CN106632660A - T cell receptor (TCR) capable of identifying NY-ESO-1 antigen short-peptides - Google Patents

Abstract

The invention provides a T cell receptor (TCR) capable of being specifically combined with short-peptide SLLMWITQC derived from a NY-ESO-1 antigen. The antigen short-peptide SLLMWITQC and HLA A0201 can form a compound and are presented to the surfaces of cells together. The invention further provides nucleic acid molecules for encoding the TCR and a carrier comprising the nucleic acid molecules. In addition, the invention further provides cells for transducing the TCR.

Description

TCR recognizing short peptide of NY-ESO-1 antigen

technical field

The present invention relates to a TCR capable of recognizing short peptides derived from the NY-ESO-1 antigen. The present invention also relates to NY-ESO-1 specific T cells obtained by transducing the above TCR, and their role in the prevention and treatment of NY-ESO- 1 Use in related diseases.

Background technique

NY-ESO-1 belongs to the cancer-testis antigen (Cancer-Testis Antigen, CTA) family, which can be expressed in testis, ovarian tissue and many different types of tumor tissues, but not in other normal tissues. strong tumor antigens. NY-ESO-1 is an endogenous antigen, which is degraded into small molecular polypeptides after being produced in cells, and combines with MHC (major histocompatibility complex) molecules to form a complex, which is presented to the cell surface. SLLMWITQC is a short peptide derived from NY-ESO-1 antigen, which is a target for the treatment of NY-ESO-1 related diseases. Studies have shown that NY-ESO-1 is expressed in various tumor tissues, including neuroblastoma (Rodolfo M, et al., Cancer Res, 2003, 63(20):6948-6955), sarcoma (Jungbhth A A et al., Int J Cancer, 200l, 94 (2): 252-256), malignant melanoma (Barrow C, et al., Clin Cancer Res, 2006, 12 (3Pt 1): 764-771) have very Highly expressed in prostate cancer, bladder cancer, breast cancer, multiple myeloma, hepatocellular carcinoma, oral squamous cell carcinoma (Ries J, et al., Anticancer RES, 2009,29(12):5125-5130), And esophageal cancer (Fujita S, Clin Cancer Res, 2004, 10(19):6551-6558) also has higher expression. For the treatment of the above diseases, methods such as chemotherapy and radiotherapy can be used, but all of them will cause damage to their own normal cells.

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 a TCR specific for the NY-ESO-1 antigen short peptide, and transducing the TCR to T cells to obtain T cells specific for the NY-ESO-1 antigen short peptide , so that they play a role in cellular immunotherapy.

Contents of the invention

The purpose of the present invention is to provide a T cell receptor that recognizes NY-ESO-1 antigen short peptide.

The first aspect of the present invention provides a T cell receptor (TCR), which can bind to the SLLMWITQC-HLAA0201 complex.

In another preferred example, the TCR comprises a TCR α-chain variable domain and a TCR β-chain variable domain, and the amino acid sequence of CDR3 of the TCR α-chain variable domain is ATDANGKII (SEQ ID NO: 12); and/or Or the amino acid sequence of CDR3 of the TCR β chain variable domain is ASSLGSNEQY (SEQ ID NO: 15).

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

α CDR1-TSINN (SEQ ID NO: 10)

α CDR2-IRSNERE (SEQ ID NO: 11)

α CDR3-ATDANGKII (SEQ ID NO: 12); and/or

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

β CDR1-SGHDY (SEQ ID NO: 13)

β CDR2-FNNNVP (SEQ ID NO: 14)

β CDR3-ASSLGSNEQY (SEQ ID NO: 15).

In another preferred example, the TCR comprises a TCR α chain variable domain and a TCR β chain variable domain, and the TCR α chain variable domain is an amino acid sequence having at least 90% sequence identity with SEQ ID NO:1 and/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 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 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 amino acid sequence of the α chain variable domain of the TCR comprises SEQ ID NO:32 and/or the amino acid sequence of the TCR β chain variable domain comprises SEQ ID NO:34.

In another preferred example, the amino acid sequence of the TCR is SEQ ID NO:30.

In another preferred example, the TCR includes (a) all or part of the TCR alpha chain except the transmembrane domain; and (b) all or part of the TCR beta 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.

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;

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 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 example, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 33 encoding the variable domain of the TCR alpha chain.

In another preferred example, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 35 encoding the variable domain of the TCR β chain.

In another preferred example, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 4 encoding the TCR α chain and/or comprises the nucleotide sequence 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 or a stem cell .

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 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, the nucleic acid molecule described in the third aspect of the present invention, the fourth aspect of the present invention The use of the carrier according to the aspect, or the cell according to the sixth aspect of the present invention, is used to prepare a drug for treating tumor or autoimmune disease.

The ninth 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 , 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, or the pharmaceutical composition of the seventh aspect of the present invention;

Preferably, the disease is a tumor, and preferably the tumor includes neuroblastoma, sarcoma, melanoma, prostate cancer, bladder cancer, breast cancer, multiple myeloma, hepatocellular carcinoma, oral squamous cell carcinoma, esophageal cancer And gastric cancer, lung cancer, head and neck squamous cell carcinoma, colon cancer, ovarian cancer, etc.

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 of the TCR α chain variable domain, the nucleotide sequence of the TCR α chain variable domain, the amino acid sequence of the TCR α chain, and the nucleosides of the TCR α chain, respectively Acid sequence, TCR α chain amino acid sequence with leader sequence and TCR α 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 TCR β chain variable domain, the nucleotide sequence of the TCR β chain variable domain, the amino acid sequence of the TCR β chain, and the nucleosides of the TCR β chain, respectively. Acid sequence, TCR β chain amino acid sequence with leader sequence and TCR β chain nucleotide sequence with leader sequence.

