CN110272482B - T cell receptor recognizing PRAME antigen short peptide - Google Patents

Abstract

The present invention provides a T cell receptor (TCR) that can specifically recognize the short peptide EVLVDLFLK derived from the PRAME antigen, specifically, the present invention provides a T cell receptor, the TCR can interact with the presentation on the cell surface The EVLVDLFLK‑HLA‑A*1101 complex binds. The present invention also provides a nucleic acid molecule sequence encoding the TCR and a vector comprising the nucleic acid molecule sequence.

Description

T cell receptors that recognize short peptides from the PRAME antigen

technical field

The present invention relates to TCRs that can specifically recognize PRAME antigenic short peptides. The present invention also relates to PRAME-specific T cells obtained by transducing the above-mentioned TCRs, and their use in preventing and treating PRAME-related diseases.

Background technique

As an endogenous tumor antigen, RPAME 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 EVLVDLFLK is a short peptide derived from PRAME. In addition to the expression of PRAME antigen in solid tumors such as melanoma, renal cell carcinoma, lung cancer, breast cancer, and medulloblastoma, it is also expressed in some hematological malignancies, such as acute myeloid leukemia, chronic myeloid leukemia, acute lymphoid Cell carcinoma, myeloma, etc. (Kessler JH, et al. J Exp Med, 2001, 193(1):73-88. ChangAY, et al. J Clin Invest. 2017, 127(7):2705-2718). For the treatment of the above diseases, chemotherapy and radiation therapy can be used, but they will cause damage to their own normal cells. Therefore, using short peptides derived from PRAME antigens as targets for the above-mentioned various cancers can not only be used as markers for the diagnosis of the above-mentioned diseases, but also can be used to produce preventive and/or therapeutic agents for the above-mentioned diseases, such as antibodies or T cells. receptor.

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 a TCR with specific recognition ability for PRAME antigen short peptide, making it work, or transducing the TCR into T cells to obtain TCR with specific recognition ability for PRAME antigen short peptide cells, making them useful in cellular immunotherapy.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a T cell receptor that can specifically recognize the PRAME antigen short peptide.

A first aspect of the present invention provides a T cell receptor (TCR) capable of binding to EVLVDLFLK-HLA-A*1101 complex.

In another preferred embodiment, the TCR comprises a TCRα chain variable domain and a TCRβ chain variable domain, and the amino acid sequence of CDR3 of the TCRα chain variable domain is AATSGGSYIPT (SEQ ID NO: 12); and/or the The amino acid sequence of CDR3 of the variable domain of the TCR beta chain is ASSLSGRLGEQF (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-AATSGGSYIPT (SEQ ID NO: 12); and/or

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

βCDR1-MNHNY (SEQ ID NO: 13)

βCDR2-SVGAGI (SEQ ID NO: 14)

βCDR3-ASSLSGRLGEQF (SEQ ID NO: 15).

In another preferred embodiment, the TCR comprises a TCRα chain variable domain and a TCRβ chain variable domain, and the TCRα chain variable domain is an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1; and/ Or the TCR beta chain variable domain is an amino acid sequence with at least 90% sequence identity to SEQ ID NO:2.

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

In another preferred embodiment, the TCR comprises a beta chain variable domain amino acid sequence of SEQ ID NO:2.

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, 5, 25 and/or the amino acid sequence of the β chain of the TCR is SEQ ID NO: 4, 6, 27.

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 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 the 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: 3, 5, 25 and/or the amino acid sequence of the β chain of the TCR is SEQ ID NO: 4, 6, 27.

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: 7 encoding the variable domain of the TCRα chain.

In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 8 encoding the variable domain of the TCRβ chain.

In another preferred embodiment, the nucleic acid molecule comprises nucleotide sequences encoding TCRα chains of SEQ ID NOs: 9, 16, 26 and/or nucleotide sequences encoding TCRβ chains: SEQ ID NOs: 17, 18, 28 .

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 lentiviral 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 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, and the first aspect of the present invention. The nucleic acid molecule described in the third aspect, the vector described in the fourth aspect of the present invention, or the cell described in the sixth aspect of the present 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, and the fourth aspect of the present invention. Use of the vector described above, or the cell described in the sixth aspect of the present invention, for preparing a medicine 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, the present 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 is selected from the group consisting of melanoma, renal cell carcinoma, lung cancer, breast cancer, medulloblastoma, hematological malignancies, or a combination thereof.

