the TCR molecule of the present invention can be "TRAC*01", and the constant domain sequence of the β chain of the TCR molecule can be "TRBC1*01" or "TRBC2*01". The 53rd position of the amino acid sequence given in TRAC*01 of IMGT is Arg, which is expressed herein as: Arg53 of TRAC*01 exon 1, and the same applies to the others. Preferably, the amino acid sequence of the α chain of the TCR molecule of the present invention is SEQ ID NO: 3, and / or the amino acid sequence of the β chain is SEQ ID NO: 7.

[0125] Naturally occurring TCRs are membrane proteins that are stabilized by their transmembrane regions. Just as immunoglobulins (antibodies) serve as antigen recognition molecules, TCRs can also be developed for diagnostic and therapeutic applications, requiring the production of soluble TCR molecules. Soluble TCR molecules do not include their transmembrane regions. Soluble TCRs have a wide range of uses. They can be used not only to study the interaction between TCRs and pMHC, but also as diagnostic tools for detecting infections or as markers for autoimmune diseases. Similarly, soluble TCRs can be used to deliver therapeutic agents (such as cytotoxic compounds or immunostimulatory compounds) to cells presenting specific antigens. In addition, soluble TCRs can be combined with other molecules (such as anti-CD3 antibodies) to redirect T cells, thereby targeting cells presenting specific antigens. The present invention also obtains soluble TCRs that are specific for short peptides of the MAGE-A4 antigen.

[0126] To obtain a soluble TCR, in one aspect, the TCR of the present invention can be a TCR that has artificial disulfide bonds introduced between residues in its α and β chain constant domains. Cysteine ​​residues form artificial interchain disulfide bonds between the α and β chain constant domains of the TCR. Cysteine ​​residues can replace other amino acid residues at appropriate sites in the native TCR to form artificial interchain disulfide bonds. For example, cysteine ​​residues can be substituted for Thr48 in exon 1 of TRAC*01 and Ser57 in exon 1 of TRBC1*01 or TRBC2*01 to form disulfide bonds. Other sites for introducing cysteine ​​residues to form disulfide bonds can also be:

[0127] Thr45 of exon 1 of TRAC*01 and Ser77 of exon 1 of TRBC1*01 or TRBC2*01;

[0128] Tyr10 of exon 1 of TRAC*01 and Ser17 of exon 1 of TRBC1*01 or TRBC2*01;

[0129] Thr45 of exon 1 of TRAC*01 and Asp59 of exon 1 of TRBC1*01 or TRBC2*01;

[0130] Ser15 of exon 1 of TRAC*01 and Glu15 of exon 1 of TRBC1*01 or TRBC2*01;

[0131] Arg53 of exon 1 of TRAC*01 and Ser54 of exon 1 of TRBC1*01 or TRBC2*01;

[0132] Pro89 of exon 1 of TRAC*01 and Ala19 of exon 1 of TRBC1*01 or TRBC2*01;

[0133] or Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01.

[0134] 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 of the TCR constant domains of the present invention can be truncated by up to 50, or up to 30, or up to 15, or up to 10, or up to 8 or fewer amino acids to eliminate the cysteine ​​residues and thereby eliminate the native disulfide bonds. This can also be achieved by mutating the cysteine ​​residues that form the native disulfide bonds to another amino acid.

[0135] As described above, the TCRs of the present invention may include artificial 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 natural disulfide bond present in the TCR.

[0136] To obtain a soluble TCR, on the other hand, the TCR of the present invention also includes a TCR in which a mutation occurs in its hydrophobic core region, and the mutations in these hydrophobic core regions are preferably mutations that can improve the stability of the soluble TCR of the present invention, as described in the patent document with publication number WO2014 / 206304. Such a 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.

[0137] The TCR with a mutation in the hydrophobic core region of the present invention can be a stable, soluble single-chain TCR composed of a flexible peptide chain connecting the variable domains of the α and β chains of the TCR. 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 α and β chains of the TCR.

[0138] In addition, with regard to stability, patent document 201680003540.2 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 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:

[0139] amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01;

[0140] amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01;

[0141] amino acid 46 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01;

[0142] or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01.

[0143] Preferably, such a 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 forms a heterodimer with the β chain.

[0144] More preferably, such a 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] Therapeutic agents that can be combined or coupled to the TCR of the present invention include, but are not limited to:

[0149] 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539);

[0150] 2. Biological toxicity (Chaudhary et al., 1989, Nature 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy 51, 565);

[0151] 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);

[0152] 4. Antibody Fc fragment (Mosquera et al., 2005, The Journal of Immunology 174, 4381);

[0153] 5. Antibody scFv fragment (Zhu et al., 1995, International Journal of Cancer 62, 319);

[0154] 6. Gold nanoparticles / nanorods (Lapotko et al., 2005, Cancer letters 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115);

[0155] 7. Viral particles (Peng et al., 2004, Gene therapy 11, 1234);

[0156] 8. Liposomes (Mamot et al., 2005, Cancer Research 65, 11631);

[0157] 9. Nanomagnetic particles;

[0158] 10. Prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL));

[0159] 11. Chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, etc.

[0160] In addition, the TCR of the present invention can also be a hybrid TCR containing sequences derived from more than one species. For example, studies have shown that murine TCRs can be expressed more effectively in human T cells than human TCRs. Therefore, the TCR of the present invention can contain human variable domains and murine constant domains. The disadvantage of this method is that it may trigger an immune response. Therefore, when it is used in adoptive T cell therapy, a regulatory program should be included to perform immunosuppression to allow the implantation of murine T cells expressing murine T cells.

[0161] It should be understood that the amino acid names herein are represented by single letters or three letters in the internationally accepted English alphabet, and the correspondence between single letters and three letters of the amino acid names is as follows: 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), Val (V).