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

Figure 4a and Figure 4b are the amino acid sequence and nucleotide sequence of the soluble TCR α chain, respectively.

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

Figure 6 is the gel image of the purified soluble TCR. The leftmost lane is the reducing gel, the middle lane is the molecular weight marker (marker), and the rightmost lane is the non-reducing gel.

Figure 7a and Figure 7b are the amino acid sequence and nucleotide sequence of the single-chain TCR, respectively.

Figure 8a and Figure 8b are the amino acid sequence and nucleotide sequence of the single-chain TCR α chain, respectively.

Figure 9a and Figure 9b are the amino acid sequence and nucleotide sequence of the single-chain TCR β chain, respectively.

Figure 10a and Figure 10b are the amino acid sequence and nucleotide sequence of the single-chain TCR linker sequence (linker), respectively.

Figure 11 is the gel image of the purified soluble single-chain TCR.

Fig. 12 is a BIAcore kinetic map of the binding of the soluble TCR of the present invention to the SLLMWITQC-HLA A0201 complex.

Fig. 13 is a BIAcore kinetic map of the binding of the soluble single-chain TCR of the present invention to the SLLMWITQC--HLA A0201 complex.

Figure 14 shows that cells transduced with the TCR of the present invention have a killing effect on target cells expressing relevant antigens, but basically have no killing effect on target cells not expressing relevant antigens.

detailed description

After extensive and in-depth research, the inventors have found a TCR that can specifically bind to the NY-ESO-1 antigen short peptide SLLMWITQC (SEQ IDNO: 9), and the antigen short peptide SLLMWITQC can form a complex with HLA A0201 and together presented to the 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. Accordingly, the first aspect of the present invention provides a TCR molecule capable of binding the SLLMWITQC-HLA A0201 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:

α CDR1-TSINN (SEQ ID NO: 10)

α CDR2-IRSNERE (SEQ ID NO: 11)

α CDR3-ATDANGKII (SEQ ID NO: 12); and/or

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

β CDR1-SGHDY (SEQ ID NO: 13)

β CDR2-FNNNVP (SEQ ID NO: 14)

β CDR3-ASSLGSNEQY (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 TCR α 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 β chain variable domain of the present invention is an amino acid sequence identical to SEQ ID NO:1 SEQ ID NO: 5 has an amino acid sequence of 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 NY-ESO-1 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 TCR α and β chains. For the single-chain soluble TCR constructed in Example 4 of the present invention, the amino acid sequence of the variable domain of the α chain is SEQ ID NO: 32, and the encoded nucleotide sequence is SEQ ID NO: 33; the amino acid sequence of the variable domain of the β chain is It is SEQ ID NO:34, and the encoded nucleotide sequence is SEQ ID NO:35.

In addition, in terms of stability, patent document 201510260322.4 also discloses that the introduction of an artificial interchain disulfide bond 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 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) Both 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 SLLMWITQC-HLA A0201 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. (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 (Cancer Immunology and Immunotherapy) 53, 345; Halin et al., 2003, Cancer Research (Cancer Research) 63, 3202); 4. Antibody Fc fragment (Mosquera et al., 2005, 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/nanorods ( 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 therapy ( Gene therapy) 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research (Cancer research) 65, 11631); 9. Nanomagnetic particles; 10. Prodrug activating enzymes (for example, DT-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:

α CDR1- actagtataaacaat (SEQ ID NO: 16)

α CDR2- atacgttcaaatgaaagagag (SEQ ID NO: 17)

α CDR3- gctacggacgcaaacggcaagatcatc (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:

β CDR1- tcaggacacgactac (SEQ ID NO: 19)

β CDR2- tttaacaacaacgttccg (SEQ ID NO: 20)

β CDR3- gccagcagtttagggagcaacgagcagtac (SEQ ID NO: 21)

Therefore, the nucleotide sequence of the nucleic acid molecule of the present invention encoding the TCR α chain of the present invention includes SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and/or the nucleic acid of the present invention encoding the TCR β chain of the present invention The nucleotide sequence of the molecule includes 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 TCR α chain variable domain 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 TCR beta chain variable domain of the present invention includes SEQ ID NO:6. Alternatively, the nucleotide sequence of the nucleic acid molecule encoding the TCR α chain variable domain of the present invention comprises SEQ ID NO: 33 and/or the nucleotide sequence of the nucleic acid molecule encoding the TCR β chain variable domain of the present invention comprises SEQ ID NO: 33 ID NO: 35. More preferably, the nucleotide sequence of the nucleic acid molecule of the present invention comprises SEQ ID NO:4 and/or SEQ ID NO:8. Alternatively, the nucleotide sequence of the nucleic acid molecule of the present invention is SEQ ID NO:31.

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 NY-ESO-1 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, 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).

NY-ESO-1 antigen-related diseases

The present invention also relates to a method for treating and/or preventing NY-ESO-1 related diseases in a subject, which comprises the step of adoptively transferring NY-ESO-1 specific T cells to the subject. The NY-ESO-1-specific T cells recognize the SLLMWITQC-HLA A0201 complex.

The NY-ESO-1-specific T cells of the present invention can be used to treat any NY-ESO-1-related diseases presenting the NY-ESO-1 antigen short peptide SLLMWITQC-HLA A0201 complex, including but not limited to tumors, preferably all The above tumors include neuroblastoma, sarcoma, melanoma, prostate cancer, bladder cancer, breast cancer, multiple myeloma, hepatocellular carcinoma, oral squamous cell carcinoma, esophageal cancer, gastric cancer, lung cancer, squamous cell carcinoma of the head and neck, Colon cancer, ovarian cancer, etc.

treatment method

It can be carried out by isolating T cells of patients or volunteers suffering from diseases related to NY-ESO-1 antigen, introducing the TCR of the present invention into the above T cells, and then returning these genetically modified cells to the patient. treat. Therefore, the present invention provides a method for treating NY-ESO-1-related diseases, comprising infusing isolated T cells expressing the TCR of the present invention, preferably, the T cells are derived from the patient himself, 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 be combined with the NY-ESO-1 antigen short peptide complex SLLMWITQC-HLA A0201, 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 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 NY-ESO-1 Antigen Short Peptide-Specific T Cells

Peripheral blood lymphocytes (PBL) from healthy volunteers with genotype HLA-A0201 were stimulated with a synthetic short peptide SLLMWITQC (SEQ ID NO.:9; Beijing Saibaisheng Gene Technology Co., Ltd.). The SLLMWITQC short peptide was annealed with biotin-labeled HLA-A0201 to prepare pHLA 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.

