CN115677847A - A T-cell receptor recognizing SSX2 and its coding sequence - Google Patents

Abstract

The invention relates to a T cell receptor for identifying SSX2 and a coding sequence thereof. The invention provides a T Cell Receptor (TCR) capable of specifically binding a short peptide AQIPEKIQK derived from SSX2 antigen, said antigen short peptide AQIPEKIQK can form a complex with HLA A1101 and be presented together to the cell surface. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules. In addition, the invention provides cells that transduce a TCR of the invention.

Description

A T-cell receptor recognizing SSX2 and its coding sequence

technical field

The present invention relates to a TCR capable of recognizing short peptides derived from the SSX2 antigen and its coding sequence. The present invention also relates to SSX2-specific T cells obtained by transducing the above-mentioned TCR, and their use in preventing and treating SSX2-related diseases.

Background technique

SSX2 is the synovial sarcoma X breakpoint, also known as HOM-MEL-40. SSX2 is one of ten highly homologous nucleic acid proteins of the SSX family. The SSX protein is a tumor-testis antigen expressed only in tumor cells and testicular germ cells without MHC expression. SSX2 is expressed in a variety of human cancer cells, including but not limited to, liver cancer, lung cancer, fibrosarcoma, breast cancer, colon cancer, prostate cancer. After SSX2 is produced in cells, it is degraded into small molecular polypeptides, and combines with MHC (major histocompatibility complex) molecules to form a complex, which is presented to the cell surface. AQIPEKIQK (SEQ ID NO:9) is a short peptide derived from the SSX2 antigen and is a target for the treatment of SSX2-related diseases.

T cell adoptive immunotherapy is the transfer of reactive T cells specific to target cell antigens into the patient's body so that they can act against the target cells. T cell receptor (TCR) is a membrane protein on the surface of T cells, which can recognize short antigenic peptides on the surface of corresponding target cells. In the immune system, direct physical contact between T cells and antigen-presenting cells (APCs) is triggered by the combination of antigenic short peptide-specific TCR and short peptide-major histocompatibility complex (pMHC complex), and then T cells and The other cell membrane surface molecules of APC and APC interact, causing a series of subsequent cell signal transmission and other physiological responses, so that T cells with different antigen specificities exert immune effects on their target cells. Therefore, those skilled in the art are committed to isolating TCRs specific to SSX2 antigen short peptides, and transducing T cells with the TCR to obtain T cells specific to SSX2 antigen short peptides, so that they can be used in cellular immunotherapy. play a role in.

Contents of the invention

The purpose of the present invention is to provide a T cell receptor that recognizes short peptides of SSX2 antigen.

The first aspect of the present invention provides a T cell receptor (TCR), which can bind to the AQIPEKIQK-HLAA1101 complex.

In another preferred example, the three complementarity determining regions (CDRs) of the variable domain of the TCRα chain are:

αCDR1-SSYSPS (SEQ ID NO: 10)

αCDR2-YTSAATLV (SEQ ID NO: 11)

αCDR3-VVSPGNTPLV (SEQ ID NO: 12); and/or

The three complementarity-determining regions of the TCRβ chain variable domain are:

βCDR1-SGHVS (SEQ ID NO: 13)

βCDR2-FQNEAQ (SEQ ID NO: 14)

βCDR3-ASSPTVGTSAYNEQF (SEQ ID NO: 15).

In another preferred example, the TCR comprises a TCRα chain variable domain and a TCRβ chain variable domain, and the TCRα chain variable domain is an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1; and/or Or the TCR beta chain variable domain is an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5.

In another preferred example, the TCR comprises the amino acid sequence of the α-chain variable domain SEQ ID NO:1.

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

In another preferred embodiment, the TCR is an αβ heterodimer, which comprises a TCR α chain constant region TRAC*01 and a TCR β chain constant region TRBC1*01 or TRBC2*01.

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.

In another preferred example, the TCR is of human origin.

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

In another preferred embodiment, the TCR is soluble.

In another preferred example, the TCR is a single chain.

In another preferred example, the TCR is formed by linking the α-chain variable domain and the β-chain variable domain through a peptide linker sequence.

In another preferred example, the constant regions of the α and β chains of the TCR are the constant regions of the murine α and β chains, respectively.

In another preferred example, the TCR is at the 11th, 13th, 19th, 21st, 53rd, 76, 89, 91st, or 94th amino acid position in the α-chain variable region, and/or at the reciprocal amino acid position of the J gene short peptide of the α-chain There are one or more mutations in the 3rd, penultimate 5th or penultimate 7th position; and/or the TCR is in the 11th, 13th, 19th, 21st, 53rd, 76th, 89th, 91st amino acid of the β-chain variable region , or position 94, and/or one or more mutations in the second-to-last, fourth-to-last or sixth-to-last amino acid positions of the short peptide of the β-chain J gene, wherein the amino acid positions are numbered according to IMGT (International Immunogenetics Information system) at the location number listed.

In another preferred example, the TCR includes (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;

And (a) and (b) each comprise a functional variable domain, or comprise a functional variable domain and at least a part of said TCR chain constant domain.

In another preferred embodiment, the cysteine residue forms an artificial disulfide bond between the α and β chain constant domains of the TCR.

In another preferred example, the cysteine residues forming artificial disulfide bonds in the TCR are substituted for one or more groups of sites selected from the following:

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

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

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

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

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

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

Pro89 of TRAC*01 exon 1 and Ala19 of TRBC1*01 or TRBC2*01 exon 1; and Tyr10 of TRAC*01 exon 1 and Glu20 of TRBC1*01 or TRBC2*01 exon 1.

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:28. The nucleotide sequences encoding them are respectively shown in SEQ ID NO:27 and SEQ ID NO:29.

In another preferred example, the TCR contains an artificial interchain disulfide bond between the α-chain variable region and the β-chain constant region.

In another preferred example, it is characterized in that the cysteine residues forming artificial interchain disulfide bonds in the TCR are substituted for one or more groups of sites selected from the following:

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

The 47th amino acid of TRAV and the 61st amino acid of TRBC1*01 or TRBC2*01 exon 1;

Amino acid 46 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01; or

Amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01.

In another preferred example, the TCR comprises an α-chain variable domain and a β-chain variable domain and all or part of the β-chain constant domain except the transmembrane domain, but it does not contain an α-chain constant domain, and the TCR The α-chain variable domain forms a heterodimer with the β-chain.

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

In another preferred embodiment, the conjugate that binds to the T cell receptor is a detectable marker, a therapeutic agent, a PK modifying moiety or any combination of these substances. Preferably, the therapeutic agent is an anti-CD3 antibody.

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

The third aspect of the present invention provides a nucleic acid molecule comprising the nucleic acid sequence encoding the TCR molecule described in the first aspect of the present invention or its complementary sequence.

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

In another preferred example, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 2 encoding the variable domain of the TCRα chain.

In another preferred example, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 6 encoding the variable domain of the TCRβ chain.

In another preferred example, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 4 encoding the TCRα chain and/or comprises the nucleotide sequence of SEQ ID NO: 8 encoding the TCR β chain.

The fourth aspect of the present invention provides a vector, the vector contains the nucleic acid molecule described in the third aspect of the present invention; preferably, the vector is a viral vector; more preferably, the vector is a lentivirus Viral vector.

The fifth aspect of the present invention provides an isolated host cell containing the vector of the fourth aspect of the present invention or the exogenous nucleic acid molecule of the third aspect of the present invention integrated in the genome .

The sixth aspect of the present invention provides a cell transduced with the nucleic acid molecule described in the third aspect of the present invention or the vector described in the fourth aspect of the present invention; preferably, the cell is a T cell, NK cells, NKT cells or stem cells.

The seventh aspect of the present invention provides a pharmaceutical composition, which contains a pharmaceutically acceptable carrier and the TCR described in the first aspect of the present invention, the TCR complex described in the second aspect of the present invention, the The nucleic acid molecule of the third aspect of the invention, the vector of the fourth aspect of the invention, or the cell of the sixth aspect of the invention.

The eighth aspect of the present invention provides the use of the T cell receptor described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, or the cells described in the sixth aspect of the present invention, for To prepare medicines for treating tumors or autoimmune diseases, preferably, the tumors are SSX2 positive tumors.

The ninth aspect of the present invention provides the T cell receptor described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, or the cells described in the sixth aspect of the present invention for treating tumors or Drugs for autoimmune diseases; preferably, the tumor is a SSX2 positive tumor.