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 (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

Description of drawings

Figure 1a, Figure 1b, Figure 1c, Figure 1d, Figure 1e and Figure 1f show the TCRα chain variable domain amino acid sequence, TCRα chain variable domain nucleotide sequence, TCRα chain amino acid sequence, TCRα chain nucleotide sequence, TCRα chain amino acid sequence with leader sequence and TCRα chain nucleotide sequence with leader sequence.

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

Figure 3 shows the CD8 ⁺ and tetramer-PE double positive staining results 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 show the amino acid sequence and nucleotide sequence of the soluble TCR beta chain, respectively.

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

Figure 7 shows a Biacore kinetic profile of the binding of the soluble TCR of the present invention to the EVLVDLFLK-HLA-A*1101 complex.

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

Figure 9 shows the results of the ELISPOT assay.

Figure 10 shows 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 PRAME antigenic short peptide EVLVDLFLK (SEQ ID NO: 29), which can form a complex with HLA-A*1101 and be presented to the cell surface. The present invention also provides a nucleic acid molecule sequence encoding the TCR and a vector comprising the nucleic acid molecule sequence. 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

As used herein, the term "PRAME TCR-T" refers to cells that are reactive to the PRAME antigenic peptides of the present invention, but not to other antigenic peptides.

As used herein, the terms "non-PRAME TCR-transduced T cells" and "Non-PRAME TCR-T" are used interchangeably, both refer to T cells expressing other TCRs, and can also be understood as targeting the PRAME antigenic peptides of the present invention , but the poorly functioning TCR, these T cells combined with the PRAME antigen peptide of the present invention could not elicit a response.

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 to the EVLVDLFLK-HLA-A*1101 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-AATSGGSYIPT (SEQ ID NO: 12); and/or

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

βCDR1-MNHNY (SEQ ID NO: 13)

βCDR2-SVGAGI (SEQ ID NO: 14)

βCDR3-ASSLSGRLGEQF (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 to SEQ ID NO: 1; and/or the TCRβ chain variable domain of the present invention is an amino acid sequence with SEQ ID NO: 1 NO:2 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:2. More preferably, the amino acid sequence of the beta chain variable domain of the TCR molecule is SEQ ID NO:2.

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:2. More preferably, the amino acid sequence of the beta chain variable domain of the single-chain TCR molecule is SEQ ID NO:2.

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, 5, 25, and/or the amino acid sequence of the β chain is SEQ ID NO: 4, 6, 27.

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 PRAME antigen short peptide. In a preferred embodiment, the amino acid sequence of the soluble TCRα chain of the present invention is shown in SEQ ID NO.:25, and the nucleotide sequence is shown in SEQ ID NO.:26; the amino acid sequence of the TCRβ chain of the present invention is shown in SEQ ID NO.:26 As shown in ID NO.:27, the nucleotide sequence is shown as SEQ ID NO.:28.

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 alpha chain J gene (TRAJ) short peptide amino acid position 3,5,7 from the bottom, and/or the beta chain J gene (TRBJ) short peptide amino acid position 2,4,6 from the bottom, where 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 beta 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 EVLVDLFLK-HLA-A*1101 complex), a therapeutic agent, a PK (protein kinase) modification moiety, or any Combinations of the above substances are combined or coupled.

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/nanorods ( 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, Gene therapy ( Gene therapy) 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research 65, 11631); 9. Nanomagnetic particles; 10. Prodrug-activating enzymes (eg, DT-diaphorase) (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:

αCDR1- gatagcgctatttacaac (SEQ ID NO: 19)

αCDR2- attcagtcaagtcagagagag (SEQ ID NO: 20)

αCDR3- gctgctacatcaggaggaagctacatacctaca (SEQ ID NO: 21).

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:

βCDR1- atgaaccataactac (SEQ ID NO:22)

βCDR2- tcagttggtgctggtatc (SEQ ID NO:23)

βCDR3- gccagcagccttagcgggaggttgggtgagcagttc (SEQ ID NO: 24).

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: 19, SEQ ID NO: 20 and SEQ ID NO: 21, 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:22, SEQ ID NO:23 and SEQ ID NO:24.

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 TCR alpha chain of the present invention includes SEQ ID NO: 7 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 comprises SEQ ID NO:8. More preferably, the nucleotide sequence of the nucleic acid molecule of the invention comprises SEQ ID NO: 9, 16 and/or SEQ ID NO: 17, 18.

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 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: 7.

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 a TCR specific for the PRAME 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).

