CN109890839B - T cell receptors that recognize SAGE1 antigenic short peptides - Google Patents

Abstract

Provided is a T Cell Receptor (TCR) capable of specifically binding the short peptide VFSTVPPAFI derived from SAGE1 antigen, a nucleic acid molecule encoding the TCR, a vector comprising the nucleic acid molecule, and a cell transducing the TCR. The antigen short peptide VFSTVPPAFI can form a complex with HLA A2402 and is presented on the surface of a cell together.

Description

T cell receptors that recognize SAGE1 antigenic short peptides

technical field

The present invention relates to TCRs capable of recognizing short peptides derived from SAGE1 antigen, the present invention also relates to SAGE1-specific T cells obtained by transducing the above-mentioned TCRs, and their use in preventing and treating SAGE1-related diseases.

Background technique

As an endogenous tumor antigen, SAGE1 is degraded into small molecule polypeptides after being generated in cells, and combined with MHC (major histocompatibility complex) molecules to form complexes, which are presented to the cell surface. Studies have shown that VFSTVPPAFI is a short peptide derived from SAGE1. SAGE1 antigen is expressed in tumor tissues such as melanoma, bladder cancer, liver cancer, epidermoid carcinoma, non-small cell lung cancer and squamous cell carcinoma, but not in most normal tissues except testis (Martelange V1, De Smet C, De Plaen E, Lurquin C, Boon T. Cancer Res. 2000; 60(14): 3848-55; Atanackovic D, et al., Cancer Biol Ther. 2006; 5(9): 1218-25). For the treatment of the above diseases, chemotherapy and radiation therapy can be used, but they will cause damage to their own normal cells.

T cell adoptive immunotherapy is the transfer of T cells specific for target cell antigens into the patient's body, so that they can play a role against the target cells. T cell receptor (TCR) is a membrane protein on the surface of T cells that can recognize short antigenic peptides on the surface of the corresponding target cells. In the immune system, direct physical contact between T cells and antigen presenting cells (APCs) is triggered by the binding of antigen peptide-specific TCRs to the peptide-major histocompatibility complex (pMHC complex), and then T cells and The other cell membrane surface molecules of the two APCs interact, causing a series of subsequent cell signaling and other physiological responses, so that T cells with different antigen specificities can exert immune effects on their target cells. Therefore, those skilled in the art are devoted to isolating TCRs specific for SAGE1 antigen short peptides, making them work, or transducing the TCRs into T cells to obtain T cells specific for SAGE1 antigen short peptides, so that the They play a role in cellular immunotherapy.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a T cell receptor that recognizes the SAGE1 antigenic short peptide.

The first aspect of the present invention provides a T cell receptor (TCR), the TCR can bind to the VFSTVPPAFI-HLAA2402 complex.

In another preferred embodiment, the TCR comprises a TCRα chain variable domain and a TCR chain variable domain, and the amino acid sequence of CDR3 of the TCRα chain variable domain is CAVLYTGANSKLTF (SEQ ID NO. 12); and/or the The amino acid sequence of CDR3 of the variable domain of the TCR chain is CASSLVGKQPQHF (SEQ ID NO. 15).

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

αCDR1-DSAIYN (SEQ ID NO. 10)

αCDR2-IQSSQRE (SEQ ID NO. 11)

αCDR3-CAVLYTGANSKLTF (SEQ ID NO. 12); and/or

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

CDR1-SGHDT (SEQ ID NO. 13)

CDR2-YYEEEE (SEQ ID NO. 14)

CDR3-CASSLVGKQPQHF (SEQ ID NO. 15).

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

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

In another preferred embodiment, the TCR comprises a beta chain variable domain amino acid sequence of SEQ ID NO.5.

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

In another preferred embodiment, 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 embodiment, the TCR is soluble.

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

In another preferred embodiment, 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 embodiment, the TCR is at the 11th, 13th, 19th, 21st, 53rd, 76th, 89th, 91st, or 94th amino acid position of the α chain variable region, and/or the reciprocal amino acid of the α chain J gene short peptide has one or more mutations in position 3, 5 from the bottom, or 7 from the bottom; and/or the TCR is at amino acids 11, 13, 19, 21, 53, 76, 89, 91 of the beta chain variable region , or position 94, and/or one or more mutations in the penultimate amino acid position 2, 4 or 6 of the β-chain J gene short peptide, wherein the amino acid positions are numbered according to IMGT (International Information on Immunogenetics) system) listed in the position number.

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

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

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

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:

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

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

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

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

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

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

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

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

In another preferred embodiment, 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.

In another preferred embodiment, an artificial interchain disulfide bond is contained between the variable region of the α chain and the constant region of the β chain of the TCR.

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

Amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*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 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 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 except the transmembrane domain, but it does not comprise an α-chain constant domain, and the TCR The α chain variable domain forms a heterodimer with the β chain.

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

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

The second aspect of the present invention provides a multivalent TCR complex comprising 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 a nucleic acid sequence encoding the TCR molecule described in the first aspect of the present invention or a complementary sequence thereof.

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

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

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

The fourth aspect of the present invention provides a vector, which 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 viral vector viral vector.

The fifth aspect of the present invention provides an isolated host cell, wherein the host cell contains the vector described in the fourth aspect of the present invention or the exogenous nucleic acid molecule described in the third aspect of the present invention is integrated into the genome .

The sixth aspect of the present invention provides a cell that transduces 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 or a stem cell .

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

The eighth aspect of the present invention provides the T cell receptor of the first aspect of the present invention, or the TCR complex of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, and the fourth aspect of the present invention. The use of the vector described in the aspect, or the cell described in the sixth aspect of the present invention, is used to prepare a medicament for treating tumors or autoimmune diseases.

The ninth aspect of the present invention provides a method for treating a disease, 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 , the nucleic acid molecule described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, or the cell described in the sixth aspect of the present invention, or the pharmaceutical composition described in the seventh aspect of the present invention;

Preferably, the disease is a tumor, preferably the tumor includes melanoma, and other tumors such as gastric cancer, lung cancer (eg, lung squamous cell carcinoma), esophagus cancer, bladder cancer, head and neck tumors (eg, head and neck cancers). Neck squamous cell carcinoma), prostate cancer, breast cancer, colon cancer, ovarian cancer, renal cell carcinoma, Hodgkin's lymphoma, sarcoma, medulloblastoma, leukemia, etc.

In another preferred embodiment, the tumor includes melanoma, bladder cancer, liver cancer, epidermoid carcinoma, non-small cell lung cancer and squamous cell carcinoma.

It should be understood that within the scope of the present invention, the above -mentioned technical characteristics of the present invention and the technical characteristics described in the following (implementation examples) can be combined with each other to form a new or preferred technical solution. Due to space limitations, it is not repeated here.

Description of drawings

Fig. 1a, Fig. 1b, Fig. 1c, Fig. 1d, Fig. 1e and Fig. 1f are respectively the amino acid sequence of TCRα chain variable domain, the nucleotide sequence of TCRα chain variable domain, the amino acid sequence of TCRα chain, the nucleotide sequence of TCRα chain, with The amino acid sequence of the TCRα chain of the leader sequence and the nucleotide sequence of the TCRα chain with the leader sequence.