[0162] Nucleic acid molecules

[0163] The second aspect of the invention provides a nucleic acid molecule encoding the TCR molecule of the first aspect of the invention or a portion thereof, which portion may be one or more CDRs, the variable domains of the α and / or β chains, and the α chain and / or β chain.

[0164] The nucleotide sequence encoding the CDR region of the α chain of the TCR molecule of the first aspect of the present invention is as follows:

[0165] CDR1α-gtcagcggtttaagaggg SEQ ID NO: 16,

[0166] CDR2α-ctgtattcagctggggaagaa SEQ ID NO: 17, and,

[0167] CDR3α-gctgtgcacccattgaattatggaggaagccaaggaaatctcatc SEQ ID NO: 18.

[0168] The nucleotide sequence encoding the CDR region of the β chain of the TCR molecule of the first aspect of the present invention is as follows:

[0169] CDR1β-gactttcaggccacaact SEQ ID NO:19,

[0170] CDR2β-tccaatgagggctccaaggcc SEQ ID NO: 20, and,

[0171] CDR3β-agtgctagagaatggcggggcactgaagctttc SEQ ID NO: 21.

[0172] Therefore, the nucleotide sequences of nucleic acid molecules encoding the TCR α chain of the present invention include SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and / or the nucleotide sequences of nucleic acid molecules encoding the TCR β chain of the present invention include SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.

[0173] The nucleic acid molecules of the present invention may be single-stranded or double-stranded, may be RNA or DNA, and may or may not contain introns.

[0174] Preferably, the nucleic acid molecule of the present invention does not contain an intron but can encode a polypeptide of the present invention, for example, the nucleotide sequence of the nucleic acid molecule encoding the TCR α chain variable domain of the present invention includes SEQ ID NO: 2, and / or the nucleotide sequence of the nucleic acid molecule encoding the TCR β chain variable domain of the present invention includes SEQ ID NO: 6. Alternatively, the nucleotide sequence of the nucleic acid molecule encoding the TCR α chain variable domain of the present invention includes SEG ID NO: 31, and / or the nucleotide sequence of the nucleic acid molecule encoding the TCR β chain variable domain of the present invention includes SEG ID NO: 33.

[0175] More preferably, the nucleotide sequence of the nucleic acid molecule of the present invention comprises SEQ ID NO: 4 and / or SEQ ID NO: 8. Alternatively, the nucleotide sequence of the nucleic acid molecule of the present invention is SEQ ID NO: 29.

[0176] It should be understood that due to the degeneracy of the genetic code, different nucleotide sequences can encode the same polypeptide. Therefore, the nucleic acid sequence encoding the TCR of the present invention may be identical to the nucleic acid sequence set forth herein or a degenerate variant. For example, a "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes the protein sequence of SEQ ID NO: 1 but differs from the nucleotide sequence of SEQ ID NO: 2.

[0177] The nucleotide sequence may be codon-optimized. Different cells utilize different codons, and expression can be increased by altering the codons in the sequence depending on the cell type. Codon usage tables for mammalian cells and various other organisms are well known to those skilled in the art.

[0178] 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. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. The DNA can be either a coding strand or a non-coding strand.

[0179] carrier

[0180] The present invention also relates to vectors comprising the nucleic acid molecules of the present invention, including expression vectors, i.e. constructs capable of in vivo or in vitro expression. Commonly used vectors include bacterial plasmids, bacteriophages, and animal and plant viruses.

[0181] Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors.

[0182] Preferably, the vector can transfer the nucleic acid of the present invention into cells, such as T cells, so that the cells express TCR specific for the MAGE-A4 antigen. Ideally, the vector should be able to be expressed at a high level in T cells.

[0183] cell

[0184] The present invention also relates to host cells produced by genetic engineering using the vector or coding sequence of the present invention. The host cells contain the vector of the present invention or have the nucleic acid molecule of the present invention integrated into their chromosomes. The host cells are selected from prokaryotic and eukaryotic cells, such as Escherichia coli, yeast cells, or CHO cells.

[0185] In addition, the present invention also includes isolated cells expressing the TCR of the present invention, which may be, but are not limited to, T cells, NK cells, NKT cells, stem cells, and in particular T cells. The T cells may be derived from T cells isolated from a subject, or may be a mixed cell population isolated from a subject, such as a portion of a peripheral blood lymphocyte (PBL) population. For example, the cells may be isolated from peripheral blood mononuclear cells (PBMCs), which may be CD4 + Helper T cells or CD8 + Cytotoxic T cells. These cells can be found in CD4 + Helper T cells / CD8 + Typically, the cells can be activated with antibodies (e.g., anti-CD3 or anti-CD28 antibodies) to render them more receptive to transfection, for example, with a vector comprising a nucleotide sequence encoding a TCR molecule of the invention.

[0186] Alternatively, the cells of the present invention may also be or be derived from stem cells, such as hematopoietic stem cells (HSCs). Gene transfer to HSCs will not result in TCR expression on the cell surface because CD3 molecules are not expressed on the stem cell surface. However, when the stem cells differentiate into lymphoid precursors that migrate to the thymus, the expression of CD3 molecules will initiate expression of the introduced TCR molecules on the surface of the thymocytes.

[0187] There are many methods suitable for transfecting T cells with DNA or RNA encoding the TCR of the present invention (e.g., Robbins et al., (2008) J. Immunol. 180:6116-6131). T cells expressing the TCR of the present invention can be used for adoptive immunotherapy. Those skilled in the art will be aware of many suitable methods for adoptive therapy (e.g., Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).