The tenth aspect of the present invention provides a method for treating diseases, comprising administering an appropriate amount of the T cell receptor described in the first aspect of the present invention or the TCR complex described in the second aspect of the present invention to a subject in need of treatment , or the cell according to the sixth aspect of the present invention, or the pharmaceutical composition according to the seventh aspect of the present invention; preferably, the disease is a tumor, preferably, the tumor is a SSX2-positive tumor.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1a, Figure 1b, Figure 1c, Figure 1d, Figure 1e and Figure 1f are the amino acid sequence (SEQ ID NO: 1) and the nucleotide sequence (SEQ ID NO: 2) of the variable domain of the TCRα chain, respectively. , TCRα chain amino acid sequence (SEQ ID NO:3), TCRα chain nucleotide sequence (SEQ ID NO:4), TCRα chain amino acid sequence with leader sequence (SEQ ID NO:22) and TCRα chain core with leader sequence Nucleotide sequence (SEQ ID NO: 23).

Figure 2a, Figure 2b, Figure 2c, Figure 2d, Figure 2e and Figure 2f are the amino acid sequence (SEQ ID NO:5) and the nucleotide sequence (SEQ ID NO:6) of the TCRβ chain variable domain respectively , TCR β chain amino acid sequence (SEQ ID NO: 7), TCR β chain nucleotide sequence (SEQ ID NO: 8), TCR β chain amino acid sequence (SEQ ID NO: 24) with leader sequence and TCR β chain core with leader sequence Nucleotide sequence (SEQ ID NO: 25).

Figure 3 shows the double positive staining results of CD8 ⁺ -APC and tetramer-PE of monoclonal cells.

Figure 4a and Figure 4b are the amino acid sequence (SEQ ID NO:26) and nucleotide sequence (SEQ ID NO:27) of the soluble TCRα chain, respectively.

Figure 5a and Figure 5b are the amino acid sequence (SEQ ID NO:28) and nucleotide sequence (SEQ ID NO:29) of the soluble TCRβ chain, respectively.

Figure 6a and Figure 6b are gel images of purified soluble TCR. Wherein, the right swimming lanes of Figures 6a and 6b are reducing gels and non-reducing gels respectively, and the left swimming lanes are molecular weight markers.

Fig. 7 is a BIAcore kinetic map of the binding of the soluble TCR of the present invention to the AQIPEKIQK-HLA A1101 complex.

Figure 8 shows the results of the ELISPOT activation function verification of the obtained T cell clones.

Fig. 9 is the result of ELISPOT activation function verification of effector cells transfected with TCR of the present invention for T2 cells loaded with gradient concentrations of short peptides.

Fig. 10 is the result of ELISPOT activation function verification of effector cells transfected with TCR of the present invention for tumor cell lines.

Detailed ways

After extensive and in-depth research, the inventors have found a TCR that can specifically bind to the SSX2 antigen short peptide AQIPEKIQK (SEQ ID NO: 9), and the antigen short peptide AQIPEKIQK can form a complex with HLA A1101 and be presented together to cell surface. The present invention also provides the nucleic acid molecule encoding the TCR and the vector comprising the nucleic acid molecule. In addition, the present invention also provides cells transduced with the TCR of the present invention.

the term

MHC molecules are proteins of the immunoglobulin superfamily and can be class I or class II MHC molecules. Therefore, it is specific for the presentation of antigens, and different individuals have different MHCs, which can present different short peptides in a protein antigen to the surface of their respective APC cells. Human MHC is often referred to as HLA genes or HLA complexes.

T cell receptor (TCR), is the sole receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC). In the immune system, the combination of antigen-specific TCR and pMHC complexes triggers direct physical contact between T cells and antigen-presenting cells (APCs), and then other cell membrane surface molecules of T cells and APCs interact. It causes a series of subsequent cell signal transmission and other physiological responses, so that T cells with different antigen specificities exert immune effects on their target cells.

TCR is a glycoprotein on the cell membrane surface that exists in the form of a heterodimer of α chain/β chain or γ chain/δ chain. TCR heterodimers consist of alpha and beta chains in 95% of T cells, whereas 5% of T cells have TCRs consisting of gamma and delta chains. The native αβ heterodimeric TCR has an α chain and a β chain, which constitute the subunits of the αβ heterodimeric TCR. Broadly speaking, each chain of α and β contains a variable region, a connecting region, and a constant region, and the β chain usually also contains a short variable region between the variable region and the connecting region, but this variable region is often regarded as the connecting region a part of. Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, fitted in framework regions. The CDR region determines the combination of the TCR and the pMHC complex, in which CDR3 is recombined from the variable region and the linker region, known as the hypervariable region. The α and β chains of a TCR are generally regarded as having two "domains" each, namely a variable domain and a constant domain, the variable domains are composed of linked variable and linker regions. The sequence of the TCR constant domain can be found in the public database of the International Immunogenetics Information System (IMGT). For example, the constant domain sequence of the α chain of the TCR molecule is "TRAC*01", and the constant domain sequence of the β chain of the TCR molecule is "TRBC1* 01" or "TRBC2*01". In addition, the α and β chains of TCR also contain a transmembrane region and a cytoplasmic region, which is very short.

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.

Natural interchain disulfide bonds vs. artificial interchain disulfide bonds

There is a group of disulfide bonds between the Cα and Cβ chains in the near-membrane region of the natural TCR, which is called "natural inter-chain disulfide bonds" in the present invention. In the present invention, artificially introduced interchain covalent disulfide bonds whose positions are different from those of natural interchain disulfide bonds are called "artificial interchain disulfide bonds".

In order 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 sequence from the N-terminal to the C-terminal, such as TRBC1*01 or TRBC2*01 , the 60th amino acid in the order from N-terminal to C-terminal is P (proline), then it can be described as Pro60 of exon 1 of TRBC1*01 or TRBC2*01 in the present invention, and can also be described as It is expressed as the 60th amino acid of exon 1 of TRBC1*01 or TRBC2*01, and for example in TRBC1*01 or TRBC2*01, the 61st amino acid in the order from N-terminal to C-terminal is Q (glutamine Amide), then it can be described as Gln61 of TRBC1*01 or TRBC2*01 exon 1 in the present invention, and it can also be expressed as the 61st amino acid of TRBC1*01 or TRBC2*01 exon 1, other 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 a certain amino acid in TRAV is numbered 46 in IMGT, it will be described as the 46th amino acid in TRAV in the present invention, and so on. In the present invention, if there are special instructions for the sequence position numbers of other amino acids, follow the special instructions.

Detailed description of the invention

TCR molecule

During antigen processing, antigens are degraded inside the cell and then carried to the cell surface by MHC molecules. T cell receptors recognize peptide-MHC complexes on the surface of antigen-presenting cells. Therefore, the first aspect of the present invention provides a TCR molecule capable of binding the AQIPEKIQK-HLA A1101 complex. Preferably, said TCR molecule is isolated or purified. The α and β chains of this TCR each have 3 complementarity determining regions (CDRs).

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

αCDR1-SSYSPS (SEQ ID NO: 10)

αCDR2-YTSAATLV (SEQ ID NO: 11)

αCDR3-VVSPGNTPLV (SEQ ID NO: 12); and/or

The three complementarity-determining regions of the TCRβ chain variable domain are:

βCDR1-SGHVS (SEQ ID NO: 13)

βCDR2-FQNEAQ (SEQ ID NO: 14)

βCDR3-ASSPTVGTSAYNEQF (SEQ ID NO: 15).

Chimeric TCR can be prepared by embedding the amino acid sequence of the above CDR region of the present invention into any suitable framework structure. 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 region 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 sequence and any suitable framework structure. The TCRα 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:1; and/or the TCRβ chain variable domain of the present invention is an amino acid sequence identical to SEQ ID NO:1 NO: 5 amino acid sequences having at least 90%, preferably 95%, more preferably 98% sequence identity.