Figure 2a, Figure 2b, Figure 2c, Figure 2d, Figure 2e and Figure 2f are the amino acid sequence of the variable domain of the TCRβ chain, the nucleotide sequence of the variable domain of the TCRβ chain, the amino acid sequence of the TCRβ chain, and the nucleotide sequence of the TCRβ chain, respectively. The amino acid sequence of the TCR beta chain of the leader sequence and the nucleotide sequence of the TCR beta chain with the leader sequence.

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

Figure 4a and Figure 4b show the amino acid sequence and nucleotide sequence of the soluble TCRα chain, respectively.

Figure 5a and Figure 5b are the amino acid sequence and nucleotide sequence of the soluble TCR beta chain, respectively.

Figure 6 is a gel image of the soluble TCR obtained after purification. The leftmost lane is the reducing gel, the middle lane is the molecular weight marker, and the rightmost lane is the non-reducing gel.

Figure 7 is the ProteOn binding of soluble TCR of the present invention to VFSTVPPAFI-HLA A2402 complex

Kinetic diagram.

Figure 8 shows the detection results of primary T cells transduced by tetramer staining TCR.

Figure 9 shows the detection results of the ELISPOT test.

Fig. 10 is a graph showing the killing effect of T cells transduced by the TCR of the present invention on specific target cells.

Detailed ways

After extensive and in-depth research, the inventors found a TCR that can specifically bind to the SAGE1 antigen short peptide VFSTVPPAFI (SEQ ID NO. 9), which can form a complex with HLA A2402 and be presented to cells together surface. The present invention also provides a nucleic acid molecule encoding the TCR and a 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 antigen presentation, and different individuals have different MHCs, which can present different short peptides in a protein antigen to their respective APC cell surfaces. The human MHC is often referred to as the HLA gene or HLA complex.

The T cell receptor (TCR) is the only receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC). In the immune system, direct physical contact between T cells and antigen-presenting cells (APCs) is triggered by the binding of antigen-specific TCRs to pMHC complexes, and then other cell membrane surface molecules of both T cells and APCs interact. It causes a series of subsequent cell signaling 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 surface of the cell membrane that exists in the form of heterodimers 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 composed of gamma and delta chains. A native αβ heterodimeric TCR has an α chain and a β chain, and the α chain and the β chain constitute the subunits of the αβ heterodimeric TCR. Broadly speaking, the alpha and beta chains each contain a variable region, a linker region, and a constant region, and the beta chain usually also contains a short variable region between the variable region and the linker region, but the variable region is often regarded as the linker region a part of. Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, chimeric in framework regions. The CDR region determines the binding of TCR to the pMHC complex, and CDR3 is recombined from the variable region and the linker region, which is called the hypervariable region. The alpha and beta chains of a TCR are generally viewed as having two "domains" each, a variable domain and a constant domain, the variable domains being composed of linked variable and linking regions. The sequences of the TCR constant domains can be found in the public database of the International Immunogenetics Information System (IMGT). 01" or "TRBC2*01". In addition, the α and β chains of TCR also contain a transmembrane region and a cytoplasmic region, and the cytoplasmic region is very short.

In the present invention, the terms "polypeptide of the present invention", "TCR of the present invention", "T cell receptor of the present invention" are used interchangeably.

Natural interchain disulfide bonds and artificial interchain disulfide bonds

There is a set of disulfide bonds between the Cα and Cβ chains in the near-membrane region of the native TCR, which are referred to as "native interchain disulfide bonds" in the present invention. In the present invention, the artificially introduced interchain covalent disulfide bond whose position is different from that of the natural interchain disulfide bond is referred to as "artificial interchain disulfide bond".

For the convenience of describing 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 sequentially from the N-terminus to the C-terminus, such as TRBC1*01 or TRBC2*01 , the 60th amino acid in the sequence from the N-terminus to the C-terminus is P (proline), then in the present invention, it can be described as Pro60 of exon 1 of TRBC1*01 or TRBC2*01, or it can be described as Pro60 of exon 1 of TRBC1*01 It is expressed as the 60th amino acid of exon 1 of TRBC1*01 or TRBC2*01, and in TRBC1*01 or TRBC2*01, the 61st amino acid in the sequence from the N-terminus to the C-terminus is Q (glutamate). amide), in the present invention, it can be described as Gln61 of TRBC1*01 or TRBC2*01 exon 1, or it can be described as the 61st amino acid of TRBC1*01 or TRBC2*01 exon 1, other And so on. In the present invention, the position numbers of the amino acid sequences of the variable regions TRAV and TRBV are numbered according to the position numbers listed in IMGT. For example, for a certain amino acid in TRAV, the position number listed in IMGT is 46, then it is described as the 46th amino acid of TRAV in the present invention, and so on. In the present invention, if the sequence position numbering of other amino acids has special instructions, the special instructions are followed.

Detailed description of the invention

TCR molecule

During antigen processing, the antigen is degraded within 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. Accordingly, a first aspect of the present invention provides a TCR molecule capable of binding the VFSTVPPAFI-HLA A2402 complex. Preferably, the TCR molecule is isolated or purified. The alpha and beta chains of this TCR each have three complementarity determining regions (CDRs).

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

αCDR1-DSAIYN (SEQ ID NO. 10)

αCDR2-IQSSQRE (SEQ ID NO. 11)

αCDR3-CAVLYTGANSKLTF (SEQ ID NO. 12); and/or

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

βCDR1-SGHDT (SEQ ID NO. 13)

βCDR2-YYEEEE (SEQ ID NO. 14)

CDR3-CASSLVGKQPQHF (SEQ ID NO. 15).

Chimeric TCRs can be prepared by inserting the above-described amino acid sequences of the CDR regions of the present invention into any suitable framework structure. As long as the framework structure is compatible with the CDR regions 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. Accordingly, the TCR molecule of the present invention refers to a TCR molecule comprising the above-mentioned alpha and/or beta chain CDR region sequences 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 TCR chain variable domain of the present invention is an amino acid sequence with SEQ ID NO. NO:5 has an amino acid sequence of at least 90%, preferably 95%, more preferably 98% sequence identity.

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 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) and CDR2 (SEQ ID NO: 10) of the above-mentioned α chain. ID NO: 11) and CDR3 (SEQ ID NO. 12). Preferably, the TCR molecule comprises the alpha chain variable domain amino acid sequence 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 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. 13) of the above-mentioned β chain NO: 14) and CDR3 (SEQ ID NO. 15). Preferably, the TCR molecule comprises the beta chain variable domain amino acid sequence of SEQ ID NO.5. More preferably, the amino acid sequence of the beta 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. A description of single-chain TCR molecules can be found in Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-12658. Those skilled in the art can readily construct single-chain TCR molecules comprising the CDRs regions of the present invention as described in the literature. Specifically, the single-chain TCR molecule comprises Vα, Vβ and Cβ, preferably linked in order from the N-terminus to the C-terminus.

The α-chain variable domain amino acid sequence 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 alpha chain variable domain amino acid sequence 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 beta chain variable domain amino acid sequence 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 beta chain. Preferably, the single-chain TCR molecule comprises the beta chain variable domain amino acid sequence of SEQ ID NO.5. More preferably, the amino acid sequence of the beta chain variable domain of the single-chain TCR molecule is SEQ ID NO.5.