[0188] MAGE-A4 antigen-associated diseases

[0189] The present invention also relates to a method for treating and / or preventing MAGE-A4-related diseases, including melanoma, and other solid tumors such as gastric cancer, lung cancer, esophageal cancer, bladder cancer, head and neck squamous cell carcinoma, etc., in a subject, comprising adoptively transferring MAGE-A4-specific T cells into the subject. The MAGE-A4-specific T cells can recognize the GVYDGREHTV-HLA A0201 complex.

[0190] The MAGE-A4-specific T cells of the present invention can be used to treat any MAGE-A4-related disease that presents the MAGE-A4 antigen short peptide GVYDGREHTV-HLAA0201 complex, including melanoma, and other solid tumors such as gastric cancer, lung cancer, esophageal cancer, bladder cancer, head and neck squamous cell carcinoma, etc.

[0191] Treatment

[0192] Treatment can be achieved by isolating T cells from patients or volunteers suffering from diseases associated with the MAGE-A4 antigen, including melanoma, and other solid tumors such as gastric cancer, lung cancer, esophageal cancer, bladder cancer, head and neck squamous cell carcinoma, and the like, and introducing the TCR of the present invention into the aforementioned T cells, and then returning these genetically modified cells to the patient. Therefore, the present invention provides a method for treating MAGE-A4-related diseases, including melanoma, and other solid tumors such as gastric cancer, lung cancer, esophageal cancer, bladder cancer, head and neck squamous cell carcinoma, and the like, comprising infusing isolated T cells expressing the TCR of the present invention, preferably derived from the patient, into the patient. Generally, the method comprises (1) isolating T cells from the patient, (2) transducing the T cells in vitro with the nucleic acid molecule of the present invention or a nucleic acid molecule capable of encoding the TCR molecule of the present invention, and (3) infusing the genetically modified T cells into the patient. The number of cells isolated, transfected, and returned can be determined by the physician.

[0193] The main advantages of the present invention are

[0194] The TCR of the present invention can specifically bind to the MAGE-A4 antigen short peptide complex GVYDGREHTV-HLA A0201, and the effector cells transduced with the TCR of the present invention can be specifically activated.

[0195] 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 for which specific conditions are not specified in the following examples are generally performed under conventional conditions, such as those described in (Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (Molecular Cloning-A Laboratory Manual) (3rd edition) (2001) CSHL Press), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise stated.

[0196] Example 1 Cloning of MAGE-A4 antigen short peptide-specific T cells

[0197] The synthetic short peptide GVYDGREHTV (SEQ ID NO: 9, synthesized by Jiangsu KingSher Biotechnology Co., Ltd.) was used to stimulate peripheral blood lymphocytes (PBL) from healthy volunteers with the genotype of HLA-A0201. The GVYDGREHTV short peptide was renatured with HLA-A0201 labeled with biotin to prepare pMHC haploids. These haploids were combined with PE-labeled streptavidin (BD Company) to form PE-labeled tetramers, and the tetramers and anti-CD8-APC double-positive cells were sorted. The sorted cells were amplified and secondary sorted according to the above method, and then monocloned by limiting dilution. The monoclonal cells were stained with tetramers, and the double-positive clones screened were as follows. Figure 1 As shown, Figure 1 This is the result of double-positive staining of a monoclonal cell for CD8-APC and tetramer-PE. Double-positive clones obtained through multiple rounds of screening must undergo further functional testing.

[0198] IFN-γ is a powerful immunomodulatory factor produced by activated T lymphocytes. Therefore, in this example, IFN-γ levels were detected by ELISPOT assays well known to those skilled in the art to verify the activation function and antigen specificity of cells transfected with the TCR of the present invention. The function and specificity of the T cell clones were further tested by ELISPOT assays. The effector cells used in the IFN-γ ELISPOT assays in this example were T cell clones obtained in the present invention. The target cells were T2-A0201 cells (referring to T2 cells transfected with HLA-A0201) and SW620 (MAGE-A4 overexpression) loaded with GVYDGREHTV short peptides, and the control group was T2-A0201 cells and NUGC4 cells loaded with other antigenic short peptides. T2 cells were purchased from ATCC, SW620 cells were purchased from ATCC, and NUGC4 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd.

[0199] First, prepare the ELISPOT plate. The ELISPOT experimental steps are as follows: add the various components of the test to the ELISPOT plate in the following order: 20,000 target cells / well, 2,000 effector cells / well, then add 20 μL of the corresponding short peptide to the experimental group and the control group. The final concentration of the short peptide loaded with T2 is 10-5M. Add 20 μL of culture medium (test culture medium) to the blank group, and set up 2 replicate wells. Then incubate overnight (37°C, 5% CO2). The plate is then washed and subjected to secondary detection and color development. The plate is dried for 1 hour, and then the spots formed on the membrane are counted using an immunospot plate reader (ELISPOT READERsystem; AID Company). The experimental results are shown in Figure 2. Figure 6 As shown, Figure 6 The following are the results of ELISPOT activation validation of the obtained T cell clones. The obtained T cell clones highly released IFN-γ in response to T2-A0201 cells loaded with GVYDGREHTV peptide and SW620 cells (overexpressing MAGE-A4), but had little response to T2-A0201 cells loaded with other antigenic peptides and NUGC4.

[0200] Example 2 Obtaining TCR Genes of MAGE-A4 Antigen Short Peptide-Specific T Cell Clones

[0201] Using Quick-RNA TMMiniPrep (ZYMO Research) was used to extract total RNA from the HLA-A0201-restricted T cell clones specific for the short antigen peptide GVYDGREHTV identified in Example 1. cDNA was synthesized using the Clontech SMART RACE cDNA Amplification Kit with primers designed for the conserved C-terminal region of the human TCR gene. The sequence was cloned into a T vector (TAKARA) for sequencing. It should be noted that the sequence described is complementary and does not contain introns.