In a preferred example of the present invention, the TCR molecule of the present invention is a heterodimer composed of α and β chains. Specifically, on the 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: 10) of the above α chain. ID NO: 11) and CDR3 (SEQ ID NO: 12). Preferably, the TCR molecule comprises the amino acid sequence of the α-chain variable domain of SEQ ID NO:1. More preferably, the amino acid sequence of the α-chain variable domain of the TCR molecule is SEQ ID NO:1. On the other hand, the β chain of the heterogeneous dimeric TCR molecule comprises a variable domain and a constant domain, and the amino acid sequence of the variable domain of the β chain comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 13) of the above-mentioned β chain. NO: 14) and CDR3 (SEQ ID NO: 15). Preferably, the TCR molecule comprises the amino acid sequence of the β-chain variable domain of SEQ ID NO:5. More preferably, the amino acid sequence of the β chain variable domain of the TCR molecule is SEQ ID NO:5.

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 the description of single-chain TCR molecules, reference can be made to Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-12658. Those skilled in the art can easily construct single-chain TCR molecules comprising the CDRs regions of the present invention according to the literature. Specifically, the single-chain TCR molecule comprises Vα, Vβ and Cβ, preferably connected in order from N-terminus to C-terminus.

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. Preferably, the single-chain TCR molecule comprises the amino acid sequence of an α-chain variable domain of SEQ ID NO:1. More preferably, the amino acid sequence of the α-chain variable domain of the single-chain TCR molecule is SEQ ID NO:1. The amino acid sequence of the β chain variable domain of the single-chain TCR molecule includes CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 14) and CDR3 (SEQ ID NO: 15) of the above β chain. Preferably, the single-chain TCR molecule comprises the amino acid sequence of the β-chain variable domain of SEQ ID NO:5. More preferably, the amino acid sequence of the β-chain variable domain of the single-chain TCR molecule is SEQ ID NO:5.

In a preferred example 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 here as: Arg53 of exon 1 of TRAC*01, and so on. 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.

The naturally occurring TCR is a membrane protein that is stabilized by its transmembrane region. Like immunoglobulin (antibody) as an antigen recognition molecule, TCR can also be developed for diagnosis and treatment, and soluble TCR molecules need to be obtained at this time. Soluble TCR molecules do not include their transmembrane domains. Soluble TCR has a wide range of applications. It can not only be used to study the interaction between TCR and pMHC, but also can be used as a diagnostic tool for detecting infection or as a marker for autoimmune diseases. Similarly, soluble TCRs can be used to deliver therapeutic agents, such as cytotoxic or immunostimulatory compounds, to cells presenting specific antigens, and additionally, soluble TCRs can be conjugated to other molecules, such as anti-CD3 antibodies To redirect T cells so that they target cells presenting specific antigens. The present invention also obtains the soluble TCR specific to the SSX2 antigen short peptide.

To obtain a soluble TCR, on the one hand, the TCR of the present invention may be a TCR that introduces an artificial disulfide bond between the residues of the constant domains of its α and β chains. Cysteine residues form artificial interchain disulfide bonds between the alpha and beta chain constant domains of the TCR. Cysteine residues can be substituted for other amino acid residues at appropriate sites in native TCRs to form artificial interchain disulfide bonds. For example, substitution of Thr48 of TRAC*01 exon 1 and substitution of cysteine residues of Ser57 of TRBC1*01 or TRBC2*01 exon 1 to form disulfide bonds. Other sites for introducing cysteine residues to form disulfide bonds can also be: Thr45 of TRAC*01 exon 1 and Ser77 of TRBC1*01 or TRBC2*01 exon 1; TRAC*01 exon Tyr10 of 1 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; of exon 1 of TRAC*01 Ser15 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; or Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01. That is, cysteine residues replace any one group of positions in the above-mentioned α and β chain constant domains. Up to 50, or up to 30, or up to 15, or up to 10, or up to 8 or fewer amino acids may be truncated at the C-terminus of one or more TCR constant domains of the invention so that they do not include Cysteine residues can be used to delete natural disulfide bonds, or by mutating a cysteine residue that forms a natural disulfide bond to another amino acid.

As noted above, the TCRs of the invention may contain artificial disulfide bonds introduced between residues in the constant domains of their alpha and beta chains. It should be noted that the TCR of the present invention can contain both the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence, with or without the artificial disulfide bond introduced between the constant domains as described above. The TRAC constant domain sequence of the TCR and the TRBC1 or TRBC2 constant domain sequence may be linked by native disulfide bonds present in the TCR.

In order to obtain soluble TCR, on the other hand, the TCR of the present invention also includes TCRs with mutations in its hydrophobic core region, and these mutations in the hydrophobic core region are preferably mutations that can improve the stability of the soluble TCR of the present invention, as disclosed in Publication No. described in the patent literature of WO2014/206304. Such TCRs may have mutations at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable domain amino acids 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or Or the 3rd, 5th, and 7th positions from the bottom of the amino acid position of the α-chain J gene (TRAJ) short peptide, and/or the 2nd, 4th, and 6th position from the bottom of the amino acid position of the β-chain J gene (TRBJ) short peptide, where the position number of the amino acid sequence By position number as 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 numbers of the amino acid residues of different TCRs in IMGT according to the database.

In the present invention, the TCR with a mutation in the hydrophobic core region may 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 linking the variable domains of TCRα and β chains.

In addition, in terms of stability, patent document 201680003540.2 also discloses that the introduction of artificial interchain disulfide bonds between the α-chain variable region and the β-chain constant region of TCR can significantly improve the stability of TCR. Therefore, the TCR of the present invention may also contain an artificial interchain disulfide bond between the variable region of the α chain and the constant region of the β chain. Specifically, the cysteine residue that forms an artificial interchain disulfide bond between the α-chain variable region and the β-chain constant region of the TCR is substituted for: amino acid 46 of TRAV and TRBC1*01 or TRBC2* Amino acid 60 of exon 1 of 01; amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01; amino acid 46 of TRAV and exon of TRBC1*01 or TRBC2*01 Amino acid 61 of exon 1; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01. Preferably, such a TCR may comprise (i) all or part of a TCR alpha chain except for its transmembrane domain, and (ii) all or part of a TCR beta chain except for its transmembrane domain, wherein (i) and (ii ) all contain the variable domain of the TCR chain and at least a part of the constant domain, and the α chain and the β chain form a heterodimer. More preferably, such a TCR may comprise an α-chain variable domain and a β-chain variable domain and all or part of a β-chain constant domain except the transmembrane domain, but it does not contain an α-chain constant domain, and the α-chain of said TCR Chain variable domains form heterodimers with beta chains.

The TCRs of the invention may also be provided in the form of multivalent complexes. The multivalent TCR complex of the present invention comprises two, three, four or more multimers formed by the combination of TCRs of the present invention, such as the tetramerization domain of p53 can be used to produce tetramers, or multimers A complex formed by the combination of a TCR of the present invention and another molecule. The TCR complexes of the invention can be used in vitro or in vivo to track or target cells presenting specific antigens, and can also be used as intermediates for the production of other multivalent TCR complexes for such applications.

The TCR of the present invention can be used alone, and can also be combined with a conjugate in a covalent or other manner, preferably in a covalent manner. The conjugates include detectable markers (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the AQIPEKIQK-HLA A1101 complex), therapeutic agents, PK (protein kinase) modifying moieties, or any of the above combinations or couplings.

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 products capable of producing detectable enzymes.

Therapeutic agents that can be combined or coupled with the TCR of the present invention include, but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews (Cancer metastasis reviews) 24, 539); 2. Biological toxicity (Chaudhary et al., 1989 , Nature (Nature) 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy (Cancer Immunology and Immunotherapy) 51, 565); 3. Cytokines such as IL-2 etc. (Gill ies et al., 1992, National Academy of Sciences of the United States Journal (PNAS) 89, 1428; Card et al., 2004, Cancer Immunology and Immunotherapy (Cancer Immunology and Immunotherapy) 53, 345; Halin et al., 2003, Cancer Research (Cancer Research) 63, 3202); 4. Antibody Fc fragment ( Mosquera et al., 2005, The Journal Of Immunology (The Journal Of Immunology) 174, 4381); 5. Antibody scFv fragment (Zhu et al., 1995, International Journal of Cancer (International Journal of Cancer) 62,319); 6. Gold nanoparticles/nanoparticles Rod (Lapotko et al., 2005, Cancer letters (Cancer letters) 239, 36; Huang et al., 2006, Journal of the American Chemical Society (Journal of the American Chemical Society) 128, 2115); 7. Virus particles (Peng et al., 2004, Gene 8. Liposomes (Mamot et al., 2005, Cancer research (Cancer research) 65, 11631); 9. Nanomagnetic particles; 10. Prodrug activating enzymes (for example, DT-myocardial diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)); 11. Chemotherapeutic agents (eg, cisplatin) or nanoparticles in any form, etc.