Example 2 Obtaining the TCR gene and carrier construction of NY-ESO-1 antigen short peptide-specific T cell clone

The total RNA of the short antigen peptide SLLMWITQC-specific and HLA-A0201-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, Figure 1a, Figure 1b, Figure 1c, Figure 1d, Figure 1e and Figure 1f are the TCR α chain Variable domain amino acid sequence, TCR α chain variable domain nucleotide sequence, TCR α chain amino acid sequence, TCR α chain nucleotide sequence, TCR α chain amino acid sequence with leader sequence, and TCR α chain nucleotide sequence with leader sequence Sequence; Figure 2a, Figure 2b, Figure 2c, Figure 2d, Figure 2e and Figure 2f are the amino acid sequence of TCR β chain variable domain, the nucleotide sequence of TCR β chain variable domain, the amino acid sequence of TCR β chain, and the TCR β chain Nucleotide sequence, TCR beta chain amino acid sequence with leader sequence and TCR beta chain nucleotide sequence with leader sequence.

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

α CDR1-TSINN (SEQ ID NO: 10)

α CDR2-IRSNERE (SEQ ID NO: 11)

α CDR3-ATDANGKII (SEQ ID NO: 12)

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

β CDR1-SGHDY (SEQ ID NO: 13)

β CDR2-FNNNVP (SEQ ID NO: 14)

β CDR3-ASSLGSNEQY (SEQ ID NO: 15)

The full-length genes of TCR α chain and β chain were respectively cloned into the lentiviral expression vector pLenti (addgene) by overlapping PCR. Specifically, the full-length genes of the TCR α chain and the TCR β chain are connected by overlapping PCR to obtain the TCR α-2A-TCR β fragment. The lentiviral expression vector and TCR α-2A-TCR β were digested and ligated to obtain the pLenti-TRA-2A-TRB-IRES-NGFR plasmid. As a control, a lentiviral vector pLenti-eGFP expressing eGFP was also constructed. Then use 293T/17 to package the fake virus.

Example 3 Expression, refolding and purification of NY-ESO-1 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 positions of the introduced cysteine residues are Thr48 of TRAC*01 exon 1 and Ser57 of TRBC2*01 exon 1; the amino acid sequence and nucleotides of the α chain The sequences are shown in Figure 4a and Figure 4b, respectively, the amino acid sequence and nucleotide sequence of the β chain are shown in Figure 5a and Figure 5b, respectively, and the introduced cysteine residues are represented by bold and underlined letters. The target gene sequences of the above-mentioned 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 respectively transformed into expression bacteria BL21(DE3) by chemical transformation method, the bacteria were grown in LB culture medium, and induced with a final concentration of 0.5mM IPTG at OD ₆₀₀ =0.6, the α and β chains of TCR were The inclusion bodies formed after expression 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 (pH 8.1).

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, the final concentration 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 map of the soluble TCR obtained in the present invention is shown in FIG. 6 .

Example 4 Generation of soluble single-chain TCR specific for NY-ESO-1 antigen short peptide

According to the patent document WO2014/206304, the variable domains of TCR α and β chains in Example 2 were constructed by site-directed mutagenesis into a stable soluble single-chain TCR molecule linked by a flexible short peptide (linker). The amino acid sequence and nucleotide sequence of the single-chain TCR molecule are shown in Fig. 7a and Fig. 7b respectively. The amino acid sequence and nucleotide sequence of its alpha chain variable domain are shown in Figure 8a and Figure 8b respectively; the amino acid sequence and nucleotide sequence of its beta chain variable domain are shown in Figure 9a and Figure 9b respectively; its linker The amino acid sequence and nucleotide sequence of the sequence are shown in Figure 10a and Figure 10b, respectively.

The target gene was digested with Nco I and Not I, and then ligated with the pET28a vector that was digested with Nco I and Not I. The ligation product was transformed into E.coliDH5α, coated with kanamycin-containing LB plates, cultured upside down at 37°C overnight, positive clones were picked for PCR screening, and positive recombinants were sequenced. After confirming the sequence was correct, the recombinant plasmids were extracted and transformed into E. coliBL21(DE3), for expression.

Example 5 Expression, refolding and purification of NY-ESO-1 antigen short peptide-specific soluble single-chain TCR

All the BL21(DE 3) colonies containing the recombinant plasmid pET28a-template strand prepared in Example 4 were inoculated in LB medium containing kanamycin, cultivated at 37°C until the OD600 was 0.6-0.8, and added IPTG to the final concentration 0.5mM, 37 ℃ continue to culture 4h. Centrifuge at 5000rpm for 15min to harvest the cell pellet, use Bugbuster Master Mix (Merck) to lyse the cell pellet, centrifuge at 6000rpm for 15min to recover inclusion bodies, then wash with Bugbuster (Merck) to remove cell debris and membrane components, centrifuge at 6000rpm for 15min to collect inclusion bodies body. The inclusion bodies were dissolved in buffer solution (20mM Tris-HCl pH 8.0, 8M urea), and the insoluble matter was removed by high-speed centrifugation. The supernatant was quantified by BCA method, then aliquoted, and stored at -80°C for future use.