[0202] After sequencing, the TCRs expressed by double-positive clones were:

[0203] The amino acid sequence of the TCR α chain variable domain is set forth in SEQ ID NO: 1, the nucleotide sequence of the TCR α chain variable domain is set forth in SEQ ID NO: 2, the amino acid sequence of the TCR α chain is set forth in SEQ ID NO: 3, the nucleotide sequence of the TCR α chain is set forth in SEQ ID NO: 4, the amino acid sequence of the TCR α chain with a leader sequence is set forth in SEQ ID NO: 22, and the nucleotide sequence of the TCR α chain with a leader sequence is set forth in SEQ ID NO: 23;

[0204] The amino acid sequence of the TCRβ chain variable domain is shown in SEQ ID NO: 5, the nucleotide sequence of the TCRβ chain variable domain is shown in SEQ ID NO: 6, the amino acid sequence of the TCRβ chain is shown in SEQ ID NO: 7, the nucleotide sequence of the TCRβ chain is shown in SEQ ID NO: 8, the amino acid sequence of the TCRβ chain with a leader sequence is shown in SEQ ID NO: 24, and the nucleotide sequence of the TCRβ chain with a leader sequence is shown in SEQ ID NO: 25.

[0205] SEQ ID NO: 1:

[0206] EDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVHPLNYGGSQGNLIFGKGTKLSVKP.

[0207] SEQ ID NO: 2:

[0208] gaagaccaggtgacgcagagtcccgaggccctgagactccaggagggagagagtagcagtctcaactgcagttacacagtcagcggtttaagagggctgttctggtataggcaagatcctgggaaaggccctgaattcctcttcaccctgtattcagctggggaagaaaaggagaaagaaaggctaaaagccacattaacaaagaaggaaagctttctgcacatcacagcccctaaacctgaagactcagccacttatctctgtgctgtgcacccattgaattatggaggaagccaaggaaatctcatctttggaaaaggcactaaactctctgttaaacca。

[0209] SEQ ID NO:3:

[0210] EDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVHPLNYGGSQGNLIFGKGTKLSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS。

[0211] SEQ ID NO:4:

[0212] gaagaccaggtgacgcagagtcccgaggccctgagactccaggagggagagagtagcagtctcaactgcagttacacagtcagcggtttaagagggctgttctggtataggcaagatcctgggaaaggccctgaattcctcttcaccctgtattcagctggggaagaaaaggagaaagaaaggctaaaagccacattaacaaagaaggaaagctttctgcacatcacagcccctaaacctgaagactcagccacttatctctgtgctgtgcacccattgaattatggaggaagccaaggaaatctcatctttggaaaaggcactaaactctctgttaaaccaaatAtccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaagtctgtctgcctattcaccgattttgattctcaaacaaatgtgtcacaaagtaaggattctgatgtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaagagcaacagtgctgtggcctggagcaacaaatctgactttgcatgtgcaaacgccttcaacaacagcattattccagaagacaccttcttccccagcccagaaagttcctgtgatgtcaagctggtcgagaaaagctttgaaacagatacgaacctaaactttcaaaacctgtcagtgattgggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatgacgctgcggctgtggtccagc。

[0213] SEQ ID NO:22:

[0214] EKMLECAFIVLWLQLGWLSGEDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVHPLNYGGSQGNLIFGKGTKLSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS。

[0215] SEQ ID NO:23:

[0216] gagaaaatgttggagtgtgcattcatagtcttgtggcttcagcttggctggttgagtggagaagaccaggtgacgcagagtcccgaggccctgagactccaggagggagagagtagcagtctcaactgcagttacacagtcagcggtttaagagggctgttctggtataggcaagatcctgggaaaggccctgaattcctcttcaccctgtattcagctggggaagaaaaggagaaagaaaggctaaaagccacattaacaaagaaggaaagctttctgcacatcacagcccctaaacctgaagactcagccacttatctctgtgctgtgcacccattgaattatggaggaagccaaggaaatctcatctttggaaaaggcactaaactctctgttaaaccaaatatccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaagtctgtctgcctattcaccgattttgattctcaaacaaatgtgtcacaaagtaaggattctgatgtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaagagcaacagtgctgtggcctggagcaacaaatctgactttgcatgtgcaaacgccttcaacaacagcattattccagaagacaccttcttccccagcccagaaagttcctgtgatgtcaagctggtcgagaaaagctttgaaacagatacgaacctaaactttcaaaacctgtcagtgattgggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatgacgctgcggctgtggtccagc。

[0217] SEQ ID NO:5:

[0218] GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAREWRGTEAFFGQGTRLTVV。

[0219] SEQ ID NO:6:

[0220] ggtgctgtcgtctctcaacatccgagctgggttatctgtaagagtggaacctctgtgaagatcgagtgccgttccctggactttcaggccacaactatgttttggtatcgtc

[0221] agttcccgaaacagagtctcatgctgatggcaacttccaatgagggctccaaggccacatacgagcaaggcgtcgagaaggacaagtttctcatcaaccatgcaagc

[0222] ctgaccttgtccactctgacagtgaccagtgcccatcctgaagacagcagcttctacatctgcagtgctagagaatggcggggcactgaagctttctttggacaaggcaccagactcacagttgta。

[0223] SEQ ID NO:7:

[0224] GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLIN

[0225] HASLTLSTLTVTSAHPEDSSFYICSAREWRGTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKA

[0226] TLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRC

[0227] QVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF。

[0228] SEQ ID NO:8:

[0229] ggtgctgtcgtctctcaacatccgagctgggttatctgtaagagtggaacctctgtgaagatcgagtgccgttccctggactttcaggccacaactatgttttggtatcgtc