In addition, the TCRs of the invention may also be hybrid TCRs comprising sequences derived from more than one species. For example, it has been shown that murine TCRs are more efficiently expressed in human T cells than human TCRs. Thus, a TCR of the invention may comprise a human variable domain and a murine constant domain. A drawback of this approach is the potential for eliciting an immune response. Therefore, its use in adoptive T cell therapy should have regulatory protocols for immunosuppression to allow engraftment of murine-expressing T cells.

It should be understood that the names of amino acids in this article are represented by a single English letter or three English letters commonly used in the world, and the corresponding relationship between a single English letter and three English letters in an amino acid name 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).

nucleic acid molecule

A second aspect of the present invention provides a nucleic acid molecule encoding a TCR molecule of the first aspect of the present invention or a portion thereof, which may be one or more CDRs, variable domains of α and/or β chains, and α chains and/or or beta strand.

The nucleotide sequence encoding the CDR region of the alpha chain of the TCR molecule in the first aspect of the present invention is as follows:

CDR1α-tcttcttattcaccatct (SEQ ID NO: 16)

CDR2α-tacacatcagcggccaccctggtt (SEQ ID NO: 17)

CDR3α-gttgtgagccccggaaacacacctcttgtc (SEQ ID NO: 18)

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

CDR1β-tcgggtcatgtatcc (SEQ ID NO: 19)

CDR2β-ttccagaatgaagctcaa (SEQ ID NO: 20)

CDR3β-gccagcagccccacggtggggactagcgcgtacaatgagcagttc (SEQ ID NO: 21)

Accordingly, the nucleotide sequence of the nucleic acid molecule of the invention encoding the TCR alpha chain of the invention comprises SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and/or the core of the nucleic acid molecule of the invention encoding the TCR beta chain of the invention The nucleotide sequences include SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.

The nucleotide sequence of a nucleic acid molecule of the invention may be single-stranded or double-stranded, the nucleic acid molecule may be RNA or DNA, and may or may not contain introns. Preferably, the nucleotide sequence of the nucleic acid molecule of the present invention does not contain introns but can encode the polypeptide of the present invention, for example, the nucleotide sequence of the nucleic acid molecule of the present invention encoding the variable domain of the TCRα chain of the present invention includes SEQ ID NO: 2 and /or The nucleotide sequence of the nucleic acid molecule of the present invention encoding the TCRβ chain variable domain of the present invention includes SEQ ID NO:6. Preferably, the nucleotide sequence of the nucleic acid molecule of the present invention comprises SEQ ID NO:4 and/or SEQ ID NO:8.

It is understood that due to the degeneracy of the genetic code, different nucleotide sequences may encode the same polypeptide. Therefore, the nucleic acid sequence encoding the TCR of the present invention may be the same as the nucleic acid sequence shown in the figures of the present invention or a degenerate variant. As an example in the present invention, "degenerate variant" refers to a nucleic acid sequence that encodes a protein sequence having SEQ ID NO:1 but differs from the sequence of SEQ ID NO:2.

Nucleotide sequences may be codon optimized. Different cells use different codons, and the codons in the sequence can be changed to increase the expression according to the cell type. Codon usage tables for mammalian cells, as well as for a variety of other organisms, are well known to those skilled in the art.

The full-length sequence of the nucleic acid molecule of the present invention or its fragments can usually be obtained by, but not limited to, PCR amplification, recombination or artificial synthesis. At present, the DNA sequence encoding the TCR of the present invention (or its fragments, or its derivatives) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. DNA can be either the coding strand or the non-coding strand.

carrier

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

Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, herpesviral vectors, retroviral vectors, lentiviral vectors, baculoviral vectors.

Preferably, the vector can transfer the nucleotide of the present invention into cells, such as T cells, so that the cells express SSX2 antigen-specific TCR. Ideally, the vector should be consistently expressed at high levels in T cells.

cell

The present invention also relates to host cells produced by genetic engineering with the vectors or coding sequences of the present invention. The host cell contains the vector of the present invention or the nucleic acid molecule of the present invention is integrated in the chromosome. The host cell is selected from: prokaryotic cells and eukaryotic cells, such as Escherichia coli, yeast cells, CHO cells and the like.

In addition, the present invention also includes isolated cells expressing the TCR of the present invention, which can be, but not limited to, T cells, NK cells, NKT cells, stem cells, especially T cells. The T cells may be derived from T cells isolated from the subject, or may be part of a mixed cell population isolated from the subject, such as a peripheral blood lymphocyte (PBL) population. For example, the cells can be isolated from peripheral blood mononuclear cells (PBMC), and can be CD4 ⁺ helper T cells or CD8 ⁺ cytotoxic T cells. The cells may be in a mixed population of CD4 ⁺ helper T cells/CD8 ⁺ cytotoxic T cells. Typically, the cells can be activated with antibodies (e.g., anti-CD3 or anti-CD28 antibodies) to make them more receptive to transfection, for example, with a vector comprising a nucleotide sequence encoding a TCR molecule of the invention dye.

Alternatively, the cells of the invention may also be or be derived from stem cells, such as hematopoietic stem cells (HSC). Gene transfer to HSCs did not result in TCR expression on the cell surface because stem cells do not express the CD3 molecule on the surface. However, when stem cells differentiate into lymphoid precursors that migrate to the thymus, expression of the CD3 molecule will initiate expression of the introduced TCR molecule on the surface of the thymocyte.

There are a number of methods suitable for T cell transfection with DNA or RNA encoding a TCR of the invention (eg, Robbins et al., (2008) J. Immunol. 180:6116-6131). T cells expressing the TCR of the present invention can be used for adoptive immunotherapy. Many suitable methods for performing adoptive therapy are known to those skilled in the art (eg, Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).

SSX2 antigen-associated diseases

The present invention also relates to a method for treating and/or preventing SSX2-related diseases in a subject, comprising the step of adoptively transferring SSX2-specific T cells to the subject. The SSX2-specific T cells recognize the AQIPEKIQK-HLAA1101 complex.

The SSX2-specific T cells of the present invention can be used to treat any SSX2-related disease presenting the SSX2 antigen short peptide AQIPEKIQK-HLAA1101 complex, including but not limited to tumors, such as liver cancer, lung cancer, fibrosarcoma, breast cancer, colon cancer, prostate cancer .

treatment method

Treatment can be carried out by isolating T cells from patients or volunteers suffering from diseases related to SSX2 antigen, introducing the TCR of the present invention into the above T cells, and then returning these genetically modified cells to the patient. Therefore, the present invention provides a method for treating SSX2-related diseases, comprising infusing isolated T cells expressing the TCR of the present invention, preferably, the T cells are derived from the patient itself, into the patient. Generally, it includes (1) isolating T cells from the patient, (2) in vitro transducing T cells with the nucleic acid molecule of the present invention or a nucleic acid molecule capable of encoding the TCR molecule of the present invention, (3) introducing genetically engineered T cells into the patient in vivo. The number of cells to be isolated, transfected and reinfused can be determined by the physician.

The main advantages of the present invention are:

(1) The TCR of the present invention can specifically bind to the SSX2 antigen short peptide complex AQIPEKIQK-HLA A1101, and at the same time, the effector cells transduced with the TCR of the present invention can be specifically activated.

The following specific examples further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific condition in the following examples, usually according to conventional conditions, for example (Sambrook and Russell et al., Molecular Cloning: Laboratory Manual (Molecular Cloning-A Laboratory Manual) (Third Edition) (2001) CSHL Press ), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples can be obtained from commercially available channels unless otherwise specified.

Example 1, cloning SSX2 antigen short peptide-specific T cells

Peripheral blood lymphocytes (PBL) from healthy volunteers with genotype HLA-A1101 were stimulated with the synthetic short peptide AQIPEKIQK (SEQ ID NO:9; Jiangsu GenScript Biotechnology Co., Ltd.). AQIPEKIQK short peptide was annealed with biotin-labeled HLA-A1101 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. Sorted cells were expanded and subjected to secondary sorting as described above, followed by monoclonalization by limiting dilution. Monoclonal cells were stained with tetramers, and the screened double-positive clones are shown in Figure 3. The double-positive clones obtained through layers of screening still need to meet further functional tests.