Add 2.5mL of buffer (6M Gua-HCl, 50mM Tris-HCl pH 8.1, 100mM NaCl, 10mM EDTA) to 5mg of dissolved single-chain TCR inclusion body protein, then add DTT to a final concentration of 10mM, and treat at 37°C for 30min . Add the above-mentioned single-chain TCR dropwise to 125mL refolding buffer (100mM Tris-HCl pH 8.1, 0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mMβ-mercapthoethylamine, 1.87mM Cystamine) with a syringe, Stir at 4°C for 10 minutes, then put the refolding solution into a dialysis bag with a cellulose membrane with a cutoff of 4kDa, place the dialysis bag in 1L of pre-cooled water, and stir slowly overnight at 4°C. After 17 hours, the dialysate was replaced with 1L pre-cooled buffer solution (20mM Tris-HCl pH 8.0), and the dialysis was continued at 4°C for 8h, and then the dialysate was replaced with the same fresh buffer solution to continue dialysis overnight. After 17 hours, the sample was filtered through a 0.45 μm filter membrane, vacuum degassed, and then passed through an anion exchange column (HiTrap Q HP, GE Healthcare) to purify the protein with a 0-1M NaCl linear gradient eluent prepared with 20 mM Tris-HCl pH 8.0, and collected The eluted fractions were analyzed by SDS-PAGE, and the fractions containing single-chain TCR were concentrated and further purified by gel filtration column (Superdex 7510/300, GE Healthcare), and the target fractions were also analyzed by SDS-PAGE.

The eluted fractions for BIAcore analysis were further tested for purity by gel filtration. The conditions are: chromatographic column Agilent Bio SEC-3 (300A, φ7.8×300mm), the mobile phase is 150mM phosphate buffer, the flow rate is 0.5mL/min, the column temperature is 25°C, and the ultraviolet detection wavelength is 214nm.

The SDS-PAGE gel map of the soluble single-chain TCR obtained in the present invention is shown in FIG. 11 .

Example 6 Combination Characterization

BIAcore analysis

This example demonstrates that the soluble TCR molecules of the present invention can specifically bind to the SLLMWITQC-HLA A0201 complex.

The binding activity of the TCR molecule obtained in Example 3 and Example 5 to the SLLMWITQC-HLA A0201 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 SLLMWITQC-HLAA0201 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 SLLMWITQC-HLA A0201 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 SLLMWITQC (Beijing Saibaisheng Gene Technology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg/ml. The inclusion bodies of the light chain and heavy chain were dissolved with 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 SLLMWITQC 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, 0.2mM PMSF, cooled to 4°C), then sequentially add 20mg/L light chain and 90mg/L heavy chain (final concentration, heavy chain is added in three times, 8h/time), renaturation is carried out at 4°C for at least 3 days To 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

The biotinylated pMHC molecules were concentrated to 1ml with Millipore ultrafiltration tubes, the biotinylated pMHC was purified by gel filtration chromatography, and HiPrep was pre-equilibrated with filtered PBS using an Akta purification instrument (GE General Electric Company). ^TM 16/60 S200 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 pooled, 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 profiles of the soluble TCR molecule of the present invention and the binding of the soluble single-chain TCR molecule constructed by the present invention to the SLLMWITQC-HLA A0201 complex were obtained, as shown in Figure 12 and Figure 13, respectively. The map shows that both the soluble TCR molecule and the soluble single-chain TCR molecule obtained in the present invention can bind to the SLLMWITQC-HLA A0201 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 7 Killing experiment of cells transduced with TCR of the present invention

In this example, non-radioactive cytotoxicity experiments were used to measure the release of LDH, thereby verifying the killing function of the cells transduced with the TCR of the present invention.

Those skilled in the art are familiar with the method of using LDH release assay to detect cell function. In the LDH experiment of this example, PBL cells isolated from the blood of healthy volunteers were transfected with TCR with lentivirus as effector cells. The target cell lines were A375(3525), MEL624(1605), NCI-H1650(2) and MEL526(4) cells. According to the nanostring results, among them, A375 (3525) and MEL624 (1605) expressed NY-ESO-1 antigen. NCI-H1650 (2) and MEL526 (4) basically did not express NY-ESO-1 antigen, which were used as controls.

First prepare the LDH plate. On the first day of the experiment, each component of the experiment was added to the plate in the following order: the medium was adjusted to 2 X 10 ⁶ cells/ml for effector cells, and the medium was adjusted to 5 X 10 ⁵ cells/ml for each target cell line. After mixing evenly, take 100 μL of target cell line 5 X 10 ⁵ cells/ml (ie 50,000 cells/well), and 100 μL of effector cells 2 X 10 ⁶ cells/ml (ie 200,000 cells/well) into corresponding wells , and set up three replicate holes. Simultaneously set effector cell spontaneous wells, target cell spontaneous wells, target cell maximum wells, volume correction control wells and medium background control wells. Incubate overnight (37°C, 5% CO ₂ ). On the second day of the experiment, the color development was detected, and the absorbance value was recorded at 490 nm with a microplate reader (Bioteck) after the reaction was terminated. The experimental results are shown in Figure 14. The cells transduced with the TCR of the present invention have a killing effect on target cells expressing relevant antigens, but basically have no killing effect on target cells not expressing relevant antigens.

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.

-1435 sequence listing

<110> Guangzhou Xiangxue Pharmaceutical Co., Ltd.