PRAME antigen-related diseases

The present invention also relates to a method of treating and/or preventing a PRAME-related disease in a subject comprising the step of adoptively transferring PRAME-specific T cells to the subject. The PRAME-specific T cells can recognize EVLVDLFLK complex with HLA-A*1101.

The PRAME-specific T cells of the present invention can be used to treat any PRAME-related disease that presents the PRAME antigen short peptide EVLVDLFLK-HLA-A*1101 complex. Including, but not limited to, solid tumors such as melanoma, renal cell carcinoma, lung cancer, breast cancer, medulloblastoma, and hematological malignancies, such as acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic carcinoma, myeloma, etc. .

treatment method

Treatment can be performed by isolating T cells from patients or volunteers with PRAME antigen-related diseases, introducing the TCRs 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 PRAME-related diseases, comprising infusing isolated T cells expressing the TCR of the present invention, preferably, the T cells are derived from the patient itself, into the patient. 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 include:

(1) The TCR of the present invention can specifically bind to the PRAME antigen short peptide complex EVLVDLFLK-HLA-A*1101, and the cells transduced with the TCR of the present invention can be specifically activated and have obvious killing effects on specific target cells effect.

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 not 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 PRAME antigen short peptide-specific T cells

Peripheral blood lymphocytes (PBL) from healthy volunteers with HLA-A11 genotype were stimulated with the synthetic short peptide PRAME 176-184EVLVDLFLK (Beijing Saibaisheng Gene Technology Co., Ltd.). The PRAME176-184EVLVDLFLK peptide was renatured with biotin-labeled HLA-A*1101 to prepare pHLA monomer. 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 tetramer and anti-CD8 antibody, and the screened double-positive clones were shown in Figure 3.

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

The total RNA of the PRAME 176-184EVLVDLFLK-specific, HLA-A11-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 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-AATSGGSYIPT (SEQ ID NO: 12)

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

βCDR1-MNHNY (SEQ ID NO: 13)

βCDR2-SVGAGI (SEQ ID NO: 14)

βCDR3-ASSLSGRLGEQF (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-PRAME TRA-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 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 expressing 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 in 10 times the volume of deionized water (4°C), and after 12 hours, the deionized water was changed to buffer (20 mM Tris, pH 8.0) and dialyzed at 4°C for 12 hours. After the dialysis was completed, the solution was filtered through a 0.45 μM 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 determined by SDS-PAGE to be more than 90%, 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 can specifically bind to the EVLVDLFLK-HLA-A*1101 complex.

The binding activity of the TCR molecule obtained in Example 3 to the EVLVDLFLK-HLA-A*1101 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 antibody-coated chip surface, and then the EVLVDLFLK-HLA-A*1101 complex was flowed through the detection channel, and the other channel was used as a reference channel, and 0.05mM biotin was added to the detection channel. A flow rate of 10 μL/min was flowed through the chip for 2 min to block the remaining binding sites of streptavidin. The preparation process of the above EVLVDLFLK-HLA-A*1101 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 EVLVDLFLK (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. LVLGTLEEV 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 μm cellulose acetate filter and loaded onto a HiTrap Q HP (GE) anion exchange column (5 ml bed volume). The protein was eluted using Akta purifier (GE), 0-400 mM NaCl linear gradient prepared in 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 detected 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). /60S200HR 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 PRAME-specific T cell receptor lentiviral packaging and primary T cell transfection with PRAME 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-PRAME TRA-2A-TRB-IRES-NGFR described in Example 2) were used using Express-In-mediated transient transfection (OpenBiosystems). 293T/17 cells were transfected with a lentiviral vector, and 3 plasmids containing other components necessary for the construction of 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 plasmids, package pLenti-PRAME TRA-2A-TRB-IRES-NGFR and pLenti-eGFP pseudoviruses, mix the above expression plasmids with the packaging plasmids pMDLg/pRRE, pRSV-REV and pMD.2G, one 15 The dosage for centimeter diameter dishes 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 μg. 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; take the corresponding The amount of PEI was mixed with 1800 microliters of OPTI-MEM medium, and the mixture was allowed to stand for 5 minutes at room temperature to become a PEI mixture. Mix the DNA mixture and PEI mixture together and let stand for 30 minutes at room temperature, then add 3150 μl OPTI-MEM medium, mix well and add it to the 293T/17 cells that have been converted into 11.25 ml OPTI-MEM, Gently shake the Petri dish to mix the medium well and incubate at 37°C/5% CO ₂ . 5-7 hours after transfection, the transfection medium was removed and replaced with DMEM ((Gibco, cat. no. C11995500bt)) complete medium containing 10% fetal bovine serum at 37°C/5% _CO nourish. Media supernatants containing packaged lentiviruses were collected on days 3 and 4. To harvest the packaged lentivirus, the collected culture supernatant was centrifuged at 3000g for 15 minutes to remove cell debris, filtered through a 0.45 micron filter (Merck Millipore, catalog number SLGP033RB), and finally with a 50KD cutoff. Concentration tubes (Merck Millipore, catalog number UFC905096) were used to concentrate, most of the supernatant was removed, and finally concentrated to 1 mL, aliquoted and frozen at -80°C. Pseudovirus samples were taken for virus titer determination, and the steps were referred to the instructions of the p24ELISA (Clontech, catalog number 632200) kit. As a control, the pseudovirus of pLenti-eGFP was also included.