IFN-γ is a powerful immunoregulatory factor produced by activated T lymphocytes. Therefore, in this example, the number of IFN-γ is detected by the ELISPOT experiment well known to those skilled in the art to verify the activation function and activation function of cells transfected with the TCR of the present invention. Antigen specificity. The function and specificity of the T cell clone were further detected by ELISPOT experiment. The effector cells used in the IFN-γELISPOT experiment of this example are the T cell clones obtained in the present invention, and the target cells are T2-A11 (referring to T2 cells transfected with HLA-A1101), K562-A11- SSX2 (referring to K562 cells transfected with HLA-A1101 and SSX2), and the control group were T2-A11 and K562 loaded with other antigen short peptides. Among them, T2 cells and K562 cells were purchased from ATCC.

First prepare the ELISPOT plate, and the ELISPOT experiment steps are as follows: Add the components of the test to the ELISPOT plate in the following order: 20,000 target cells/well and 2,000 effector cells/well, then add 20 μl of corresponding short peptides to the experimental group and the control group , so that the final concentration of the short peptide was 10 ^-5 M, 20 μl medium (test medium) was added to the blank group, and 2 replicate wells were set. It was then incubated overnight (37°C, 5% CO ₂ ). Then the plate was washed for secondary detection and color development, and the plate was dried for 1 hour, and the spots formed on the membrane were counted by an immunospot plate reader (ELISPOT READER system; AID company). The experimental results are shown in Figure 8. The obtained T cell clones highly released IFN-γ to T2-A11 and K562-A11-SSX2 loaded with AQIPEKIQK short peptides, but released little IFN-γ to T2-A11 and K562 loaded with other antigen short peptides. No reaction.

Example 2. Obtaining the TCR gene of the SSX2 antigen short peptide-specific T cell clone

The total RNA of the AQIPEKIQK-specific and HLA-A1101-restricted T cell clones screened in Example 1 was extracted with Quick-RNA ^™ MiniPrep (ZYMO research). The cDNA was synthesized using clontech's SMART RACE cDNA amplification kit, and the primers used were designed at the C-terminal conserved region of the human TCR gene. The sequence was cloned into T vector (TAKARA) for sequencing. It should be noted that this sequence is complementary and does not contain introns. After sequencing, the sequence structures of the TCR α chain and β chain expressed by the double-positive clone are shown in Figure 1 and Figure 2, respectively. Variable domain amino acid sequence, TCRα chain variable domain nucleotide sequence, TCRα chain amino acid sequence, TCRα chain nucleotide sequence, TCRα chain amino acid sequence with leader sequence and TCRα chain nucleotide sequence with leader sequence; Figure 2a, Figure 2b, Figure 2c, Figure 2d, Figure 2e and Figure 2f are the amino acid sequence of the TCRβ chain variable domain, the nucleotide sequence of the TCRβ chain variable domain, the amino acid sequence of the TCRβ chain, the nucleotide sequence of the TCRβ chain, and the TCRβ chain amino acid sequence and TCRβ chain nucleotide sequence with leader sequence.

The alpha chain was identified to contain CDRs with the following amino acid sequence:

αCDR1-SSYSPS (SEQ ID NO: 10)

αCDR2-YTSAATLV (SEQ ID NO: 11)

αCDR3-VVSPGNTPLV (SEQ ID NO: 12)

The beta strand contains CDRs with the following amino acid sequence:

βCDR1-SGHVS (SEQ ID NO: 13)

βCDR2-FQNEAQ (SEQ ID NO: 14)

βCDR3-ASSPTVGTSAYNEQF (SEQ ID NO: 15).

Example 3, Expression, refolding and purification of SSX2 antigen short peptide-specific soluble TCR

In order to obtain a soluble TCR molecule, the α and β chains of the TCR molecule of the present invention may only include their variable domains and part of the constant domains respectively, and a cysteine residue is introduced into the constant domains of the α and β chains respectively To form an artificial interchain disulfide bond, the amino acid sequence and nucleotide sequence of its α chain are shown in Figure 4a and Figure 4b, respectively, and the amino acid sequence and nucleotide sequence of its β chain are shown in Figure 5a and Figure 5b, respectively . The target gene sequences of the above-mentioned TCRα and β chains were synthesized and inserted into the expression vector pET28a+ (Novagene) by the standard method described in "Molecular Cloning a Laboratory Manual" (third edition, Sambrook and Russell) , the upstream and downstream cloning sites are NcoI and NotI, respectively. The insert was confirmed by sequencing.

The expression vectors of the TCRα and β chains were transformed into the expressing bacteria BL21(DE3) by chemical transformation method respectively, the bacteria were grown in LB culture medium, induced with a final concentration of 0.5mM IPTG at OD ₆₀₀ =0.6, and the α and β chains of TCR were expressed The inclusion bodies formed after were extracted by BugBuster Mix (Novagene), and repeatedly washed with BugBuster solution, and the inclusion bodies were finally dissolved in 6M guanidine hydrochloride, 10mM dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA ), 20mM Tris (pH8.1).

The dissolved TCR α and β chains were quickly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine, 6.6mM β-mercapoethylamine (4°C) at a mass ratio of 1:1, and the final concentration was 60mg/mL. After mixing, the solution was placed in 10 times the volume of deionized water for dialysis (4°C). After 12 hours, the deionized water was replaced with buffer solution (20mM Tris, pH 8.0) and the dialysis was continued at 4°C for 12 hours. After the dialysis was completed, the solution was filtered through a 0.45 μM filter membrane, and then purified by an anion exchange column (HiTrap Q HP, 5 ml, GE Healthcare). The elution peaks containing TCRs of successfully refolded α and β dimers were confirmed by SDS-PAGE. TCR was then further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was determined by SDS-PAGE to be greater than 90%, and the concentration was determined by the BCA method. The SDS-PAGE gel images of the soluble TCR obtained in the present invention are shown in Figures 6a and 6b.

Embodiment 4, combining characterization

This example proves that the soluble TCR molecule of the present invention can specifically bind to the AQIPEKIQK-HLAA1101 complex through BIAcore analysis.

The binding activity between the TCR molecule obtained in Example 3 and the AQIPEKIQK-HLAA1101 complex was detected using a BIAcore T200 real-time analysis system. Add the anti-streptavidin antibody (GenScript) to the coupling buffer (10mM sodium acetate buffer, pH 4.77), and then flow the antibody through the CM5 chip activated with EDC and NHS to immobilize the antibody on the chip surface , and finally the unreacted activated surface was blocked with ethanolamine hydrochloric acid solution to complete the coupling process, and the coupling level was about 15,000 RU.

Let a low concentration of streptavidin flow over the surface of the antibody-coated chip, then flow the AQIPEKIQK-HLAA1101 complex through the detection channel, and the other channel as a reference channel, and then 0.05mM biotin at 10μL/min The flow rate flowed through the chip for 2 minutes to block the remaining binding sites of streptavidin.

The preparation process of the above-mentioned AQIPEKIQK-HLA A1101 complex is as follows:

a.Purification

Collect 100ml of E.coli bacteria liquid induced to express heavy chain or light chain, centrifuge at 8000g at 4°C for 10min, wash the bacteria once with 10ml PBS, then resuspend the bacteria with 5ml BugBuster Master Mix Extraction Reagents (Merck) vigorously shake, and place in Rotate and incubate at room temperature for 20 min, then centrifuge at 6000 g for 15 min at 4 °C, discard the supernatant, and collect inclusion bodies.