[0230] agttcccgaaacagagtctcatgctgatggcaacttccaatgagggctccaaggccacatacgagcaaggcgtcgagaaggacaagtttctcatcaaccatgcaagc

[0231] ctgaccttgtccactctgacagtgaccagtgcccatcctgaagacagcagcttctacatctgcagtgctagagaatggcggggcactgaagctttctttggacaaggca

[0232] ccagactcacagttgtaGaggacctgaacaaggtgttcccacccgaggtcgctgtgtttgagccatcagaagcagagatctcccacacccaaaaggccacactggt

[0233] gtgcctggccacaggcttcttccccgaccacgtggagctgagctggtgggtgaatgggaaggaggtgcacagtggggtcagcacggacccgcagcccctcaagg

[0234] agcagcccgccctcaatgactccagatactgcctgagcagccgcctgagggtctcggccaccttctggcagaacccccgcaaccacttccgctgtcaagtccagttc

[0235] tacgggctctcggagaatgacgagtggacccaggatagggccaaacccgtcacccagatcgtcagcgccgaggcctggggtagagcagactgtggctttacctcg

[0236] gtgtcctaccagcaaggggtcctgtctgccaccatcctctatgagatcctgctagggaaggccaccctgtatgctgtgctggtcagcgcccttgtgttgatggccatggtcaagagaaaggatttc。

[0237] SEQ ID NO:24:

[0238] LLLLLLLGPGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKAT

[0239] YEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAREWRGTEAFFGQGTRLTVVEDLNKVFPPEVAV

[0240] FEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSA

[0241] TFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF。

[0242] SEQ ID NO:25:

[0243] ctgctgcttctgctgcttctggggccaggctccgggcttggtgctgtcgtctctcaacatccgagctgggttatctgtaagagtggaacctctgtgaagatcgagtgccg

[0244] ttccctggactttcaggccacaactatgttttggtatcgtcagttcccgaaacagagtctcatgctgatggcaacttccaatgagggctccaaggccacatacgagcaag

[0245] gcgtcgagaaggacaagtttctcatcaaccatgcaagcctgaccttgtccactctgacagtgaccagtgcccatcctgaagacagcagcttctacatctgcagtgctag

[0246] agaatggcggggcactgaagctttctttggacaaggcaccagactcacagttgtaGaggacctgaacaaggtgttcccacccgaggtcgctgtgtttgagccatcag

[0247] aagcagagatctcccacacccaaaaggccacactggtgtgcctggccacaggcttcttccccgaccacgtggagctgagctggtgggtgaatgggaaggaggtgc

[0248] acagtggggtcagcacggacccgcagcccctcaaggagcagcccgccctcaatgactccagatactgcctgagcagccgcctgagggtctcggccaccttctggc

[0249] agaacccccgcaaccacttccgctgtcaagtccagttctacgggctctcggagaatgacgagtggacccaggatagggccaaacccgtcacccagatcgtcagcg

[0250] ccgaggcctggggtagagcagactgtggctttacctcggtgtcctaccagcaaggggtcctgtctgccaccatcctctatgagatcctgctagggaaggccaccctgtatgctgtgctggtcagcgcccttgtgttgatggccatggtcaagagaaaggatttc。

[0251] SEQ ID NO: 9: GVYDGREHTV.

[0252] The α chain was identified to contain a CDR with the following amino acid sequence:

[0253] αCDR1-VSGLRG SEQ ID NO: 10,

[0254] αCDR2-LYSAGEE SEQ ID NO:11, and

[0255] αCDR3-AVHPLNYGGSQGNLI SEQ ID NO: 12;

[0256] The beta chain contains a CDR with the following amino acid sequence:

[0257] βCDR1-DFQATT SEQ ID NO: 13,

[0258] βCDR2-SNEGSKA SEQ ID NO: 14, and

[0259] βCDR3-SAREWRGTEAF SEQ ID NO:15.

[0260] Example 3 Expression, refolding and purification of MAGE-A4 antigen short peptide-specific soluble TCR

[0261] To obtain soluble TCR molecules, the α and β chains of the TCR molecules of the present invention can each contain only their variable domains and a portion of their constant domains, and a cysteine ​​residue is introduced into the constant domains of the α and β chains to form an artificial interchain disulfide bond. The amino acid sequence of the α chain is shown in SEQ ID NO: 26; the amino acid sequence of the β chain is shown in SEQ ID NO: 27. The target gene sequences of the TCR α and TCR β chains 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. The inserts were confirmed to be correct by sequencing.

[0262] SEQ ID NO: 26:

[0263] EDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVHPLNYGGSQGNLIF GKGTKLSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFCSPESS.

[0264] SEQ ID NO: 27:

[0265] GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAREWRGTEAFFGQGTRLTVVEDLKNVFP PEVAVFEPSECEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD.

[0266] 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).

[0267] 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 an 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 BCA assay. Figure 2 As shown, Figure 2 a and Figure 2 The right lanes in b are reducing gel and non-reducing gel, respectively, and the left lanes are molecular weight markers.

[0268] Example 4 Generation of Soluble Single-Chain TCR Specific for MAGE-A4 Antigen Peptide

[0269] According to the patent document WO2014 / 206304, the variable domains of the TCR α chain and the TCR β chain in Example 2 were constructed into a stable soluble single-chain TCR molecule connected by a flexible short peptide (linker) by using a site-directed mutagenesis method. The amino acid sequence of the single-chain TCR molecule is shown in SEQ ID NO: 28, and the nucleotide sequence of the single-chain TCR molecule is shown in SEQ ID NO: 29; the amino acid sequence and nucleotide sequence of its linker are marked with a single underline. The amino acid sequence of its α chain variable domain is shown in SEQ ID NO: 30, and the nucleotide sequence of its α chain variable domain is shown in SEQ ID NO: 31; the amino acid sequence of its β chain variable domain is shown in SEQ ID NO: 32, and the nucleotide sequence of its β chain variable domain is shown in SEQ ID NO: 33.