<120> TCR recognizing short peptide of NY-ESO-1 antigen

<130> P2016-1815

<150> CN201510751225.5

<151> 2015-11-04

<160> 37

<170> PatentIn version 3.5

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agaggccttg tccacctaat tttaatacgt tcaaatgaaa gagagaaaca cagtggaaga 180

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Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser Arg

65 70 75 80

Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Thr Asp Ala Asn Gly Lys

85 90 95

Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro Asn Ile Gln

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

245 250

<210> 4

<211> 750

<212>DNA

<213> artificial sequence (artificial)

<220>

<223> TCR alpha chain

<400> 4

agtcaacagg gagaagagga tcctcaggcc ttgagcatcc aggagggtga aaatgccacc 60

atgaactgca gttacaaaac tagtataaac aatttacagt ggtatagaca aaattcaggt 120

agaggccttg tccacctaat tttaatacgt tcaaatgaaa gagagaaaca cagtggaaga 180

ttaagagtca cgcttgacac ttccaagaaa agcagttcct tgttgatcac ggcttcccgg 240

gcagcagaca ctgcttctta cttctgtgct acggacgcaa acggcaagat catctttgga 300

aaagggacac gacttcatat tctccccaat atccagaacc ctgaccctgc cgtgtaccag 360

ctgagagact ctaaatccag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 420

acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaaac tgtgctagac 480

atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacaa atctgacttt 540

gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca 600

gaaagttcct gtgatgtcaa gctggtcgag aaaagctttg aaacagatac gaacctaaac 660

tttcaaaacc tgtcagtgat tgggttccga atcctcctcc tgaaagtggc cgggtttaat 720

ctgctcatga cgctgcggct gtggtccagc 750

<210> 5

<211> 112

<212> PRT

<213> artificial sequence (artificial)

<220>

<223> TCR beta chain variable domain

<400> 5

Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met Gly

1 5 10 15

Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu

20 25 30

Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile Tyr

35 40 45

Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg

50 55 60

Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln

65 70 75 80

Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Leu

85 90 95

Gly Ser Asn Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr

100 105 110

<210> 6

<211> 336

<212>DNA

<213> artificial sequence (artificial)

<220>

<223> TCR beta chain variable domain

<400> 6

gatgctggag ttatccagtc accccggcac gaggtgacag agatgggaca agaagtgact 60

ctgagatgta aaccaatttc aggacacgac taccttttct ggtacagaca gaccatgatg 120

cggggactgg agttgctcat ttactttaac aacaacgttc cgatagatga ttcagggatg 180

cccgaggatc gattctcagc taagatgcct aatgcatcat tctccactct gaagatccag 240

ccctcagaac ccagggactc agctgtgtac ttctgtgcca gcagtttagg gagcaacgag 300

cagtacttcg ggccgggcac caggctcacg gtcaca 336

<210> 7

<211> 291

<212> PRT

<213> artificial sequence (artificial)

<220>

<223> TCR beta chain

<400> 7

Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met Gly

1 5 10 15

Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu

20 25 30

Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile Tyr

35 40 45

Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg

50 55 60

Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln

65 70 75 80

Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Leu

85 90 95

Gly Ser Asn Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr

100 105 110

Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro

115 120 125

Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu

130 135 140

Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

145 150 155 160

Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys

165 170 175

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

180 185 190

Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys

195 200 205

Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp

210 215 220

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

225 230 235 240

Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser

245 250 255

Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala

260 265 270

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

275 280 285

Ser Arg Gly

290

<210> 8

<211> 873

<212>DNA

<213> artificial sequence (artificial)

<220>

<223> TCR beta chain

<400> 8

gatgctggag ttatccagtc accccggcac gaggtgacag agatgggaca agaagtgact 60

ctgagatgta aaccaatttc aggacacgac taccttttct ggtacagaca gaccatgatg 120

cggggactgg agttgctcat ttactttaac aacaacgttc cgatagatga ttcagggatg 180

cccgaggatc gattctcagc taagatgcct aatgcatcat tctccactct gaagatccag 240

ccctcagaac ccagggactc agctgtgtac ttctgtgcca gcagtttagg gagcaacgag 300

cagtacttcg ggccgggcac caggctcacg gtcacagagg acctgaaaaa cgtgttccca 360

cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 420

ctggtgtgcc tggccacagg cttctacccc gaccacgtgg agctgagctg gtgggtgaat 480

gggaaggagg tgcacagtgg ggtcagcaca gacccgcagc ccctcaagga gcagcccgcc 540

ctcaatgact ccagatactg cctgagcagc cgcctgaggg tctcggccac cttctggcag 600

aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 660

tggacccagg atagggccaa acctgtcacc cagatcgtca gcgccgaggc ctggggtaga 720

gcagactgtg gcttcacctc cgagtcttac cagcaagggg tcctgtctgc caccatcctc 780

tatgagatct tgctagggaa ggccaccttg tatgccgtgc tggtcagtgc cctcgtgctg 840

atggccatgg tcaagagaaa ggattccaga ggc 873

<210> 9

<211> 9

<212> PRT

<213> artificial sequence (artificial)

<220>

<223> Antigen short peptide

<400> 9

Ser Leu Leu Met Trp Ile Thr Gln Cys

1 5

<210> 10

<211> 5

<212> PRT

<213> artificial sequence (artificial)

<220>

<223>α CDR1

<400> 10

Thr Ser Ile Asn Asn

1 5

<210> 11

<211> 7

<212> PRT

<213> artificial sequence (artificial)

<220>

<223>α CDR2

<400> 11

Ile Arg Ser Asn Glu Arg Glu

1 5

<210> 12

<211> 9

<212> PRT

<213> artificial sequence (artificial)

<220>

<223>α CDR3

<400> 12

Ala Thr Asp Ala Asn Gly Lys Ile Ile

1 5

<210> 13

<211> 5

<212> PRT

<213> artificial sequence (artificial)

<220>

<223>β CDR1

<400> 13

Ser Gly His Asp Tyr

1 5

<210> 14

<211> 6

<212> PRT

<213> artificial sequence (artificial)