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 alpha chain of the TCR molecule of the present invention can be "TRAC*01", and the constant domain sequence of the beta 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 represented 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 immunoglobulins (antibodies) as antigen recognition molecules, TCRs can also be developed for diagnostic and therapeutic applications, where soluble TCR molecules need to be obtained. Soluble TCR molecules do not include their transmembrane domains. Soluble TCR has a wide range of uses, not only to study the interaction of TCR with pMHC, but also as a diagnostic tool to detect infection or as a marker for autoimmune diseases. Similarly, soluble TCRs can be used to deliver therapeutic agents (eg, cytotoxic or immunostimulatory compounds) to cells presenting specific antigens, and in addition, soluble TCRs can bind to other molecules (eg, anti-CD3 antibodies) to redirect T cells so that they target cells presenting specific antigens. The present invention also obtains a soluble TCR specific for the SAGE1 antigenic short peptide.

To obtain soluble TCRs, in one aspect, the TCRs of the invention may be TCRs in which artificial disulfide bonds are introduced between residues of their alpha and beta chain constant domains. Cysteine residues form artificial interchain disulfide bonds between the constant domains of the alpha and beta chains of the TCR. Cysteine residues can be substituted for other amino acid residues at appropriate sites in the native TCR to form artificial interchain disulfide bonds. For example, substitution of Thr48 of exon 1 of TRAC*01 and substitution of cysteine residues of Ser57 of exon 1 of TRBC1*01 or TRBC2*01 to form a disulfide bond. Other sites where cysteine residues are introduced to form disulfide bonds can also be: Thr45 in exon 1 of TRAC*01 and Ser77 in exon 1 of TRBC1*01 or TRBC2*01; exon 1 of TRAC*01 1 Tyr10 and TRBC1*01 or Ser17 of TRBC2*01 exon 1; Thr45 of TRAC*01 exon 1 and Asp59 of TRBC1*01 or TRBC2*01 exon 1; TRAC*01 exon 1 Ser15 and Glu15 in exon 1 of TRBC1*01 or TRBC2*01; Arg53 in exon 1 of TRAC*01 and Ser54 in exon 1 of TRBC1*01 or TRBC2*01; Pro89 and exon 1 of TRAC*01 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 are substituted for any set of sites in the constant domains of the alpha and beta chains described above. One or more C-termini of the TCR constant domains of the invention may 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 so that they do not include The purpose of deleting the natural disulfide bond can be achieved by the cysteine residue, or by mutating the cysteine residue forming the natural disulfide bond into another amino acid to achieve the above purpose.

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

In order to obtain a soluble TCR, on the other hand, the TCR of the present invention also includes a TCR with mutation in its hydrophobic core region, and the mutation of these hydrophobic core regions is preferably a mutation that can improve the stability of the soluble TCR of the present invention, such as in Publication No. Described in the patent document of WO2014/206304. Such a TCR may be mutated 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, 7th amino acid position from the bottom of the alpha chain J gene (TRAJ) short peptide, and/or the 2nd, 4th, and 6th position from the bottom of the amino acid position of the beta chain J gene (TRBJ) short peptide, wherein the position number of the amino acid sequence Numbered by position as listed in the International Information System on Immunogenetics (IMGT). Those skilled in the art are aware of the above-mentioned International Immunogenetics Information System, and can obtain the position numbers of amino acid residues of different TCRs in IMGT according to the database.

In the present invention, the mutated TCR in the hydrophobic core region can be a stable soluble single-chain TCR composed of a flexible peptide chain linking 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 chain.

In addition, in terms of stability, patent document PCT/CN2016/077680 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 high-affinity TCR of the present invention may also contain artificial interchain disulfide bonds 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* 01 amino acid 60 of exon 1; TRAV amino acid 47 and TRBC1*01 or TRBC2*01 exon 1 amino acid 61; TRAV 46 amino acid and TRBC1*01 or TRBC2*01 exon 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 the TCR alpha chain excluding its transmembrane domain, and (ii) all or part of the TCR chain excluding its transmembrane domain, wherein (i) and (ii) ) both contain the variable domain and at least a part of the constant domain of the TCR chain, and the α chain and the β chain form a heterodimer. More preferably, such a TCR may contain an alpha chain variable domain and a beta chain variable domain and all or part of the beta chain constant domain except the transmembrane domain, but it does not contain the alpha chain constant domain, the alpha chain of the TCR. The chain variable domains form heterodimers with beta chains.

The TCRs of the present invention may also be provided in the form of multivalent complexes. The multivalent TCR complexes of the present invention comprise two, three, four or more multimers formed by combining the TCRs of the present invention, for example, the tetramerization domain of p53 can be used to generate tetramers, or multiple A complex formed by combining a TCR 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, as well as to generate intermediates for other multivalent TCR complexes with such applications.

The TCR of the present invention can be used alone, or can be combined with the conjugate in a covalent or other manner, preferably in a covalent manner. The conjugate includes a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the VFSTVPPAFI-HLA A2402 complex), a therapeutic agent, a PK (protein kinase) modification moiety, or any of the above combination or conjugation.

Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing detectable products enzyme.

Therapeutic agents that can be conjugated or conjugated to the TCRs of the present invention include, but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539); 2. Biotoxicity (Chaudhary et al., 1989) , Nature 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy 51, 565); 3. Cytokines such as IL-2, etc. (Gillies et al., 1992, Proceedings of the National Academy of Sciences of the United States of America) (PNAS) 89, 1428; Card et al, 2004, Cancer Immunology and Immunotherapy 53, 345; Halin et al, 2003, Cancer Research 63, 3202); 4. Antibody Fc fragment (Mosquera et al, 2005, The Journal Of Immunology 174, 4381); 5. Antibody scFv fragments (Zhu et al, 1995, International Journal of Cancer 62, 319); 6. Gold nanoparticles/nanoparticles Rod (Lapotko et al, 2005, Cancer letters 239, 36; Huang et al, 2006, Journal of the American Chemical Society 128, 2115); 7. Viral particles (Peng et al, 2004, Genes Gene therapy 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research 65, 11631); 9. Nanomagnetic particles; 10. Prodrug-activating enzymes (eg, DT-myocardial flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. chemotherapeutic agents (eg, cisplatin) or nanoparticles in any form, etc.

Additionally, the TCRs of the present invention may also be hybrid TCRs comprising sequences derived from more than one species. For example, studies have shown that murine TCRs are more efficiently expressed in human T cells than human TCRs. Thus, the TCRs of the present invention may comprise human variable domains and murine constant domains. The downside of this approach is the potential to elicit an immune response. Therefore, there should be a regulatory regime for immunosuppression when it is used in adoptive T cell therapy to allow engraftment of murine expressing T cells.

It should be understood that the names of amino acids in this paper are represented by the international single English letter or three English letters, and the correspondence between the single English letter and the three English letters of the 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 invention provides a nucleic acid molecule encoding a TCR molecule of the first aspect of the invention, or a portion thereof, which portion may be one or more CDRs, variable domains of alpha and/or beta chains, and alpha chains and/or or beta chains.

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

(SEQ ID NO.16)αCDR1-

(SEQ ID NO. 16)

(SEQ ID NO.17)αCDR2-

(SEQ ID NO. 17)

(SEQ ID NO.18)αCDR3-

(SEQ ID NO. 18)

The nucleotide sequence encoding the beta chain CDR region of the TCR molecule according to the first aspect of the present invention is as follows:

(SEQ ID NO.19)βCDR1-

(SEQ ID NO. 19)

(SEQ ID NO.20)βCDR2-

(SEQ ID NO. 20)

(SEQ ID NO.21)βCDR3-

(SEQ ID NO. 21)

Accordingly, the nucleotide sequences of the nucleic acid molecules of the present invention encoding the TCR alpha chains of the present invention include SEQ ID NO. 16, SEQ ID NO. 17 and SEQ ID NO. 18, and/or the core of the nucleic acid molecules of the present invention encoding the TCR beta chains of the present invention The nucleotide sequences include SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21.