[0270] The target gene 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.

[0271] SEQ ID NO: 28:

[0272] EDQVTQSPEALRVPEGESVSINCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITDVKPEDSATYFCAVHPLNYGGSQGNLIFGKGTKLSVKP GGGSEGGGSEGGGSEGGGSEGGTG GAVVSQHPSHLSVKSGTSVKLECRSLDFQATTMFWYRQDPGQSLELMATSNEGSKATYEQGVEKDRFNINHASLTLSTLTITSVHPEDSSFYFCSAREWRGTEAFFGQGTRLTVD.

[0273] SEQ ID NO: 29:

[0274] gaagatcaagttactcaaagtccggaagcactgcgtgtaccggaaggcgaaagcgtgagtatcaactgcagctataccgttagcggtctgcgcggtctgttttggtatcgtcaagatccgggtaaagggccggaatttctgtttaccctgtatagcgcgggcgaagagaaa gagaaagagcgtctgaaagcgaccctgaccaaaaaagagagctttctgcacatcaccgacgttaaaccggaagatagcgcgacctacttctgcgcagttcatccgctgaattacggcggtagtcaaggtaacctgatcttcggcaaaggcaccaaactgtcagttaaaccg ggcggcggttctgaaggtggtggtagcgaa ggcggcggttccgaaggtggtggttctgaaggcggtaccggcggcgcagttgttagtcagcatccgtctcatctgagcgtgaaaagcggtaccagcgttaaactggagtgtcgtagtctggattttcaagccaccaccatgttctggtaccgtcaagatccgggtcaatctctggaactgatggcaaccagcaacgaaggtagcaaagcaacctacgaacaaggcgttgagaaagaccgcttcaacatcaaccacgcaagtctgaccctgagtaccctgaccattaccagcgtccatccggaagatagcagcttctatttctgttctgctcgcgagtggcgcggtacggaagcattcttcggccagggtacccgtctgaccgttgat。

[0275] SEQ ID NO:30:

[0276] EDQVTQSPEALRVPEGESVSINCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITDVKPEDSATYFCAVHPLNYGGSQGNLIFGKGTKLSVKP。

[0277] SEQ ID NO:31:

[0278] gaagatcaagttactcaaagtccggaagcactgcgtgtaccggaaggcgaaagcgtgagtatcaactgcagctataccgttagcggtctgcgcggtctgttttggtatcgtcaagatccgggtaaagggccggaatttctgtttaccctgtatagcgcgggcgaagagaaagagaaagagcgtctgaaagcgaccctgaccaaaaaagagagctttctgcacatcaccgacgttaaaccggaagatagcgcgacctacttctgcgcagttcatccgctgaattacggcggtagtcaaggtaacctgatcttcggcaaaggcaccaaactgtcagttaaaccg。

[0279] SEQ ID NO:32:

[0280] GAVVSQHPSHLSVKSGTSVKLECRSLDFQATTMFWYRQDPGQSLELMATSNEGSKATYEQGVEKDRFNINHASLTLSTLTITSVHPEDSSFYFCSAREWRGTEAFFGQGTRLTVD.

[0281] SEQ ID NO: 33:

[0282] ggcgcagttgttagtcagcatccgtctcatctgagcgtgaaaagcggtaccagcgttaaactggagtgtcgtagtctggattttcaagccaccaccatgttctggtaccgtcaagatccgggtcaatctctggaactgatggcaaccagcaacgaaggtagcaaagcaaccta cgaacaaggcgttgagaaagaccgcttcaacatcaaccacgcaagtctgaccctgagtaccctgaccattaccagcgtccatccggaagatagcagcttctatttctgttctgctcgcgagtggcgcggtacggaagcattcttcggccagggtacccgtctgaccgttgat.

[0283] Example 5 Expression, refolding and purification of soluble single-chain TCR specific for MAGE-A4 antigen short peptide

[0284] All BL21 (DE3) colonies containing the recombinant plasmid pET28a-template chain prepared in Example 4 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 centrifuged at 6000 rpm for 15 minutes to collect the inclusion bodies. 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.

[0285] 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 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 replaced with 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 replaced with the same fresh buffer and dialysis continued overnight. After 17 hours, the sample was filtered through a 0.45 μm filter, vacuum-degassed, 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 7510 / 300, GE Healthcare). The target fraction was also analyzed by SDS-PAGE.

[0286] The eluted fractions used for BIAcore analysis were further tested for purity by gel filtration using an Agilent Bio SEC-3 column (300A, 7.8 x 300 mm diameter), a mobile phase of 150 mM phosphate buffer, a flow rate of 0.5 mL / min, a column temperature of 25°C, and UV detection at 214 nm.

[0287] The SDS-PAGE electrophoresis gel of the soluble single-chain TCR obtained after purification in this example is shown in FIG. Figure 3 As shown, the right lane is a reducing gel, the left lane is a non-reducing gel, and the middle lane is a molecular weight marker.

[0288] Example 6 Binding Characterization

[0289] This example demonstrates through BIAcore analysis that soluble TCR molecules can specifically bind to the GVYDGREHTV-HLAA0201 complex.

[0290] The binding activity of the TCR molecule obtained in Example 3 to the GVYDGREHTV-HLAA0201 complex was detected using a BIAcore T200 real-time analysis system. An anti-streptavidin antibody (GenScript) was added to a coupling buffer (10 mM sodium acetate buffer, pH 4.77). The antibody was then passed over a CM5 chip pre-activated with EDC and NHS to immobilize the antibody on the chip surface. Finally, the unreacted activated surface was blocked with ethanolamine hydrochloride solution to complete the coupling process. The coupling level was approximately 15,000 RU.