Suspend the above inclusion bodies in 5ml of BugBuster Master Mix, incubate with rotation at room temperature for 5min; add 30ml of 10-fold diluted BugBuster, mix well, and centrifuge at 6000g at 4°C for 15min; discard the supernatant, add 30ml of 10-fold diluted BugBuster to resuspend the inclusion bodies , mix well, centrifuge at 6000g at 4°C for 15min, repeat twice, add 30ml 20mM Tris-HCl pH 8.0 to resuspend inclusion bodies, mix well, centrifuge at 6000g at 4°C for 15min, finally dissolve inclusion bodies with 20mM Tris-HCl 8M urea, SDS- The purity of inclusion bodies was detected by PAGE, and the concentration was measured by BCA kit.

b. Refolding

The synthetic short peptide AQIPEKIQK was dissolved in DMSO to a concentration of 20 mg/ml. The inclusion bodies of the light chain and heavy chain were dissolved with 8M urea, 20mM Tris pH 8.0, 10mM DTT, and further denatured by adding 3M guanidine hydrochloride, 10mM sodium acetate, and 10mM EDTA before renaturation. Add AQIPEKIQK peptide at 25mg/L (final concentration) to refolding buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 0.2 mM PMSF, cooled to 4°C), and then sequentially added 20mg/L light chain and 90mg/L heavy chain (final concentration, heavy chain was added in three times, 8h/time), renaturation was carried out at 4°C for at least 3 days to After completion, SDS-PAGE test whether the renaturation is successful.

c. Purification after renaturation

Replace the refolding buffer by dialysis against 10 volumes of 20 mM Tris pH 8.0, at least twice to sufficiently 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 General Electric Company) anion exchange column (5 ml bed volume). Using Akta Purifier (GE General Electric Company), 0-400mM NaCl linear gradient solution prepared by 20mM Tris pH 8.0 was used to elute protein, and pMHC was eluted at about 250mM NaCl, and the peak components were collected, and the purity was detected by SDS-PAGE.

d. Biotinylation

Concentrate the purified pMHC molecules with Millipore ultrafiltration tubes, and at the same time replace the buffer with 20mM Tris pH8.0, then add biotinylation reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50μM D-Biotin, 100μg/ml BirA enzyme (GST-BirA), the mixture was incubated overnight at room temperature, and SDS-PAGE was used to detect whether the biotinylation was complete.

e. Purification of biotinylated complexes

Concentrate the biotinylated pMHC molecules to 1ml with a Millipore ultrafiltration tube, purify the biotinylated pMHC by gel filtration chromatography, and pre-equilibrate HiPrep with filtered PBS using an Akta purification instrument (GE General Electric Company) ^TM 16/60S200 HR column (GE General Electric Company), loaded with 1 ml of concentrated biotinylated pMHC molecules, and then eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules elute as a single peak at about 55 ml. The protein-containing fractions were combined, concentrated with Millipore ultrafiltration tubes, and the protein concentration was determined by the BCA method (Thermo). The biotinylated pMHC molecules were subpackaged and stored at -80°C by adding protease inhibitor cocktail (Roche).

Kinetic parameters were calculated using BIAcore Evaluation software, and the kinetic maps of the binding of the soluble TCR molecule of the present invention to the AQIPEKIQK-HLA A1101 complex were obtained, respectively, as shown in FIG. 7 . The map shows that the soluble TCR molecule obtained in the present invention can combine with the AQIPEKIQK-HLA A1101 complex. At the same time, the binding activity of the soluble TCR molecules of the present invention to the complexes of several other irrelevant antigen short peptides and HLA was detected by the above method, and the results showed that the TCR molecules of the present invention had no binding to other irrelevant antigens.

Example 5. Effector cell activation experiment of transfection TCR of the present invention for T2 cells loaded with gradient concentrations of short peptides

The effector cells used in this experiment were CD3 ⁺ T cells transfected with the TCR of the present invention, and CD3 ⁺ T cells transfected with other TCR (A6) from the same volunteer were used as a control group. The target cells used are T2-A11 loaded with SSX2 antigen short peptide AQIPEKIQK.

First prepare ELISPOT plate, ELISPOT plate ethanol activation coating, 4 ℃ overnight. On the first day of the experiment, remove the coating solution, wash and block, incubate at room temperature for two hours, remove the blocking solution, and add each component of the test to the ELISPOT plate: 2× ¹⁰ cells/well for target cells, 2× for effector cells 10 ³ cells/well (calculated according to the positive rate of transfection), and set up two duplicate holes. Then AQIPEKIQK short peptide was added to the corresponding wells, so that the final concentration of the short peptide in the ELISPOT well plate was sequentially from 10 ^-6 M to 10 ^-11 , a total of 6 gradient concentrations. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, the plate was washed for secondary detection and color development, the plate was dried, and the spots formed on the membrane were counted by an immunospot plate reader (ELISPOT READER system; AID20 company).

The experimental results are shown in Figure 9. For T2-A11 loaded with AQIPEKIQK short peptide, the T cells transfected with the TCR of the present invention had a significant activation effect, while the T cells transfected with other TCRs were still inactive at the highest peptide concentration.

Example 6. Activation function experiment of effector cells transfected with TCR of the present invention for tumor cell lines

In this embodiment, the function and specificity of the TCR of the present invention in cells are also tested by ELISPOT experiment. The effector cells used were CD3 ⁺ T cells expressing the specific TCR of the SSX2 antigen short peptide of the present invention, and the CD3 + T cells transfected with other TCR (A6) from the same volunteer were used as the control group. The positive tumor cell line used was SK-MEL-28-SSX2 (SK-MEL-28 cells transfected with SSX2); the negative tumor cell lines used were SK-MEL-1, SNU423, SK-MEL-5 and HUCCT1, as control group. Among them, SK-MEL-28, SK-MEL-1, SNU423, and HUCCT1 were all purchased from Guangzhou Saiku Biotechnology Co., Ltd., and SK-MEL-5 was purchased from ATCC.

First prepare the ELISPOT plate. ELISPOT plates were activated with ethanol and coated overnight at 4°C. On the first day of the experiment, remove the coating solution, wash and block, incubate at room temperature for two hours, remove the blocking solution, and add each component of the test to the ELISPOT plate: 2× ¹⁰ cells/well for target cells, 2× for effector cells 10 ³ cells/well (calculated according to the positive rate of transfection), and set up two duplicate holes. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, the plate was washed for secondary detection and color development, the plate was dried, and the spots formed on the membrane were counted by an immunospot plate reader (ELISPOT READER system; AID20 company).

The experimental results are shown in Figure 10, the effector cells transfected with other TCRs were inactive against all cell lines; the effector cells transfected with the TCR of the present invention were specifically activated by positive tumor cell lines, but inactive against negative tumor cell lines.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

SEQUENCE LISTING

<110> Xiangxue Life Science Technology (Guangdong) Co., Ltd.

<120> A T-cell receptor that recognizes SSX2 and its coding sequence

<130> 215730

<160> 29

<170> PatentIn version 3.3

<210> 1

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> TCR alpha chain variable domain amino acid sequence

<400> 1

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

Lys Tyr Thr Ser Ala Ala Thr Leu Val Lys Gly Ile Asn Gly Phe Glu

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Val Val Ser Pro Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

<210> 2

<211> 336

<212>DNA

<213> Artificial Sequence

<220>

<223> TCR alpha chain variable domain nucleotide sequence

<400> 2

gcccagtcgg tgacccagct tgacagccac gtctctgtct ctgaaggaac cccggtgctg 60

ctgaggtgca actactcatc ttcttattca ccatctctct tctggtatgt gcaacaccccc 120

aacaaaggac tccagcttct cctgaagtac acatcagcgg ccaccctggt taaaggcatc 180

aacggttttg aggctgaatt taagaagagt gaaacctcct tccacctgac gaaaccctca 240

gcccatatga gcgacgcggc tgagtacttc tgtgttgtga gccccggaaa cacacctctt 300

gtctttggaa agggcacaag actttctgtg attgca 336

<210> 3

<211> 253

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of alpha chain of TCR molecule

<400> 3

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

Lys Tyr Thr Ser Ala Ala Thr Leu Val Lys Gly Ile Asn Gly Phe Glu

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Val Val Ser Pro Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp

195 200 205

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

210 215 220

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

225 230 235 240

Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

245 250

<210> 4

<211> 759

<212>DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence of alpha chain of TCR molecule

<400> 4

gcccagtcgg tgacccagct tgacagccac gtctctgtct ctgaaggaac cccggtgctg 60

ctgaggtgca actactcatc ttcttattca ccatctctct tctggtatgt gcaacaccccc 120

aacaaaggac tccagcttct cctgaagtac acatcagcgg ccaccctggt taaaggcatc 180

aacggttttg aggctgaatt taagaagagt gaaacctcct tccacctgac gaaaccctca 240

gcccatatga gcgacgcggc tgagtacttc tgtgttgtga gccccggaaa cacacctctt 300

gtctttggaa agggcacaag actttctgtg attgcaaata tccagaaccc tgaccctgcc 360

gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 420

gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 480

gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540

tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600

cccagcccag aaagttcctg tgatgtcaag ctggtcgaga aaagctttga aacagatacg 660

aacctaaact ttcaaaacct gtcagtgatt gggttccgaa tcctcctcct gaaagtggcc 720

gggtttaatc tgctcatgac gctgcggctg tggtccagc 759

<210> 5

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> TCR beta chain variable domain amino acid sequence