(b) Transduction of primary T cells with lentiviruses containing the PRAME-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 measured by the p24 ELISA kit, the concentrated PRAME-specific T cell receptor gene lentivirus was added at a ratio of MOI=10, and the cells were centrifuged at 32°C and 900g for 1 hour of infection. 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

The PRAME 176-184EVLVDLFLK peptide was renatured with biotin-labeled HLA-A*1101 to prepare pHLA monomers. These monomers were combined with PE-labeled streptavidin (BD) into PE-labeled tetramers, called PRAME-tetramer-PE. This tetramer can label T cells expressing the PRAME-specific T cell receptor gene as positive cells. The transduced T cell samples in (b) were mixed with PRAME-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. The samples were washed twice with PBS containing 2% FBS and then detected or sorted with Milliporeguava or BD Arial for PRAME-tetramer-PE and anti-mouse β-chain-APC double positive T cells expressing the PRAME-specific T cell receptor gene , data analysis using guavaSoft 3.1 software (Merck Millipore (MerckMillipore)) or FlowJo software (Tree Star Inc, Ashland, OR) analysis.

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

Example 6 Verification of PRAME-specific TCR function

6.1 ELISPOT scheme

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 2×10 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 after about 30 days, the culture was further expanded and subjected to flow cytometry, in which CD19 ⁺ CD23 ^hi CD58 ⁺ were immortalized lymphoblastoid cell lines (LCLs). This ELISPOT assay uses HLA-A11 as a specific target cell.

Effector cell preparation

The effector cells (T cells) in this experiment were the CD8 ⁺ T cells expressing PRAME-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, Life Technologies), transduced with lentiviruses carrying PRAME-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 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 µl of the diluted capture antibody to 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.

PRAME T cells (PRAME TCR-transduced T cells, effector cells), T cells (T cells expressing other non-EVLVDLFLK peptide-specific TCRs) and LCLs A24/A11 (target cells) were prepared as described in Example 3, and Corresponding short peptides were added to the corresponding experimental groups, wherein P _PRAME 176-184EVLVDLFLK short peptides, P _N1 , P _N2 , and P _N3 were non-PRAME 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 PRAME TCR double positive T cells).

20 microliters of ^10-5 mol/liter PRAME 176-184EVLVDLFLK/nonspecific 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.

Dilute streptavidin-alkaline phosphatase 1:100 in PBS containing 10% FBS, add 100 microliters of diluted streptavidin-alkaline phosphatase to each well and incubate the plate 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

PRAME TCR-transduced T cells were examined for IFN-[gamma] release in response to target cells loaded with PRAME176-184EVLVDLFLK short peptides and nonspecific short peptides by 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, PRAME T Cell cells (effector cells) alone and LCL cells (target cells) release very little IFN-γ.

PRAME T Cells (effector cells) released more IFN-γ in response to LCL cells supplemented with P _PRAME . It shows that the TCR of the present invention can recognize the short peptide of P _PRAME , so it can mediate the activation of T cells.

PRAME T Cells (effector cells) released little IFN-γ when LCL cells were supplemented with non-specific short peptides.

LCL cells to which P _{PRAME was} supplemented with T Cells (negative control effector cells) released little IFN-γ. This indicates that other non-specific TCRs cannot recognize P _PRAME short peptides and therefore cannot mediate T cell activation.

6.2 Nonradioactive cytotoxicity test protocol

This assay is a colorimetric alternative to the ⁵¹ Cr 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), 95% RPMI 1640 without phenol red (Gibco, cat. no. 11835-030).