The nucleotide sequence of the 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 is capable of encoding 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 variable domain of the TCR beta chain of the present invention includes SEQ ID NO.6. 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. It will be appreciated that due to the degeneracy of the genetic code, different nucleotide sequences can encode the same polypeptide. Accordingly, the nucleic acid sequences encoding the TCRs of the present invention may be identical or degenerate variants of the nucleic acid sequences shown in the figures of the present invention. To illustrate with one of the examples in the present invention, "degenerate variant" refers to a nucleic acid sequence that encodes a protein sequence of SEQ ID NO. 1, but differs from the sequence of SEQ ID NO. 2.

The nucleotide sequence may be codon-optimized. Different cells differ in the use of specific codons. Depending on the type of cell, the codons in the sequence can be changed to increase the amount of expression. Codon usage tables for mammalian cells, as well as various 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 a fragment thereof can generally be obtained by, but not limited to, PCR amplification method, recombinant method or artificial synthesis method. At present, the DNA sequences encoding the TCRs of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by 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 the coding or non-coding strand.

carrier

The present invention also relates to vectors comprising the nucleic acid molecules of the present invention, including expression vectors, ie constructs capable of in vivo or in vitro expression. 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 virus (AAV) vectors, herpesvirus vectors, retroviral vectors, lentiviral vectors, baculovirus vectors.

Preferably, the vector can transfer the nucleotides of the invention into cells, such as T cells, such that the cells express TCRs specific for the SAGE1 antigen. Ideally, the vector should be able to express consistently high levels in T cells.

cell

The present invention also relates to host cells genetically engineered 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 into the chromosome. The host cell is selected from: prokaryotic cells and eukaryotic cells, such as E. coli, yeast cells, CHO cells, and the like.

In addition, the present invention also includes isolated cells, in particular T cells, expressing the TCRs of the present invention. The T cells can be derived from T cells isolated from the subject, or can be part of a mixed population of cells isolated from the subject, such as a peripheral blood lymphocyte (PBL) population. For example, the cells can be isolated from peripheral blood mononuclear cells (PBMCs) and can be CD4 ⁺ helper T cells or CD8 ⁺ cytotoxic T cells. The cells can be in a mixed population of CD4 ⁺ helper T cells/CD8 ⁺ cytotoxic T cells. Typically, the cells can be activated with antibodies (eg, anti-CD3 or anti-CD28 antibodies) to render them more receptive to transfection, eg, 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 derived from stem cells, such as hematopoietic stem cells (HSCs). Gene transfer to HSCs does not result in TCR expression on the cell surface because the CD3 molecule is not expressed on the surface of stem cells. 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 thymocytes.

There are a number of methods suitable for transfection of T cells with DNA or RNA encoding the TCRs 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. Those skilled in the art are aware of many suitable methods for adopting adoptive therapy (eg, Rosenberg et al., (2008) Nat RevCancer 8(4):299-308).

SAGE1 antigen-related diseases

The present invention also relates to a method of treating and/or preventing a disease associated with SAGE1 in a subject comprising the step of adoptively transferring SAGE1-specific T cells to the subject. The SAGE1-specific T cells recognize the VFSTVPPAFI-HLAA2402 complex.

The SAGE1-specific T cells of the present invention can be used to treat any SAGE1-related disease that presents the SAGE1 antigen short peptide VFSTVPPAFI-HLA A2402 complex. Including but not limited to tumors, such as melanoma, and other solid tumors such as gastric, lung, esophageal, bladder, head and neck squamous cell carcinoma, prostate, breast, colon, ovarian, and the like.

treatment method

Treatment can be performed by isolating T cells from patients or volunteers suffering from a disease associated with the SAGE1 antigen, introducing the TCR of the present invention into the above T cells, and then infusing these genetically engineered cells back into the patient. Therefore, the present invention provides a method for treating SAGE1-related diseases, comprising infusing the isolated T cells expressing the TCR of the present invention, preferably, the T cells are derived from the patient itself, into the patient. Typically, this involves (1) isolating T cells from a patient, (2) transducing T cells in vitro with a nucleic acid molecule of the invention or a nucleic acid molecule capable of encoding a TCR molecule of the invention, and (3) infusing genetically engineered T cells into a patient in vivo. The number of cells 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 bind to the SAGE1 antigen short peptide complex VFSTVPPAFI-HLA A2402, and the cells transduced with the TCR of the present invention can be specifically activated and have a strong killing effect on target cells.

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 of unreceipted specific conditions in the following examples, usually according to conventional conditions, such as (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 commercial sources unless otherwise specified.

Example 1 Cloning of antigen short peptide-specific T cells

Peripheral blood lymphocytes (PBL) from healthy volunteers with HLA-A2402 genotype were stimulated with the synthetic short peptide SAGE1PX149 597-606 VFSTVPPAFI (Beijing Saibaisheng Gene Technology Co., Ltd.). The SAGE1 PX149597-606 VFSTVPPAFI short peptide was renatured with biotin-labeled HLA-A*2402 to prepare pHLA monomers. These monomers were combined with PE-labeled streptavidin (BD) to form PE-labeled tetramers, and the tetramers and anti-CD8-APC double positive cells were sorted. The sorted cells were expanded and subjected to secondary sorting as described above, followed by monoclonal culture by limiting dilution. Monoclonal cells were stained with tetramers, and the screened double-positive clones were shown in Figure 3.

Example 2 Construction of TCR gene and vector for obtaining antigen short peptide-specific T cell clones of the present invention

The total RNA of the SAGE1 PX149597-606VFSTVPPAFI-specific, HLA-A2402-restricted T cell clone 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 in the C-terminal conserved region of the human TCR gene. The sequence was cloned into T vector (TAKARA) for sequencing. After sequencing, the sequence structures of the α chain and β chain of the TCR expressed by the double-positive clone are shown in Figure 1 and Figure 2, respectively. Figure 1a, Figure 1b, Figure 1c, and Figure 1d are the amino acid sequence of the variable domain of TCRα chain, TCRα Chain variable domain nucleotide sequence, TCRα chain amino acid sequence and TCRα chain nucleotide sequence; Figure 2a, Figure 2b, Figure 2c and Figure 2d are the TCRβ chain variable domain amino acid sequence, TCRβ chain variable domain nucleotide sequence, respectively Sequence, TCRβ chain amino acid sequence and TCRβ chain nucleotide sequence.

The alpha chain was identified as comprising CDRs with the following amino acid sequence:

αCDR1-DSAIYN (SEQ ID NO. 10)

αCDR2-IQSSQRE (SEQ ID NO. 11)

αCDR3-CAVLYTGANSKLTF (SEQ ID NO. 12)

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

βCDR1-SGHDT (SEQ ID NO. 13)

βCDR2-YYEEEE (SEQ ID NO. 14)

βCDR3-CASSLVGKQPQHF (SEQ ID NO. 15).