<220>

<223>β CDR2

<400> 14

Phe Asn Asn Asn Val Pro

1 5

<210> 15

<211> 10

<212> PRT

<213> artificial sequence (artificial)

<220>

<223>β CDR3

<400> 15

Ala Ser Ser Leu Gly Ser Asn Glu Gln Tyr

1 5 10

<210> 16

<211> 15

<212>DNA

<213> artificial sequence (artificial)

<220>

<223>α CDR1

<400> 16

actagtataa acaat 15

<210> 17

<211> 21

<212>DNA

<213> artificial sequence (artificial)

<220>

<223>α CDR2

<400> 17

atacgttcaa atgaaagaga g 21

<210> 18

<211> 27

<212>DNA

<213> artificial sequence (artificial)

<220>

<223>α CDR3

<400> 18

gctacggacg caaacggcaa gatcatc 27

<210> 19

<211> 15

<212>DNA

<213> artificial sequence (artificial)

<220>

<223>β CDR1

<400> 19

tcaggacacg actac 15

<210> 20

<211> 18

<212>DNA

<213> artificial sequence (artificial)

<220>

<223>β CDR2

<400> 20

tttaacaaca acgttccg 18

<210> 21

<211> 30

<212>DNA

<213> artificial sequence (artificial)

<220>

<223>β CDR3

<400> 21

gccagcagtt tagggagcaa cgagcagtac 30

<210> 22

<211> 270

<212> PRT

<213> artificial sequence (artificial)

<220>

<223> TCRα chain with leader sequence

<400> 22

Met Glu Thr Leu Leu Gly Val Ser Leu Val Ile Leu Trp Leu Gln Leu

1 5 10 15

Ala Arg Val Asn Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser

20 25 30

Ile Gln Glu Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser

35 40 45

Ile Asn Asn Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val

50 55 60

His Leu Ile Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg

65 70 75 80

Leu Arg Val Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile

85 90 95

Thr Ala Ser Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Thr Asp

100 105 110

Ala Asn Gly Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

<210> 23

<211> 810

<212>DNA

<213> artificial sequence (artificial)

<220>

<223> TCRα chain with leader sequence

<400> 23

atggaaactc tcctgggagt gtctttggtg attctatggc ttcaactggc tagggtgaac 60

agtcaacagg gagaagagga tcctcaggcc ttgagcatcc aggagggtga aaatgccacc 120

atgaactgca gttacaaaac tagtataaac aatttacagt ggtatagaca aaattcaggt 180

agaggccttg tccacctaat tttaatacgt tcaaatgaaa gagagaaaca cagtggaaga 240

ttaagagtca cgcttgacac ttccaagaaa agcagttcct tgttgatcac ggcttcccgg 300

gcagcagaca ctgcttctta cttctgtgct acggacgcaa acggcaagat catctttgga 360

aaagggacac gacttcatat tctccccaat atccagaacc ctgaccctgc cgtgtaccag 420

ctgagagact ctaaatccag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 480

acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaaac tgtgctagac 540

atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacaa atctgacttt 600

gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca 660

gaaagttcct gtgatgtcaa gctggtcgag aaaagctttg aaacagatac gaacctaaac 720

tttcaaaacc tgtcagtgat tgggttccga atcctcctcc tgaaagtggc cgggtttaat 780

ctgctcatga cgctgcggct gtggtccagc 810

<210> 24

<211> 310

<212> PRT

<213> artificial sequence (artificial)

<220>

<223> TCRβ chain with leader sequence

<400> 24

Met Asp Ser Trp Thr Leu Cys Cys Val Ser Leu Cys Ile Leu Val Ala

1 5 10 15

Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr

20 25 30

Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His

35 40 45

Asp Tyr Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu

50 55 60

Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro

65 70 75 80

Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu

85 90 95

Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala

100 105 110

Ser Ser Leu Gly Ser Asn Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys

290 295 300

Arg Lys Asp Ser Arg Gly

305 310

<210> 25

<211> 930

<212>DNA

<213> artificial sequence (artificial)

<220>

<223> TCRβ chain with leader sequence

<400> 25

atggactcct ggaccctctg ctgtgtgtcc ctttgcatcc tggtagcaaa gcacacagat 60

gctggagtta tccagtcacc ccggcacgag gtgacagaga tgggacaaga agtgactctg 120

agatgtaaac caatttcagg acacgactac cttttctggt acagacagac catgatgcgg 180

ggactggagt tgctcattta ctttaacaac aacgttccga tagatgattc agggatgccc 240

gaggatcgat tctcagctaa gatgcctaat gcatcattct ccactctgaa gatccagccc 300

tcagaaccca gggactcagc tgtgtacttc tgtgccagca gtttagggag caacgagcag 360

tacttcgggc cgggcaccag gctcacggtc acagaggacc tgaaaaacgt gttcccaccc 420

gaggtcgctg tgtttgagcc atcagaagca gagatctccc acacccaaaa ggccaacactg 480

gtgtgcctgg ccacaggctt ctaccccgac cacgtggagc tgagctggtg ggtgaatggg 540

aaggaggtgc acagtggggt cagcacagac ccgcagcccc tcaaggagca gcccgccctc 600

aatgactcca gatactgcct gagcagccgc ctgagggtct cggccacctt ctggcagaac 660

ccccgcaacc acttccgctg tcaagtccag ttctacgggc tctcggagaa tgacgagtgg 720

accccaggata gggccaaacc tgtcacccag atcgtcagcg ccgaggcctg gggtagagca 780

gactgtggct tcacctccga gtcttaccag caaggggtcc tgtctgccac catcctctat 840

gagatcttgc tagggaaggc caccttgtat gccgtgctgg tcagtgccct cgtgctgatg 900

gccatggtca agagaaagga ttccagaggc 930

<210> 26

<211> 203

<212> PRT

<213> artificial sequence (artificial)