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

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

method

target cell preparation

Four tumor cell lines K562-A24, K562-A11, SK-MEL-5 and A375 were used as target cells in this experiment. Prepare target cells in assay medium: adjust target cell concentration to 200 cells/ml and take 100 microliters per well to obtain ² x 104 cells/well.

Effector cell preparation

Effector cells (T cells) in this assay were CD8 ⁺ T cells expressing PRAME-specific TCRs as analyzed by flow cytometry in Example 3. The ratio of effector cells to target cells was 2.5:1. and 1.25:1. The CD8 ⁺ T cells plus target cells expressing other non-PRAME peptide-specific TCRs were set as the control group (2.5:1).

Assay of CD8 ⁺ T-cell Specific Killing of Tumor Cells Transduced by PRAME-specific TCR

Test preparation

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

- 100ul of target cells (prepared as described above) were added to each well

- 100ul of effector cells (prepared as described above) were added to each well

A control group was prepared as follows:

- Spontaneous release of effector cells: 100ul of effector cells.

- Spontaneous release of target cells: 100ul of target cells.

- Maximum release of target cells: 100ul of target cells.

- Medium control: 200ul medium.

-Volume correction control: 200ul medium + 20ul lysate (10X) (added before detection)

All wells were prepared in triplicate with a final volume of 200ul (not enough was made up with medium).

Incubate for 24 hours at 37°C. Before collecting the supernatant from all wells, add target cell maximum release control wells to 10× lysis buffer and leave to incubate for about 45 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 flat-bottom 96-well 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, and the maximum release of target cells was subtracted from the volume correction control to correct for the volume change due to the addition of lysate (10X).

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

PRAME TCR-transduced T cells were examined for LDH release in response to loading of specific target cells by a nonradioactive cytotoxicity assay (as described above). Use graphpad prism6 to draw the cytotoxicity percentage value calculated by the 490nm visible light absorbance value in each well after substituting it into the formula.

The statistical results of the experimental data are shown in Figure 10. As the ratio of effector targets increases, the CD8 ⁺ T cells (PRAME T Cells) transduced by PRAME TCR kill HLA A11 and PRAME antigen-expressing K562-A11 and SK-MEL-5 cells Increased effect; no killing effect on non-specific cells K562-A24 and A375. The killing rate of CD8 ⁺ T cells expressing other non-PRAME peptide-specific TCRs against HLA A11 and K562-A11 expressing PRAME antigen was significantly lower than that of the PRAME TCR-transduced T cell group. This test result shows that the TCR of the present invention can mediate the specific killing of target cells by T cells, but other non-PRAME peptide-specific TCRs cannot mediate the killing of target cells by T cells.

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.

sequence listing

<110> Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences

<120> T cell receptors that recognize short peptides of the PRAME antigen

<130> P2017-2277

<160> 29

<170> PatentIn version 3.5

<210> 1

<211> 112

<212> PRT

<213> Artificial sequence

<400> 1

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

1 5 10 15

Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala Ile Tyr Asn

20 25 30

Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr Ser Leu Leu

35 40 45

Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg Leu Asn Ala

50 55 60

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

65 70 75 80

Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Ala Thr Ser Gly Gly

85 90 95

Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His Pro

100 105 110

<210> 2

<211> 113

<212> PRT

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<400> 2

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

1 5 10 15

Gln Ser Met Thr Leu Gln Cys Thr Gln Asp Met Asn His Asn Tyr Met

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Lys Leu Ile Tyr Tyr

35 40 45

Ser Val Gly Ala Gly Ile Thr Asp Lys Gly Glu Val Pro Asn Gly Tyr

50 55 60

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

65 70 75 80

Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Ser Leu Ser

85 90 95

Gly Arg Leu Gly Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val

100 105 110

Leu

<210> 3

<211> 253

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<400> 3

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

1 5 10 15

Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala Ile Tyr Asn

20 25 30

Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr Ser Leu Leu

35 40 45

Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg Leu Asn Ala

50 55 60

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

65 70 75 80

Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Ala Thr Ser Gly Gly

85 90 95

Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His Pro

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250

<210> 4

<211> 292

<212> PRT

<213> Artificial sequence

<400> 4

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

1 5 10 15

Gln Ser Met Thr Leu Gln Cys Thr Gln Asp Met Asn His Asn Tyr Met

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Lys Leu Ile Tyr Tyr

35 40 45

Ser Val Gly Ala Gly Ile Thr Asp Lys Gly Glu Val Pro Asn Gly Tyr

50 55 60

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

65 70 75 80

Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Ser Leu Ser

85 90 95

Gly Arg Leu Gly Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Asp Ser Arg Gly