The variable domains of TCR alpha chain and beta chain, respectively, and the conserved domains of mouse TCR alpha chain and beta chain, respectively, were spliced into full-length genes by overlapping PCR and ligated into the lentiviral expression vector pLenti (addgene). Specifically, the TCRα-2A-TCRβ fragment was obtained by linking the full-length genes of the TCRα chain and the TCRβ chain by overlapPCR. The pLenti-SAGE1TRA-2A-TRB-IRES-NGFR plasmid was obtained by ligating the lentiviral expression vector and TCRα-2A-TCRβ restriction enzyme. As a control, a lentiviral vector pLenti-eGFP expressing eGFP was also constructed. Afterwards, 293T/17 was used to package the pseudovirus.

Example 3 Expression, refolding and purification of the antigen short peptide-specific soluble TCR of the present invention

In order to obtain a soluble TCR molecule, the α and β chains of the TCR molecule of the present invention can respectively contain only their variable domains and part of their constant domains, and a cysteine residue is introduced into the constant domains of the α and β chains respectively. In order to form an artificial interchain disulfide bond, the positions of introducing cysteine residues are Thr48 of exon 1 of TRAC*01 and Ser57 of exon 1 of TRBC2*01; the amino acid sequence of its α chain is the same as that of nucleotides. The sequences are shown in Fig. 4a and Fig. 4b, respectively, the amino acid sequence and nucleotide sequence of the β chain are shown in Fig. 5a and Fig. 5b, respectively, and the introduced cysteine residues are shown in bold and underlined letters. The target gene sequences of the above TCR α and β chains were synthesized and inserted into the expression vector pET28a+ (Novagene ), the upstream and downstream cloning sites are NcoI and NotI, respectively. The insert was confirmed by sequencing.

The expression vectors of TCR α and β chains were transformed into the expression bacteria BL21 (DE3) by chemical transformation, and the bacteria were grown in LB medium and induced with a final concentration of 0.5 mM IPTG at OD600 = 0.6. After the expression of TCR α and β chains The formed inclusion bodies were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution. The inclusion bodies were finally dissolved in 6M guanidine hydrochloride, 10mM dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA) , 20mM Tris (pH8.1).

The dissolved TCRα and β chains were rapidly 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, the final concentration was 60mg /mL. After mixing, the solution was dialyzed against 10 volumes of deionized water (4°C). After 12 hours, the deionized water was changed to buffer (20 mM 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 0.45M filter membrane and purified by an anion exchange column (HiTrap Q HP, 5 ml, GE Healthcare). The eluted peaks of TCR containing successfully renatured α and β dimers were confirmed by SDS-PAGE gel. 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 more than 90% determined by SDS-PAGE, and the concentration was determined by BCA method. Figure 6 shows the SDS-PAGE gel chart of the soluble TCR obtained by the present invention.

Example 4 Binding Characterization

BIAcore analysis

This example demonstrates that soluble TCR molecules of the invention are capable of specifically binding to the VFSTVPPAFI-HLAA2402 complex.

The binding activity of the TCR molecule obtained in Example 3 to the VFSTVPPAFI-HLA A2402 complex was detected using the BIAcore T200 real-time analysis system. Anti-streptavidin antibody (GenScript) was added to coupling buffer (10 mM sodium acetate buffer, pH 4.77), and the antibody was then immobilized on the chip surface by flowing through a CM5 chip preactivated with EDC and NHS , and finally blocked the unreacted activated surface with ethanolamine hydrochloric acid solution to complete the coupling process with a coupling level of about 15,000RU.

A low concentration of streptavidin was flowed over the surface of the chip coated with the antibody, then the VFSTVPPAFI-HLAA2402 complex was flowed through the detection channel, the other channel was used as a reference channel, and 0.05mM biotin was added at 10 μL/min. The flow rate was flowed through the chip for 2 min to block the remaining binding sites of streptavidin.

The preparation process of above-mentioned VFSTVPPAFI-HLA A2402 complex is as follows:

a. Purification

Collect 100ml of E.coli bacteria that induces the expression of heavy or light chains, centrifuge at 8000g at 4°C for 10min, wash the cells once with 10ml PBS, and then use 5ml of BugBuster Master Mix Extraction Reagents (Merck) to vigorously shake the cells to resuspend the cells. Incubate with rotation at room temperature for 20 min, then centrifuge at 6000g for 15 min at 4°C, discard the supernatant, and collect the inclusion bodies.

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

b. Refolding

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

c. Purification after renaturation

The renaturation buffer was exchanged 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 m cellulose acetate filter and loaded onto a HiTrap Q HP (GE) anion exchange column (5 ml bed volume). The protein was eluted with Akta purifier (GE), 0-400 mM NaCl linear gradient prepared with 20 mM Tris pH 8.0, pMHC was eluted at about 250 mM NaCl, the peak fractions were collected, and the purity was checked by SDS-PAGE.

d. Biotinylation

Purified pMHC molecules were concentrated with Millipore ultrafiltration tubes while buffer exchanged to 20 mM Tris pH 8.0, followed by addition of biotinylation reagents 0.05 M Bicine pH 8.3, 10 mM ATP, 10 mM MgOAc, 50 μM D-Biotin, 100 μg/ml BirA enzyme (GST-BirA), the mixture was incubated overnight at room temperature, and the complete biotinylation was checked by SDS-PAGE.

e. Purification of biotinylated complexes

The biotinylated pMHC molecules were concentrated to 1 ml with a Millipore ultrafiltration tube, and the biotinylated pMHC was purified by gel filtration chromatography. HiPrepTM16 was pre-equilibrated with filtered PBS using an Akta purifier (GE). /60 S200 HR column (GE), loaded with 1 ml of concentrated biotinylated pMHC molecules, and eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules eluted as a single peak at about 55 ml. The fractions containing protein were combined, concentrated with Millipore ultrafiltration tube, the protein concentration was determined by BCA method (Thermo), and the protease inhibitor cocktail (Roche) was added to store the biotinylated pMHC molecules in aliquots at -80°C.

Using the BIAcore Evaluation software to calculate the kinetic parameters, the kinetic diagram of the binding of the soluble TCR molecule of the present invention to the complex is obtained as shown in Figure 7 .

Example 5 T cell receptor lentiviral packaging and primary T cell transfection with SAGE1 TCR

(a) Preparation of lentivirus by Express-In-mediated transient transfection of 293T/17 cells

Lentiviruses containing the genes encoding the desired TCRs were packaged using a third-generation lentiviral packaging system. Four plasmids (containing one of the pLenti-SAGE1TRA-2A-TRB-IRES-NGFR described in Example 2) were used using Express-In-mediated transient transfection (OpenBiosystems). 293T/17 cells were transfected with lentiviral vectors, and 3 plasmids containing other components necessary to construct infectious but non-replicative lentiviral particles).