<400> 5

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ser Pro

85 90 95

Thr Val Gly Thr Ser Ala Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr

100 105 110

Arg Leu Thr Val Leu

115

<210> 6

<211> 351

<212>DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence of TCR beta chain variable domain

<400> 6

ggtgctggag tctcccagtc ccctaggtac aaagtcgcaa agagaggaca ggatgtagct 60

ctcaggtgtg atccaatttc gggtcatgta tccctttttt ggtaccaaca ggccctgggg 120

caggggccag agtttctgac ttatttccag aatgaagctc aactagacaa atcggggctg 180

cccagtgatc gcttctttgc agaaaggcct gagggatccg tctccactct gaagatccag 240

cgcacacagc aggaggactc cgccgtgtat ctctgtgcca gcagccccac ggtggggact 300

agcgcgtaca atgagcagtt cttcgggcca gggacacggc tcaccgtgct a 351

<210> 7

<211> 296

<212> PRT

<213> Artificial Sequence

<220>

<223> beta chain amino acid sequence of TCR

<400> 7

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ser Pro

85 90 95

Thr Val Gly Thr Ser Ala Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Val Lys Arg Lys Asp Ser Arg Gly

290 295

<210> 8

<211> 888

<212>DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence of TCR beta chain variable domain

<400> 8

ggtgctggag tctcccagtc ccctaggtac aaagtcgcaa agagaggaca ggatgtagct 60

ctcaggtgtg atccaatttc gggtcatgta tccctttttt ggtaccaaca ggccctgggg 120

caggggccag agtttctgac ttatttccag aatgaagctc aactagacaa atcggggctg 180

cccagtgatc gcttctttgc agaaaggcct gagggatccg tctccactct gaagatccag 240

cgcacacagc aggaggactc cgccgtgtat ctctgtgcca gcagccccac ggtggggact 300

agcgcgtaca atgagcagtt cttcgggcca gggacacggc tcaccgtgct agaggacctg 360

aaaaacgtgt tcccacccga ggtcgctgtg tttgagccat cagaagcaga gatctcccac 420

acccaaaagg ccaacactggt gtgcctggcc acaggcttct accccgacca cgtggagctg 480

agctggtggg tgaatgggaa ggaggtgcac agtggggtca gcacagaccc gcagcccctc 540

aaggagcagc ccgccctcaa tgactccaga tactgcctga gcagccgcct gagggtctcg 600

gccaccttct ggcagaaccc ccgcaaccac ttccgctgtc aagtccagtt ctacgggctc 660

tcggagaatg acgagtggac ccaggatagg gccaaacctg tcacccagat cgtcagcgcc 720

gaggcctggg gtagagcaga ctgtggcttc acctccgagt cttaccagca aggggtcctg 780

tctgccacca tcctctatga gatcttgcta gggaaggcca ccttgtatgc cgtgctggtc 840

agtgccctcg tgctgatggc catggtcaag agaaaggatt ccagaggc 888

<210> 9

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Short Peptides

<400> 9

Ala Gln Ile Pro Glu Lys Ile Gln Lys

1 5

<210> 10

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> CDRs

<400> 10

Ser Ser Tyr Ser Pro Ser

1 5

<210> 11

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDRs

<400> 11

Tyr Thr Ser Ala Ala Thr Leu Val

1 5

<210> 12

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDRs

<400> 12

Val Val Ser Pro Gly Asn Thr Pro Leu Val

1 5 10

<210> 13

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> CDRs

<400> 13

Ser Gly His Val Ser

1 5

<210> 14

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> CDRs

<400> 14

Phe Gln Asn Glu Ala Gln

1 5

<210> 15

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> CDRs

<400> 15

Ala Ser Ser Pro Thr Val Gly Thr Ser Ala Tyr Asn Glu Gln Phe

1 5 10 15

<210> 16

<211> 18

<212>DNA

<213> Artificial Sequence

<220>

<223> CDRs

<400> 16

tcttcttatt caccatct 18

<210> 17

<211> 24

<212>DNA

<213> Artificial Sequence

<220>

<223> CDRs

<400> 17

tacacatcag cggccaccct ggtt 24

<210> 18

<211> 30

<212>DNA

<213> Artificial Sequence

<220>

<223> CDRs

<400> 18

gttgtgagcc ccggaaacac acctcttgtc 30

<210> 19

<211> 15

<212>DNA

<213> Artificial Sequence

<220>

<223> CDRs

<400> 19

tcgggtcatg tatcc 15

<210> 20

<211> 18

<212>DNA

<213> Artificial Sequence

<220>

<223> CDRs

<400> 20

ttccagaatg aagctcaa 18

<210> 21

<211> 45

<212>DNA

<213> Artificial Sequence

<220>

<223> CDRs

<400> 21

gccagcagcc ccacggtggg gactagcgcg tacaatgagc agttc 45

<210> 22

<211> 272

<212> PRT

<213> Artificial Sequence

<220>

<223> TCR alpha chain amino acid sequence with leader sequence

<400> 22

Met Leu Leu Leu Leu Val Pro Val Leu Glu Val Ile Phe Thr Leu Gly

1 5 10 15

Gly Thr Arg Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val

20 25 30

Ser Glu Gly Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Ser Tyr

35 40 45

Ser Pro Ser Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln

50 55 60

Leu Leu Leu Lys Tyr Thr Ser Ala Ala Thr Leu Val Lys Gly Ile Asn

65 70 75 80

Gly Phe Glu Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr

85 90 95

Lys Pro Ser Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Val Val

100 105 110

Ser Pro Gly Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser

115 120 125

Val Ile Ala Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

<210> 23

<211> 816

<212>DNA

<213> Artificial Sequence

<220>

<223> TCR alpha chain nucleotide sequence with leader sequence

<400> 23

atgctcctgc tgctcgtccc agtgctcgag gtgattttta ctctgggagg aaccagagcc 60

cagtcggtga cccagcttga cagccacgtc tctgtctctg aaggaaccccc ggtgctgctg 120

aggtgcaact actcatcttc ttattcacca tctctcttct ggtatgtgca acaccccaac 180

aaaggactcc agcttctcct gaagtacaca tcagcggcca ccctggttaa aggcatcaac 240

ggttttgagg ctgaatttaa gaagagtgaa acctccttcc acctgacgaa accctcagcc 300

catatgagcg acgcggctga gtacttctgt gttgtgagcc ccggaaacac acctcttgtc 360

tttggaaagg gcacaagact ttctgtgatt gcaaatatcc agaaccctga ccctgccgtg 420

taccagctga gagactctaa atccagtgac aagtctgtct gcctattcac cgattttgat 480

tctcaaacaa atgtgtcaca aagtaaggat tctgatgtgt atatcacaga caaaactgtg 540

ctagacatga ggtctatgga cttcaagagc aacagtgctg tggcctggag caacaaatct 600

gactttgcat gtgcaaacgc cttcaacaac agcattattc cagaagacac cttcttcccc 660

agcccagaaa gttcctgtga tgtcaagctg gtcgagaaaa gctttgaaac agatacgaac 720

ctaaactttc aaaacctgtc agtgattggg ttccgaatcc tcctcctgaa agtggccggg 780

tttaatctgc tcatgacgct gcggctgtgg tccagc 816

<210> 24

<211> 315

<212> PRT

<213> Artificial Sequence

<220>

<223> TCR beta chain amino acid sequence with leader sequence

<400> 24

Met Gly Thr Arg Leu Leu Cys Trp Val Val Leu Gly Phe Leu Gly Thr

1 5 10 15

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

20 25 30

Lys Arg Gly Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His

35 40 45

Val Ser Leu Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe

50 55 60

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

65 70 75 80

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

85 90 95

Lys Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala

100 105 110

Ser Ser Pro Thr Val Gly Thr Ser Ala Tyr Asn Glu Gln Phe Phe Gly

115 120 125

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

130 135 140

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

145 150 155 160

Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His

165 170 175

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

180 185 190

Ser Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser

195 200 205

Arg Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu

290 295 300

Met Ala Met Val Lys Arg Lys Asp Ser Arg Gly

305 310 315

<210> 25

<211> 945

<212>DNA

<213> Artificial Sequence

<220>

<223> TCR beta chain nucleotide sequence with leader sequence

<400> 25

atgggcacca ggctcctctg ctgggtggtc ctgggtttcc tagggacaga tcacacaggt 60

gctggagtct cccagtcccc taggtacaaa gtcgcaaaga gaggacagga tgtagctctc 120

aggtgtgatc caatttcggg tcatgtatcc cttttttggt accaacaggc cctggggcag 180