[0291] A low concentration of streptavidin was passed through the antibody-coated chip surface, and then the GVYDGREHTV-HLAA0201 complex was passed through the detection channel. The other channel was used as a reference channel, and 0.05 mM biotin was passed through the chip at a flow rate of 10 μL / min for 2 minutes to block the remaining binding sites of streptavidin.

[0292] The preparation process of the above-mentioned GVYDGREHTV-HLA A0201 complex is as follows:

[0293] (a) Purification

[0294] 100 mL of E. coli culture induced to express heavy or light chain was collected and centrifuged at 8000 g for 10 min at 4°C. The cells were washed once with 10 mL of PBS and then resuspended in 5 mL of BugBuster Master Mix Extraction Reagents (Merck) by vigorous shaking. The cells were incubated with rotation at room temperature for 20 min and then centrifuged at 6000 g for 15 min at 4°C. The supernatant was discarded and the inclusion bodies were collected.

[0295] 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 the inclusion bodies were resuspended in 30 mL of 20 mM Tris-HCl pH 8.0, mixed, and centrifuged at 4°C and 6000 g for 15 min. Finally, the inclusion bodies were solubilized with 20 mM Tris-HCl 8 M urea. The purity of the inclusion bodies was detected by SDS-PAGE, and the concentration was measured by BCA kit.

[0296] (b) Renaturation

[0297] The synthesized short peptide GVYDGREHTV was dissolved in DMSO to a concentration of 20 mg / mL. Inclusion bodies of the light and heavy chains 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 a 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, and 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 concentrations, with the heavy chain added in three separate additions over 8 hours). Renaturation was continued at 4°C for at least 3 days until complete. Refolding success was assessed by SDS-PAGE.

[0298] (c) Purification after renaturation

[0299] The refolding buffer was exchanged by dialysis with 10 volumes of 20 mM Tris pH 8.0, with the buffer 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 then loaded onto a HiTrap Q HP (GE General Electric) anion exchange column (5 mL bed volume). The protein was eluted using an Akta purifier (GE General Electric) 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 purity was determined by SDS-PAGE.

[0300] (d) Biotinylation

[0301] The purified pMHC molecules were concentrated using Millipore ultrafiltration tubes, and the buffer was replaced with 20 mM Tris pH 8.0. Then, biotinylation reagent 0.05 M Bicine pH 8.3, 10 mM ATP, 10 mM MgOAc, 50 μM D-Biotin, and 100 μg / mL BirA enzyme (GST-BirA) were added. The mixture was incubated at room temperature overnight, and SDS-PAGE was used to detect whether the biotinylation was complete.

[0302] (e) Purification of the biotinylated complex

[0303] The biotinylated pMHC molecules were concentrated to 1 mL using Millipore ultrafiltration tubes, and the biotinylated pMHC was purified by gel filtration chromatography. The Akta purifier (GE General Electric) was used to pre-equilibrate HiPrep with filtered PBS. TMA 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.

[0304] The kinetic parameters were calculated using BIAcore Evaluation software to obtain a kinetic profile of binding to the GVYDGREHTV-HLA A0201 complex, where: Figure 4 This is the BIAcore kinetic profile of the binding of soluble TCR to the GVYDGREHTV-HLA A0201 complex. Figure 5 This is a BIAcore kinetic profile of the binding of a soluble single-chain TCR to the GVYDGREHTV-HLA A0201 complex. The profile demonstrates that the soluble TCR molecules obtained by the present invention are capable of binding to the GVYDGREHTV-HLA A0201 complex. The binding activity of the soluble TCR molecules of the present invention to several other unrelated antigenic short peptides and HLA complexes was also tested using the above method. The results showed that the TCR molecules of the present invention did not bind to any of the other unrelated antigens.

[0305] Example 7 Effector cell activation experiment on T2 cells loaded with short peptides and transfected with the TCR of the present invention

[0306] The effector cells used in this example are CD3 TCR transfected with the present invention. + T cells. The target cells used were T2-A0201 cells loaded with the MAGE-A4 antigen peptide GVYDGREHTV, and T2-A0201 cells loaded with other antigen peptides and empty cells were used as controls. The components of the test were added to the ELISPOT well plate: target cells 1×10 4 cells / well, effector cells 2×10 3 Each well was filled with GVYDGREHTV peptide (calculated by the positive transfection rate) and two replicate wells were set. Then GVYDGREHTV peptide was added to the corresponding wells to make the final concentration of the peptide in the ELISPOT well plate 10 -6 M.

[0307] ELISPOT plates were activated with ethanol and coated at 4°C overnight. On the first day of the experiment, the coating solution was removed, the cells were washed and blocked, and the cells were incubated at room temperature for two hours. The blocking solution was removed and the components of the test were added to the ELISPOT plates: 1×10 target cells 4 cells / well, effector cells: 2×103 Count the number of cells / well (calculated based on the transfection positive 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).

[0308] Figure 7 The results of ELISPOT activation function verification of T2-A11 cells loaded with short peptides and effector cells transfected with the TCR of the present invention are shown in Figure 2. Figure 7 It can be seen that T2-A0201 cells loaded with the GVYDGREHTV short peptide and transfected with the TCR of the present invention have a significant activation effect, while T2-A0201 cells loaded with other antigen short peptides or empty cells and transfected with the TCR of the present invention have basically no activation effect.