For transfection, cells were seeded on day 0 by seeding 1.7 x 107 293T/ ¹⁷ cells in a 15 cm dish so that the cells were evenly distributed on the dish and slightly more than 50% confluent. On day 1, transfect the plasmids, package pLenti-SAGE1TRA-2A-TRB-IRES-NGFR and pLenti-eGFP pseudoviruses, mix the above expression plasmids with the packaging plasmids pMDLg/pRRE, pRSV-REV and pMD.2G, each 15 cm The amount used for the diameter plate is as follows: 22.5 micrograms: 15 micrograms: 15 micrograms: 7.5 micrograms. The ratio of transfection reagent PEI-MAX to plasmid was 2:1, and the amount used per plate was 114.75 micrograms. The specific operation is as follows: add the expression plasmid and the packaging plasmid into 1800 microliters of OPTI-MEM ((Gibco, catalog number 31985-070) medium, mix well, and let stand for 5 minutes at room temperature to become a DNA mixture; The corresponding amount of PEI was mixed with 1800 microliters of OPTI-MEM medium, and stood for 5 minutes at room temperature to become the PEI mixture. The DNA mixture and the PEI mixture were mixed together and allowed to stand at room temperature for 30 minutes, and then 3150 microliters of OPTI was added. -MEM medium, mix well and add to 293T/17 cells that have been converted into 11.25 ml OPTI-MEM, shake the dish gently to mix the medium well, and culture at 37°C/5% CO _2. Transfection 5 -7 hours, remove the transfection medium and replace it with DMEM ((Gibco, cat. no. C11995500bt)) complete medium containing 10% fetal bovine serum, and cultivate at 37°C/5% _CO2 . The culture supernatants containing the packaged lentiviruses were collected on days 3 and 4. To harvest the packaged lentiviruses, the collected culture supernatants were centrifuged at 3000 g for 15 minutes to remove cell debris, and then passed through a 0.22 micron filter (Merckmidt Libo (Merck Millipore, cat. no. SLGP033RB) was filtered, and finally concentrated with a 50KD cut-off concentrator tube (Merck Millipore, cat. no. UFC905096) to remove most of the supernatant, and finally concentrated to 1 ml, After the aliquots were packaged and frozen at -80 °C .

(b) Transduction of primary T cells with lentiviruses containing the SAGE1-specific T cell receptor gene

CD8 ⁺ T cells were isolated from the blood of healthy volunteers and transduced with packaged lentivirus. These cells were counted in 48-well plates in 1640 (Gibco, Inc.) containing 50 IU/ml IL-2 and 10 ng/ml IL-7 in 10% FBS (Gibco, Cat. No. C10010500BT). (Gibco), cat. no. C11875500bt) medium at ¹ x 106 cells/ml (0.5 ml/well) with pre-washed anti-CD3/CD28 antibody-coated beads (T cell amplicon, lifetechnologies, catalogue No. 11452D) co-incubation overnight stimulation, cells:beads=3:1.

After overnight stimulation, according to the virus titer detected by the p24 ELISA kit, the concentrated SAGE1-specific T cell receptor gene lentivirus was added at the ratio of MOI=10, and the cells were centrifuged at 32°C and 900g for 1 hour. After infection, the lentivirus infection solution was removed, and the cells were resuspended in 1640 medium containing 10% FBS with 50IU/ml IL-2 and 10ng/ml IL-7 added, and cultured at 37°C/5% CO ₂ for 3 days. Cells were counted 3 days after transduction and diluted to 0.5 x ¹⁰⁶ cells/ml. Count cells every two days and replace or add fresh medium containing 50 IU/ml IL-2 and 10 ng/ml IL-7 to maintain cells at 0.5 x 10 ⁶ -1 x 10 ⁶ cells/ml. Cells were analyzed by flow cytometry from day 3 and used for functional assays (eg, ELISPOT of IFN-γ release and non-radioactive cytotoxicity assays) from day 5. From day 10 onwards or as cells slow down and shrink in size, store aliquots frozen, at least 4 x ¹⁰ cells/tube (1 x ¹⁰ cells/ml, 90% FBS/10% DMSO) .

(c) Tetramer stained TCR-transduced primary T cells

SAGE1 PX149 597-606 VFSTVPPAFI short peptide was renatured with biotin-labeled HLA-A*2402 to prepare pHLA monomer. These monomers were combined with PE-labeled streptavidin (BD) to form a PE-labeled tetramer, termed PX149-tetramer-PE. This tetramer can label T cells expressing the SAGE1-specific T cell receptor gene as positive cells. The transduced T cell samples in (b) were mixed with PX149-tetramer-PE and incubated on ice for 30 minutes, then anti-mouse beta chain-APC antibody was added and the incubation continued on ice for 15 minutes. Samples were washed twice with PBS containing 2% FBS, and then BDCalibur or BD Arial were used to detect or sort T cells that express SAGE1-specific T cell receptor gene PX149-tetramer-PE and anti-mouse β-chain-APC double-positive T cells , data analysis using CellQuest software (BD) or FlowJo software (TreeStar Inc, Ashland, OR) analysis.

After detection and analysis, the results are shown in Figure 8. After staining with PX149-tetramer-PE and anti-mouse β-chain-APC antibodies, the blank control group T cells without TCR lentivirus infection did not have PX149-tetramer-PE and anti-small cells. Murine β-chain-APC double-positive cells, while TCR lentivirus-infected T cells appeared to express TCR-expressing PX149-tetramer-PE and anti-mouse β-chain-APC double-positive cells, when treated with other tetramers other than PX149-tetramer-PE - Very few non-specific double positive cells on PE staining.

Example 6 Activation experiment of effector cells transduced with TCR of the present invention

ELISPOT solution

The following experiments were performed to demonstrate activation of TCR-transduced T cells specifically responsive to target cells. IFN-γ production detected using the ELISPOT assay was used as a readout for T cell activation.

reagent

Assay medium: 10% FBS (Gibco, cat. no. 16000-044), RPMI1640 (Gibco, cat. no. C11875500bt)

Washing buffer: 0.01M PBS/0.05% Tween 20

PBS (Gibco, catalog number C10010500BT )

PVDF ELISPOT 96-well plate (Merck Millipore, catalog number MSIPS4510 )

Human IFN-γ ELISPOT PVDF-enzyme kit (BD) contains all other reagents required (capture and detection antibodies, streptavidin-alkaline phosphatase and BCIP/NBT solution)

method

target cell preparation

The target cells in this example were immortalized lymphoblastoid cell lines (LCLs) transformed with Epstein-Barr virus (EBV). B95-8 cells were induced by phorbol myristate acetate (TPA) to produce medium supernatant containing EBV, centrifuged at 4°C/600g for 10 minutes to remove impurities, then passed through a 0.22 micron filter, and aliquoted at -70°C save. From peripheral blood lymphocytes (PBL) of healthy volunteers with genotype HLA-A11/A02/A24 (both homozygous and heterozygous), take 10 ml of PBL suspension at a concentration of 2 x 10 ⁷ /ml in 25 square centimeter culture flask, incubate for 1 hour in a 37°C/CO ₂ incubator after adding cyclosporine, quickly thaw a portion of EBV, add it to the above cells at a 1/10 dilution, shake gently and place the flask Incubate upright in a 37°C/CO ₂ incubator. After 12 days of culture, 10 ml of medium was added to continue the culture, and about 30 days later, the culture was further expanded and flow cytometry was performed. Among them, CD19 ⁺ CD23 ^hi CD58 ⁺ were immortalized lymphoblastoid cell lines (LCLs). This ELISPOT assay uses HLA-A24 as a specific target cell.

Effector cell preparation

The effector cells (T cells) in this experiment were the CD8 ⁺ T cells expressing SAGE1-specific TCR by flow cytometry analysis in Example 3, and the CD8 ⁺ T cells of the same volunteer were used as negative control effector cells. T cells were stimulated with anti-CD3/CD28-coated beads (T Cell Amplifier, LifeTechnologies), transduced with lentiviruses carrying SAGE1-specific TCR genes (according to Example 3), in cells containing 50 IU/ml IL-2 and 10 ng /ml IL-7 in 1640 medium with 10% FBS was expanded until 9-12 days after transduction, then the cells were placed in assay medium and washed by centrifugation at 300 g for 10 minutes at room temperature. Cells were then resuspended in assay medium at 2x the desired final concentration. Negative control effector cells were treated similarly.