gggccagagt ttctgactta tttccagaat gaagctcaac tagacaaatc ggggctgccc 240

agtgatcgct tctttgcaga aaggcctgag ggatccgtct ccactctgaa gatccagcgc 300

acacagcagg aggactccgc cgtgtatctc tgtgccagca gccccacggt ggggactagc 360

gcgtacaatg agcagttctt cgggccaggg acacggctca ccgtgctaga ggacctgaaa 420

aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 480

caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 540

tggtgggtga atgggaagga ggtgcacagt gggtcagca cagacccgca gcccctcaag 600

gagcagcccg ccctcaatga ctccagatac tgcctgagca gccgcctgag ggtctcggcc 660

accttctggc agaacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 720

gagaatgacg agtggaccca gtagagggcc aaacctgtca cccagatcgt cagcgccgag 780

gcctggggta gagcagactg tggcttcacc tccgagtctt accagcaagg ggtcctgtct 840

gccaccatcc tctatgagat cttgctaggg aaggccacct tgtatgccgt gctggtcagt 900

gccctcgtgc tgatggccat ggtcaagaga aaggattcca gaggc 945

<210> 26

<211> 207

<212> PRT

<213> Artificial Sequence

<220>

<223> alpha chain amino acid sequence of TCR

<400> 26

Met Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu

1 5 10 15

Gly Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro

20 25 30

Ser Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu

35 40 45

Leu Lys Tyr Thr Ser Ala Ala Thr Leu Val Lys Gly Ile Asn Gly Phe

50 55 60

Glu Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro

65 70 75 80

Ser Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Val Val Ser Pro

85 90 95

Gly Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile

100 105 110

Ala Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Ser Ile Ile Pro Glu Asp Thr Phe Phe Cys Ser Pro Glu Ser Ser

195 200 205

<210> 27

<211>621

<212>DNA

<213> Artificial Sequence

<220>

<223> Alpha chain nucleotide sequence of TCR

<400> 27

atggcgcaga gcgtgaccca gcttgacagc cacgtctctg tctctgaagg aaccccggtg 60

ctgctgaggt gcaactactc atcttcttat tcaccatctc tcttctggta tgtgcaacac 120

cccaacaaag gactccagct tctcctgaag tacacatcag cggccaccct ggttaaaggc 180

atcaacggtt ttgaggctga atttaagaag agtgaaacct ccttccacct gacgaaaccc 240

tcagcccata tgagcgacgc ggctgagtac ttctgtgttg tgagccccgg aaacacacct 300

cttgtctttg gaaagggcac aagactttct gtgattgcaa atatccagaa ccctgaccct 360

gccgtttatc agctgcgtga tagcaaaagc agcgataaaa gcgtgtgcct gttcaccgat 420

tttgatagcc agaccaacgt gagccagagc aaagatagcg atgtgtacat caccgataaa 480

accgtgctgg atatgcgcag catggatttc aaaagcaata gcgcggttgc gtggagcaac 540

aaaagcgatt ttgcgtgcgc gaacgcgttt aacaacagca tcatcccgga agatacgttc 600

ttctgcagcc cagaaagttc c 621

<210> 28

<211> 248

<212> PRT

<213> Artificial Sequence

<220>

<223> beta chain amino acid sequence of TCR

<400> 28

Met Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg

1 5 10 15

Gly Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser

20 25 30

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

35 40 45

Tyr Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp

50 55 60

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

65 70 75 80

Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ser

85 90 95

Pro Thr Val Gly Thr Ser Ala Tyr Asn Glu Gln Phe Phe Gly Pro Gly

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Ala Glu Ala Trp Gly Arg Ala Asp

245

<210> 29

<211> 744

<212>DNA

<213> Artificial Sequence

<220>

<223> TCR beta chain nucleotide sequence

<400> 29

atgggtgcag gtgttagcca gtcccctagg tacaaagtcg caaagagagg acaggatgta 60

gctctcaggt gtgatccaat ttcgggtcat gtatcccttt tttggtacca acaggccctg 120

gggcaggggc cagagtttct gacttatttc cagaatgaag ctcaactaga caaatcgggg 180

ctgcccagtg atcgcttctt tgcagaaagg cctgagggat ccgtctccac tctgaagatc 240

cagcgcacac agcaggagga ctccgccgtg tatctctgtg ccagcagccc cacggtgggg 300

actagcgcgt acaatgagca gttcttcggg ccagggacac ggctcaccgt gctagaggac 360

ctgaaaaacg tgttcccacc cgaggtcgct gtgtttgagc catcagaatg cgaaattagc 420

catacccaga aagcgaccct ggtttgtctg gcgaccggtt tttatccgga tcatgtggaa 480

ctgtcttggt gggtgaacgg caaagaagtg catagcggtg tttctaccga tccgcagccg 540

ctgaaagaac agccggcgct gaatgatagc cgttatgcgc tgtctagccg tctgcgtgtt 600

agcgcgacct tttggcaaaa tccgcgtaac cattttcgtt gccaggtgca gttttatggc 660

ctgagcgaaa acgatgaatg gacccaggat cgtgcgaagc cggttacccca gattgttagc 720

gcggaagcct ggggccgcgc agat 744

Claims (10)

1. A T cell receptor (TCR), said TCR comprises TCR α chain variable domain and TCR β chain variable domain, it is characterized in that, said TCR can be combined with AQIPEKIQK-HLA A1101 complex, and said TCR α The three complementarity determining regions (CDRs) of the chain variable domain are: αCDR1-SSYSPS (SEQ ID NO: 10) αCDR2-YTSAATLV (SEQ ID NO: 11) αCDR3-VVSPGNTPLV (SEQ ID NO: 12); and/or The three complementarity-determining regions of the TCRβ chain variable domain are: βCDR1-SGHVS (SEQ ID NO: 13) βCDR2-FQNEAQ (SEQ ID NO: 14) βCDR3-ASSPTVGTSAYNEQF (SEQ ID NO: 15). 2. The T cell receptor of claim 1, wherein it comprises a TCR α chain variable domain and a TCR β chain variable domain, the TCR α chain variable domain being at least 90% identical to SEQ ID NO: 1 an amino acid sequence with sequence identity; and/or said TCR beta chain variable domain is an amino acid sequence with at least 90% sequence identity to SEQ ID NO:5. 3. T cell receptor as claimed in claim 1, is characterized in that, the C-or N-terminus of the α chain of described TCR and/or β chain is combined with conjugate; Preferably, with described T cell The receptor-binding conjugate is a detectable label, a therapeutic agent, a PK modifying moiety, or a combination of any of these; preferably, the therapeutic agent is an anti-CD3 antibody. 4. A multivalent TCR complex, characterized in that it comprises at least two TCR molecules, and at least one of the TCR molecules is the TCR according to any one of the preceding claims. 5. A nucleic acid molecule, characterized in that, the nucleic acid molecule comprises a nucleic acid sequence encoding the TCR molecule according to any one of the preceding claims or a complementary sequence thereof; Preferably, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 2 encoding the variable domain of the TCR α chain and/or the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 6 encoding the variable domain of the TCR β chain . 6. A vector, characterized in that, the vector contains the nucleic acid molecule according to claim 5; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector. 7. An isolated host cell, characterized in that the host cell contains the vector according to claim 6 or the exogenous nucleic acid molecule according to claim 5 integrated in the chromosome. 8. A cell, characterized in that the cell is transduced with the nucleic acid molecule of claim 5 or the vector of claim 6; preferably, the cell is a T cell or a stem cell. 9. A pharmaceutical composition, characterized in that the composition contains a pharmaceutically acceptable carrier and the TCR according to any one of claims 1-3, the TCR complex described in claim 4, the The nucleic acid molecule of claim 5, or the cell of claim 8. 10. The use of the T cell receptor according to any one of claims 1-3, or the TCR complex described in claim 4, or the cells described in claim 8, characterized in that it is used for the preparation of therapeutic Drugs for tumor or autoimmune diseases.

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