[0309] Example 8 Effector cell activation experiment on T2 cells loaded with short peptides at gradient concentrations and transfected with the TCR of the present invention

[0310] The effector cells used in this example are CD3 TCR transfected with the present invention. + The target cells used were T2-A0201 cells loaded with the MAGE-A4 antigen short peptide GVYDGREHTV. The components of the test were added to the ELISPOT well plate: target cells 1×10 4 target cells / well, effector cells 2×10 3 Each well was filled with GVYDGREHTV peptide (calculated by the positive transfection rate) and two replicate wells were set. Then GVYDGREHTV peptide was added to the corresponding wells to make the final concentration of the peptide in the ELISPOT well plate 10 -6 M to 10 -12 , a total of 7 gradient concentrations.

[0311] ELISPOT plates were activated with ethanol and coated at 4°C overnight. On the first day of the experiment, the coating solution was removed, the cells were washed and blocked, and the cells were incubated at room temperature for two hours. The blocking solution was removed and the components of the test were added to the ELISPOT plates: 1×10 target cells 4 cells / well, effector cells: 2×10 3 Count the number of cells / well (calculated based on the transfection positive 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).

[0312] Figure 8The results of ELISPOT activation function verification of T2 cells loaded with short peptides at gradient concentrations and effector cells transfected with the TCR of the present invention are shown in Figure 2. Figure 8 It can be seen that T2-A0201 loaded with the GVYDGREHTV short peptide has a significant activation effect on T cells transfected with the TCR of the present invention, while T cells transfected with other TCRs are still inactive at the highest peptide concentration.

[0313] Example 9 Activation Function Experiment of IFN-γ Release by Effector Cells Transfected with TCR of the Present Invention for Tumor Cell Lines

[0314] This example also uses ELISPOT assay to detect the function and specificity of the TCR of the present invention in cells. The effector cells used are CD3 TCR expressing the MAGE-A4 antigen peptide specific TCR of the present invention. + T cells, and CD3+ T cells transfected with another TCR (A6) from the same volunteer were used as a control group. The tumor cell lines used in the examples were 293T and MCF-7. 293T and MCF-7 were purchased from ATCC. The following experiments were performed:

[0315] The MAGE-A4 positive tumor cell line used was 293T-MAGE-A4 (MAGE-A4 overexpression), and the negative cell lines were 293T and MCF-7.

[0316] First, prepare the ELISPOT plate. Activate the ELISPOT plate with ethanol and coat it at 4°C overnight. 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 are 2×10 4 cells / well, effector cells: 2×10 3 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).

[0317] Figure 9 The results of the activation function experiments of the effector cells transfected with the TCR of the present invention are shown in FIG. Figure 9 The results showed that the effector cells transfected with the TCR of the present invention had an activation effect on MAGE-A4 positive tumor cell lines, while the effector cells transfected with other TCRs had basically no activity; at the same time, the effector cells transfected with the TCR of the present invention had basically no activity on MAGE-A4 negative cell lines.

[0318] 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.

[0319] 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 TCR, characterized in that: The TCR comprises a TCR α chain variable domain and a TCR β chain variable domain, the TCR is capable of binding to the GVYDGREHTV-HLAA0201 complex, and the three complementary determining regions of the TCR α chain variable domain are: αCDR1-VSGLRG SEQ ID NO: 10, αCDR2-LYSAGEE SEQ ID NO:11, and, αCDR3-AVHPLNYGGSQGNLI SEQ ID NO: 12; and, The three complementarity determining regions of the TCRβ chain variable domain are: βCDR1-DFQATT SEQ ID NO: 13, βCDR2-SNEGSKA SEQ ID NO: 14, and βCDR3-SAREWRGTEAF SEQ ID NO:

15.

2. The TCR according to claim 1, characterized in that The sequence of the TCR α chain variable domain is an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1; and the sequence of the TCR β chain variable domain is an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

5.

3. The TCR according to claim 1, characterized in that The C- or N-terminus of the α chain of the TCR is bound to a conjugate, and / or the C- or N-terminus of the β chain of the TCR is bound to a conjugate; Preferably, the conjugate bound to the TCR comprises any one or a combination of at least two of a detectable marker, a therapeutic agent and a PK modifying moiety; Preferably, the therapeutic agent is an anti-CD3 antibody.

4. A multivalent TCR complex, characterized in that The multivalent TCR complex comprises at least two TCR molecules, and at least one of the TCR molecules is the TCR described in any one of claims 1-3.

5. A nucleic acid molecule, characterized in that The sequence of the nucleic acid molecule comprises a nucleotide sequence encoding the TCR according to any one of claims 1 to 3, and / or a complementary sequence of a nucleotide sequence encoding the TCR according to any one of claims 1 to 3; Preferably, the sequence of the nucleic acid molecule includes the nucleotide sequence SEQ ID NO:2 or SEQ ID NO:31 encoding the variable domain of the TCR α chain; and / or the sequence of the nucleic acid molecule includes the nucleotide sequence SEQ ID NO:6 or SEQ ID NO:33 encoding the variable domain of the TCR β chain.

6. A carrier, characterized in that The vector contains the nucleic acid molecule of claim 5; Preferably, the vector is a viral vector; Preferably, the viral vector is a lentiviral vector.

7. An isolated host cell, characterized in that The isolated host cell contains the vector according to claim 6, or the exogenous nucleic acid molecule according to claim 5 is integrated into the chromosome of the isolated host cell.

8. An isolated cell, characterized in that The cell is transduced with the nucleic acid molecule of claim 5 or the vector of claim 6; Preferably, the cells comprise T cells or stem cells.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises any one or a combination of at least two of the TCR according to any one of claims 1 to 3, the multivalent TCR complex according to claim 4, the nucleic acid molecule according to claim 5, and the cell according to claim 8; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

10. Use of any one or a combination of at least two of the TCR according to any one of claims 1 to 3, the multivalent TCR complex according to claim 4, and the isolated cell according to claim 8, characterized in that: The use includes preparing medicines for treating tumors or autoimmune diseases.

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