ELISPOT

Following the manufacturer's instructions, prepare the well plates as follows: Dilute the anti-human IFN-γ capture antibody 1:200 in 10 mL of sterile PBS per plate, then aliquot 100 microliters of the diluted capture antibody into each well . Plates were incubated overnight at 4°C. After incubation, the plate was washed to remove excess capture antibody. 100 microliters/well of RPMI 1640 medium containing 10% FBS was added and the wells were incubated for 2 hours at room temperature to block the wells. The medium was then washed from the well plate and any residual wash buffer was removed by flicking and tapping the ELISPOT well plate on the paper.

SAGE1 CD8 ⁺ T cells (SAGE1 TCR-transduced T cells, effector cells VF3 CD8+ T cells), CD8+ T cells (negative control effector cells), and LCL-A24/A02 (target cells) were prepared as described in Example 3, and Corresponding short peptides were added to the corresponding experimental groups, wherein PX149 was SAGE1 PX149 597-606 VFSTVPPAFI short peptide, and PA11, PA02, PA24-1, PA24-2 and PA24-3 were non-SAGE1 TCR specific binding short peptides.

The components of the assay were then added to the ELISPOT plate in the following order:

130 microliters of target cells 77000 cells/ml (resulting in a total of about 10000 target cells/well).

50 microliters of effector cells (1000 SAGE1 TCR double positive T cells).

20 microliters of ^10-5 mol/liter SAGE1 PX149 597-606 VFSTVPPAFI/non-specific short peptide solution (final concentration ^10-6 mol/liter).

All wells were prepared for addition in triplicate.

Plates were then incubated overnight (37°C/5% CO ₂ ). The next day, the medium was discarded and the plates were washed 2 times with double distilled water and 3 times with wash buffer and tapped on a paper towel to remove residues washing buffer. The primary detection antibody was then diluted in PBS containing 10% FBS and added to each well at 100 microliters/well. Plates were incubated for 2 hours at room temperature and washed 3 times with wash buffer. Excess wash buffer was removed by tapping the plate on a paper towel.

Streptavidin-alkaline phosphatase was diluted 1:100 in PBS containing 10% FBS, 100 microliters of diluted streptavidin-alkaline phosphatase was added to each well and the plate was incubated at room temperature 1 hour. Then wash 3 times with wash buffer 2 times with PBS and tap the plate on a paper towel to remove excess wash buffer and PBS. After washing, 100 μl/well of BCIP/NBT solution provided by the kit was added for development. Cover the well plate with foil during development to protect from light and let stand for 5-15 minutes. Routinely check the spots of the developing plate during this period to determine the optimal time to terminate the reaction. Remove the BCIP/NBT solution and rinse the well plate with double distilled water to stop the development reaction, spin dry, then remove the bottom of the well plate, dry the well plate at room temperature until each well is completely dry, and then use an immunospot plate counter (CTL, Cellular Technology Limited) counted the spots formed by the bottom membrane in the well plate.

result

The TCR-transduced T cells of the invention were examined for IFN-γ release in response to SAGE1 PX149597-606 VFSTVPPAFI short peptide-loaded target cells and non-specific short peptide target cells by the ELISPOT assay (described above). The number of ELSPOT spots observed in each well was plotted using GraphPad Prism6.

The experimental results are shown in Figure 9. The CD8 ⁺ T cells transduced with the TCR of the present invention have a strong activation effect on LCLs loaded with related short peptides, and release more IFN-γ, while the LCLs loaded with non-specific short peptides basically have a strong activation effect. There was no response; meanwhile, CD8 ⁺ T cells that were not transduced with the TCR of the present invention had little response to LCLs loaded with relevant short peptides, and only a small amount of IFN-γ was released.

Example 7 Non-radioactive cytotoxicity test protocol

This assay is a colorimetric alternative to the 51Cr release cytotoxicity assay and quantifies lactate dehydrogenase (LDH) released after cell lysis. LDH released in the medium was detected using a 30 min coupled enzymatic reaction in which LDH converts a tetrazolium salt (INT) to red formazan. The amount of red product produced is proportional to the number of cells lysed. Visible absorbance data at 490 nm can be collected using a standard 96-well plate reader.

Material

非放射性细胞毒性检测(普罗迈格公司，G1780)含有底物混合物、试验缓冲液、裂解溶液和终止缓冲液。CytoTox

The nonradioactive cytotoxicity assay (Promega, G1780) contains substrate mix, assay buffer, lysis solution, and stop buffer.

Assay medium: 10% FBS (heat-inactivated, Gibco, cat. no. 16000-044), 90% RPMI 1640 with phenol red (Gibco, cat. no. C11875500bt), 1% penicillin /Streptomycin (Gibco, Cat. No. 15070-063).

Microwell round bottom 96-well tissue culture plate (Nunc, catalog number 163320 )

96-well immunoplate Maxisorb (Nunc, catalog number 442404 )

method

target cell preparation

Three tumor cell lines, K562-A24, NCI-H1299-A24 and IM 9, were used as target cells in this experiment. Prepare target cells in assay medium: adjust the target cell concentration to 334 cells/ml and take 45 microliters per well to obtain 1.5 x ¹⁰⁴ cells/well.

Effector cell preparation

The effector cells (T cells) in this experiment were CD8 ⁺ T cells expressing SAGE1-specific TCR by flow cytometry analysis in Example 3. The ratios of effector cells to target cells were used at 10:1., 5:1, 2.5:1, 1.25:1 and 0.625:1. A control group (10:1) of syngeneic CD8 ⁺ T cells plus target cells was set.

CD8 ⁺ T cell-specific killing of tumor cells by SAGE1-specific TCR transduction

Test preparation

The components of the assay were added to a microwell round bottom 96-well tissue culture plate in the following order:

-45ul target cells (prepared as described above) were added to each well

- 45ul effector cells (prepared as described above) were added to each well

A control group was prepared as follows:

- Spontaneous release of effector cells: only 45ul of effector cells.

- Spontaneous release of target cells: only 45ul of target cells.

- Maximum release of target cells: only 45ul of target cells.

- Medium control: 90ul of medium only.

All wells were prepared in triplicate with a final volume of 90ul (not enough was made up with medium).

Incubate for 24 hours at 37°C. Before collecting the supernatant from all wells, the target cell maximum release control wells were placed at -70°C for approximately 30 minutes and then thawed at 37°C for 15 minutes to completely lyse the target cells.

Plates were centrifuged at 250g for 4 minutes. Transfer 50 ul of the supernatant from each well of the assay plate to the corresponding well of a 96-well immunoplate Maxisorb plate. The substrate mix was reconstituted with assay buffer (12ml), then 50ul was added to each well of the plate. The plates were covered and incubated at room temperature for 30 minutes in the dark. 50ul of stop solution was added to each well of the plate to stop the reaction. The absorbance at 490 nm was counted within 1 hour after the addition of the stop solution.

Calculation results

The absorbance value of the medium background was subtracted from all absorbance values of the experimental group, the spontaneous release group of target cells and the spontaneous release group of effector cells.