<220>

<223> Soluble TCRα chain

<400> 26

Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu Gly

1 5 10 15

Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn Leu

20 25 30

Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile Leu

35 40 45

Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val Thr

50 55 60

Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser Arg

65 70 75 80

Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Thr Asp Ala Asn Gly Lys

85 90 95

Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro Asn Ile Gln

100 105 110

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

115 120 125

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

130 135 140

Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val Leu Asp

145 150 155 160

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

165 170 175

Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro

180 185 190

Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

195 200

<210> 27

<211> 609

<212>DNA

<213> artificial sequence (artificial)

<220>

<223> Soluble TCRα chain

<400> 27

agccagcagg gcgaagaaga tcctcaggcc ttgagcatcc aggagggtga aaatgccacc 60

atgaactgca gttacaaaac tagtataaac aatttacagt ggtatagaca aaattcaggt 120

agaggccttg tccacctaat tttaatacgt tcaaatgaaa gagagaaaca cagtggaaga 180

ttaagagtca cgcttgacac ttccaagaaa agcagttcct tgttgatcac ggcttcccgg 240

gcagcagaca ctgcttctta cttctgtgct acggacgcaa acggcaagat catctttgga 300

aaagggacac gacttcatat tctccccaat atccagaacc ctgaccctgc cgtgtaccag 360

ctgagagact ctaagtcgag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 420

acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaatg tgtgctagac 480

atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacaa atctgacttt 540

gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca 600

gaaagttcc 609

<210> 28

<211> 242

<212> PRT

<213> artificial sequence (artificial)

<220>

<223> Soluble TCRβ chain

<400> 28

Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met Gly

1 5 10 15

Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu

20 25 30

Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile Tyr

35 40 45

Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg

50 55 60

Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln

65 70 75 80

Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Leu

85 90 95

Gly Ser Asn Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr

100 105 110

Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro

115 120 125

Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu

130 135 140

Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

145 150 155 160

Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys

165 170 175

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

180 185 190

Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His Phe Arg Cys

195 200 205

Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp

210 215 220

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

225 230 235 240

Ala Asp

<210> 29

<211> 726

<212>DNA

<213> artificial sequence (artificial)

<220>

<223> Soluble TCRβ chain

<400> 29

gatgcgggcg tgattcagtc accccggcac gaggtgacag agatgggaca agaagtgact 60

ctgagatgta aaccaatttc aggacacgac taccttttct ggtacagaca gaccatgatg 120

cggggactgg agttgctcat ttactttaac aacaacgttc cgatagatga ttcagggatg 180

cccgaggatc gattctcagc taagatgcct aatgcatcat tctccactct gaagatccag 240

ccctcagaac ccagggactc agctgtgtac ttctgtgcca gcagtttagg gagcaacgag 300

cagtacttcg ggccgggcac caggctcacg gtcacagagg acctgaaaaa cgtgttccca 360

cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 420

ctggtgtgcc tggccaccgg tttctacccc gaccacgtgg agctgagctg gtgggtgaat 480

gggaaggagg tgcacagtgg ggtctgcaca gacccgcagc ccctcaagga gcagcccgcc 540

ctcaatgact ccagatacgc tctgagcagc cgcctgaggg tctcggccac cttctggcag 600

gacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 660

tggacccagg atagggccaa acccgtcacc cagatcgtca gcgccgaggc ctggggtaga 720

gcagac 726

<210> 30

<211> 245

<212> PRT

<213> artificial sequence (artificial)

<220>

<223> Single-chain TCR

<400> 30

Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu Gly

1 5 10 15

Glu Asn Val Thr Ile Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn Leu

20 25 30

Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile Leu

35 40 45

Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val Thr

50 55 60

Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Glu Ile Thr Ala Val Arg

65 70 75 80

Pro Ala Asp Thr Ala Ser Tyr Phe Cys Ala Thr Asp Ala Asn Gly Lys

85 90 95

Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro Gly Gly Gly

100 105 110

Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser

115 120 125

Glu Gly Gly Thr Gly Asp Ala Gly Val Thr Gln Ser Pro Arg His Glu

130 135 140

Ser Val Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser

145 150 155 160

Gly His Asp Tyr Leu Phe Trp Tyr Arg Gln Thr Pro Lys Arg Gly Leu

165 170 175

Glu Leu Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly

180 185 190

Met Pro Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser

195 200 205

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

210 215 220

Cys Ala Ser Ser Leu Gly Ser Asn Glu Gln Tyr Phe Gly Pro Gly Thr

225 230 235 240

Arg Leu Thr Val Thr

245

<210> 31

<211> 735

<212>DNA

<213> artificial sequence (artificial)

<220>

<223> Single-chain TCR

<400> 31

tctcaacaag gtgaagaaga tccgcaggcc ctgagtattc aagaaggtga aaatgtgacc 60

atcaactgct cttacaaaac gagtatcaac aatctgcagt ggtaccgtca aaattctggc 120

cgcggtctgg ttcatctgat tctgatccgt tccaacgaac gcgaaaaaca ctcaggccgt 180

ctgcgcgtta ccctggatac cagcaaaaaa tcttctagtc tggaaatcac cgcagtccgt 240

ccggcagata cggcaagcta tttttgtgca accgacgcta atggtaaaat tatcttcggc 300

aaaggtaccc gcctgcatat tctgccgggc ggtggctccg aaggtggcgg ttcagaaggc 360

ggtggctcgg aaggtggcgg tagcgaaggc ggtaccggtg atgcgggtgt cacgcagtct 420

ccgcgtcatg aaagtgtgga aatgggccaa gaagttacgc tgcgctgcaa accgatcagc 480

ggtcacgact acctgttttg gtaccgtcag accccgaaac gcggcctgga actgctgatc 540

tacttcaaca ataacgttcc gattgatgac tcgggtatgc cggaagatcg ttttagcgcg 600

aaaatgccga atgcctcgtt cagcacgctg aaaattcagc cggtcgaacc gcgtgactcc 660

gcagtgtatt tttgtgcttc ctcactgggc agtaacgaac agtacttcgg cccgggtacc 720

cgtctgaccg tgacg 735

<210> 32

<211> 109

<212> PRT

<213> artificial sequence (artificial)