The corrected values obtained above were inserted into the formula below to calculate the percent cytotoxicity produced by each effector-to-target ratio.

% Cytotoxicity = 100 x (experimental - effector cell spontaneous - target cell spontaneous) / (target cell maximum - target cell spontaneous)

result

SAGE1 TCR-transduced T cells were examined for LDH release in response to specific target cells by a nonradioactive cytotoxicity assay (described above). Absorbance values of visible light at 490 nm in each well were plotted using graphpad prism6.

The statistical results of the experimental data are shown in Figure 10. As the ratio of effector targets increases, the T cells transduced by the TCR of the present invention have enhanced killing effects on specific target cells K562-24 and NCI H1299-A24; on non-specific target cells IM 9 The killing effect is weak. Homologous CD8 ⁺ T cells not transduced with the TCR of the present invention did not significantly kill the target cell K562-24.

All documents mentioned herein are incorporated by reference in this application as if each document 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.

Claims (29)

1. a T cell receptor (TCR) is characterized in that, described TCR can be combined with VFSTVPPAFI-HLA A2402 complex; Described TCR comprises TCRα chain variable domain and TCRβ chain variable domain, it is characterized in that, The three complementarity determining regions (CDRs) of the variable domain of the TCRα chain are: αCDR1-DSAIYN (SEQ ID NO. 10) αCDR2-IQSSQRE (SEQ ID NO. 11) αCDR3-CAVLYTGANSKLTF (SEQ ID NO. 12); and The three complementarity determining regions of the TCRβ chain variable domain are: βCDR1-SGHDT (SEQ ID NO. 13) βCDR2-YYEEEE (SEQ ID NO. 14) βCDR3-CASSLVGKQPQHF (SEQ ID NO. 15). 2. The TCR of claim 1 , comprising a variable domain of a TCRα chain and a variable domain of a TCRβ chain, the variable domain of the TCRα chain having at least 90% sequence identity with SEQ ID NO.1 and/or the TCRβ chain variable domain is an amino acid sequence with at least 90% sequence identity to SEQ ID NO:5. 3. The TCR of claim 1, wherein the TCR is an αβ heterodimer comprising a TCRα chain constant region TRAC*01 and a TCRβ chain constant region TRBC1*01 or TRBC2*01. 4 . The TCR of claim 1 , wherein 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. 5 . 5. The TCR of claim 1, wherein the TCR is soluble and/or the TCR is single-chain. 6. The TCR of claim 1, wherein the TCR is at amino acid position 11, 13, 19, 21, 53, 76, 89, 91, or 94 in the alpha chain variable region, and/or The α-chain J gene short peptide has one or more mutations in the penultimate 3rd, 5th or 7th position of the penultimate amino acid; and/or the TCR is in the β-chain variable region amino acid 11, 13, 19, 21 , 53, 76, 89, 91, or 94, and/or one or more mutations in the penultimate amino acid position 2, 4 or 6 of the β-chain J gene short peptide, wherein the amino acid positions are numbered By position number as listed in IMGT (International Information System for Immunogenetics). 7. The TCR of claim 1, wherein the TCR comprises (a) all or a portion of a TCRα chain other than the transmembrane domain; and (b) all or a portion of a TCRβ except the transmembrane domain chain; 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. 8. The TCR of claim 1, wherein cysteine residues form an artificial disulfide bond between the constant domains of the alpha and beta chains of the TCR. 9. The TCR of claim 8, wherein the cysteine residues forming artificial disulfide bonds in the TCR are substituted for one or more groups of sites selected from the group consisting of: Thr48 in exon 1 of TRAC*01 and Ser57 in exon 1 of TRBC1*01 or TRBC2*01; Thr45 in exon 1 of TRAC*01 and Ser77 in exon 1 of TRBC1*01 or TRBC2*01; Tyr10 of exon 1 of TRAC*01 and Ser17 of exon 1 of TRBC1*01 or TRBC2*01; Thr45 of exon 1 of TRAC*01 and Asp59 of exon 1 of TRBC1*01 or TRBC2*01; Ser15 of exon 1 of TRAC*01 and Glu15 of exon 1 of TRBC1*01 or TRBC2*01; Arg53 in exon 1 of TRAC*01 and Ser54 in exon 1 of TRBC1*01 or TRBC2*01; Pro89 of exon 1 of TRAC*01 and Ala19 of exon 1 of TRBC1*01 or TRBC2*01; and Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01. 10 . The TCR of claim 1 , wherein 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. 11 . 11. The TCR of claim 1, wherein an artificial interchain disulfide bond is contained between the variable region of the α chain and the constant region of the β chain of the TCR. 12. The TCR of claim 11 , wherein the cysteine residues forming artificial interchain disulfide bonds in the TCR are substituted for one or more groups selected from the group consisting of point: Amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*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 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. 13. The TCR of claim 1, wherein the TCR comprises an alpha chain variable domain and a beta chain variable domain and all or part of the beta chain constant domain except the transmembrane domain, but does not contain The alpha chain constant domain, the alpha chain variable domain of the TCR forms a heterodimer with the beta chain. 14. The TCR of claim 1, wherein a conjugate is bound to the C- or N-terminus of the alpha chain and/or beta chain of the TCR. 15. The TCR of claim 14, wherein the conjugate bound to the T cell receptor is a detectable label, a therapeutic agent, a PK modifying moiety, or a combination of any of these. 16. The TCR of claim 15, wherein the therapeutic agent is an anti-CD3 antibody. 17. A multivalent TCR complex, comprising at least two TCR molecules, and at least one of the TCR molecules is the TCR of any one of claims 1-16. 18. A nucleic acid molecule, characterized in that the nucleic acid molecule comprises a nucleic acid sequence encoding the TCR of any one of claims 1-16 or a complementary sequence thereof. 19. The nucleic acid molecule of claim 18, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 2 encoding the variable domain of the TCRα chain; and The nucleic acid molecule comprises the nucleotide sequence SEQ ID NO:6 encoding the variable domain of the TCR beta chain. 20. The nucleic acid molecule of claim 18, wherein the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO:4 encoding a TCR alpha chain and a nucleotide sequence of SEQ ID NO:8 encoding a TCR beta chain. 21 . A vector, characterized in that, the vector contains the nucleic acid molecule of claim 18 . 22. The vector of claim 21, wherein the vector is a viral vector. 23. The vector of claim 21, wherein the vector is a lentiviral vector. 24. An isolated host cell, wherein the host cell contains the vector of claim 21 or the nucleic acid molecule of claim 18 integrated into a chromosome. 25. A cell, wherein the cell is transduced with the nucleic acid molecule of claim 18 or the vector of claim 21. 26. The cell of claim 25, wherein the cell is a T cell or a stem cell. 27. A pharmaceutical composition, characterized in that the composition contains a pharmaceutically acceptable carrier and the TCR described in any one of claims 1-16, the TCR complex described in claim 17, the The nucleic acid molecule of claim 18, or the cell of claim 24. 28. Use of a T cell receptor according to any one of claims 1-16, or a TCR complex according to claim 17, or a cell according to claim 24, for the preparation of a treatment Drugs for tumors or autoimmune diseases. 29. The use of claim 28, wherein the tumor comprises melanoma, bladder cancer, liver cancer, epidermoid carcinoma, non-small cell lung cancer, and squamous cell carcinoma.

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