<220>

<223> Single-chain TCRα chain

<400> 32

Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu Gly

1 5 10 15

Glu Asn Val Thr Ile Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn Leu

20 25 30

Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile Leu

35 40 45

Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val Thr

50 55 60

Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Glu Ile Thr Ala Val Arg

65 70 75 80

Pro Ala Asp Thr Ala Ser Tyr Phe Cys Ala Thr Asp Ala Asn Gly Lys

85 90 95

Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro

100 105

<210> 33

<211> 327

<212>DNA

<213> artificial sequence (artificial)

<220>

<223> Single-chain TCRα chain

<400> 33

tctcaacaag gtgaagaaga tccgcaggcc ctgagtattc aagaaggtga aaatgtgacc 60

atcaactgct cttacaaaac gagtatcaac aatctgcagt ggtaccgtca aaattctggc 120

cgcggtctgg ttcatctgat tctgatccgt tccaacgaac gcgaaaaaca ctcaggccgt 180

ctgcgcgtta ccctggatac cagcaaaaaa tcttctagtc tggaaatcac cgcagtccgt 240

ccggcagata cggcaagcta tttttgtgca accgacgcta atggtaaaat tatcttcggc 300

aaaggtaccc gcctgcatat tctgccg 327

<210> 34

<211> 112

<212> PRT

<213> artificial sequence (artificial)

<220>

<223> Single-chain TCRβ chain

<400> 34

Asp Ala Gly Val Thr Gln Ser Pro Arg His Glu Ser Val Glu Met Gly

1 5 10 15

Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu

20 25 30

Phe Trp Tyr Arg Gln Thr Pro Lys Arg Gly Leu Glu Leu Leu Ile Tyr

35 40 45

Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg

50 55 60

Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln

65 70 75 80

Pro Val Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Leu

85 90 95

Gly Ser Asn Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr

100 105 110

<210> 35

<211> 336

<212>DNA

<213> artificial sequence (artificial)

<220>

<223> Single-chain TCRβ chain

<400> 35

gatgcgggtg tcacgcagtc tccgcgtcat gaaagtgtgg aaatgggcca agaagttacg 60

ctgcgctgca aaccgatcag cggtcacgac tacctgtttt ggtaccgtca gaccccgaaa 120

cgcggcctgg aactgctgat ctacttcaac aataacgttc cgattgatga ctcgggtatg 180

ccggaagatc gttttagcgc gaaaatgccg aatgcctcgt tcagcacgct gaaaattcag 240

ccggtcgaac cgcgtgactc cgcagtgtat ttttgtgctt cctcactggg cagtaacgaa 300

cagtacttcg gcccgggtac ccgtctgacc gtgacg 336

<210> 36

<211> 24

<212> PRT

<213> artificial sequence (artificial)

<220>

<223> Single-stranded TCR junction sequence

<400> 36

Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

1 5 10 15

Gly Gly Ser Glu Gly Gly Thr Gly

20

<210> 37

<211> 72

<212>DNA

<213> artificial sequence (artificial)

<220>

<223> Single-stranded TCR junction sequence

<400> 37

ggcggtggct ccgaaggtgg cggttcagaa ggcggtggct cggaaggtgg cggtagcgaa 60

ggcggtaccg gt 72

Claims (10)

1. A T cell receptor (TCR), characterized in that the TCR can be combined with the SLLMWITQC-HLAA0201 complex; preferably, the TCR comprises a TCR α chain variable domain and a TCR β chain variable domain, and the TCR is characterized in that In that, the amino acid sequence of CDR3 of the TCRα chain variable domain is ATDANGKII (SEQ ID NO:12); and/or the amino acid sequence of the CDR3 of the TCRβ chain variable domain is ASSLGSNEQY (SEQ ID NO:15); More preferably, the three complementarity determining regions (CDRs) of the TCRα chain variable domain are: α CDR1-TSINN (SEQ ID NO: 10) α CDR2-IRSNERE (SEQ ID NO: 11) alpha CDR3-ATDANGKII (SEQ ID NO: 12); and/or The three complementarity-determining regions of the TCRβ chain variable domain are: β CDR1-SGHDY (SEQ ID NO: 13) β CDR2-FNNNVP (SEQ ID NO: 14) β CDR3-ASSLGSNEQY (SEQ ID NO: 15). 2. The TCR of claim 1, comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, the TCR alpha chain variable domain having at least 90% sequence identity to SEQ ID NO: 1 and/or the TCRβ chain variable domain is an amino acid sequence having at least 90% sequence identity with SEQ ID NO:5. 3. The TCR according to claim 1, characterized in that, the C- or N-terminus of the α chain and/or β chain of the TCR is bound with a conjugate; preferably, it binds to the T cell receptor The conjugate is a detectable marker, a therapeutic agent, a PK modifying moiety, or a combination of any of these substances; 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 or SEQ ID NO: 33 encoding the variable domain of the TCR alpha chain; and/or The nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 35 encoding the variable domain of the TCRβ chain. 6. A carrier, characterized in that, the carrier contains the nucleic acid molecule described in claim 5; preferably, the carrier is a viral vector; more preferably, the carrier is a lentiviral vector . 7. An isolated host cell, characterized in that the host cell contains the vector according to claim 6 or the nucleic acid molecule according to any one of claim 5 integrated in the chromosome. 8. A cell, characterized in that the cell is transduced with the nucleic acid molecule according to claim 5 or the vector according to 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 according to any one of claim 5, or the cell according to 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.