290

<210> 5

<211> 272

<212> PRT

<213> Artificial sequence

<400> 5

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

1 5 10 15

Val Ser Ser Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val

20 25 30

Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala

35 40 45

Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr

50 55 60

Ser Leu Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg

65 70 75 80

Leu Asn Ala Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile

85 90 95

Ala Ala Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Ala Thr

100 105 110

Ser Gly Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

<210> 6

<211> 311

<212> PRT

<213> Artificial sequence

<400> 6

Met Ser Ile Ser Leu Leu Cys Cys Ala Ala Phe Pro Leu Leu Trp Ala

1 5 10 15

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

20 25 30

Lys Ile Gly Gln Ser Met Thr Leu Gln Cys Thr Gln Asp Met Asn His

35 40 45

Asn Tyr Met Tyr Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Lys Leu

50 55 60

Ile Tyr Tyr Ser Val Gly Ala Gly Ile Thr Asp Lys Gly Glu Val Pro

65 70 75 80

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

85 90 95

Leu Glu Leu Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Lys Arg Lys Asp Ser Arg Gly

305 310

<210> 7

<211> 336

<212> PRT

<213> Artificial sequence

<400> 7

Ala Ala Ala Cys Ala Gly Gly Ala Gly Gly Thr Gly Ala Cys Gly Cys

1 5 10 15

Ala Gly Ala Thr Thr Cys Cys Thr Gly Cys Ala Gly Cys Thr Cys Thr

20 25 30

Gly Ala Gly Thr Gly Thr Cys Cys Cys Ala Gly Ala Ala Gly Gly Ala

35 40 45

Gly Ala Ala Ala Ala Cys Thr Thr Gly Gly Thr Thr Cys Thr Cys Ala

50 55 60

Ala Cys Thr Gly Cys Ala Gly Thr Thr Thr Cys Ala Cys Thr Gly Ala

65 70 75 80

Thr Ala Gly Cys Gly Cys Thr Ala Thr Thr Thr Ala Cys Ala Ala Cys

85 90 95

Cys Thr Cys Cys Ala Gly Thr Gly Gly Thr Thr Thr Ala Gly Gly Cys

100 105 110

Ala Gly Gly Ala Cys Cys Cys Thr Gly Gly Gly Ala Ala Ala Gly Gly

115 120 125

Thr Cys Thr Cys Ala Cys Ala Thr Cys Thr Cys Thr Gly Thr Thr Gly

130 135 140

Cys Thr Thr Ala Thr Thr Cys Ala Gly Thr Cys Ala Ala Gly Thr Cys

145 150 155 160

Ala Gly Ala Gly Ala Gly Ala Gly Cys Ala Ala Ala Cys Ala Ala Gly

165 170 175

Thr Gly Gly Ala Ala Gly Ala Cys Thr Thr Ala Ala Thr Gly Cys Cys

180 185 190

Thr Cys Gly Cys Thr Gly Gly Ala Thr Ala Ala Ala Thr Cys Ala Thr

195 200 205

Cys Ala Gly Gly Ala Cys Gly Thr Ala Gly Thr Ala Cys Thr Thr Thr

210 215 220

Ala Thr Ala Cys Ala Thr Thr Gly Cys Ala Gly Cys Thr Thr Cys Thr

225 230 235 240

Cys Ala Gly Cys Cys Cys Thr Gly Gly Thr Gly Ala Cys Thr Cys Ala Gly

245 250 255

Cys Cys Ala Cys Cys Thr Ala Cys Cys Thr Cys Thr Gly Thr Gly Cys

260 265 270

Thr Gly Cys Thr Ala Cys Ala Thr Cys Ala Gly Gly Ala Gly Gly Ala

275 280 285

Ala Gly Cys Thr Ala Cys Ala Thr Ala Cys Cys Thr Ala Cys Ala Thr

290 295 300

Thr Thr Gly Gly Ala Ala Gly Ala Gly Gly Ala Ala Cys Cys Ala Gly

305 310 315 320

Cys Cys Thr Thr Ala Thr Thr Gly Thr Thr Cys Ala Thr Cys Cys Gly

325 330 335

<210> 8

<211> 339

<212> PRT

<213> Artificial sequence

<400> 8

Ala Ala Thr Gly Cys Thr Gly Gly Thr Gly Thr Cys Ala Cys Thr Cys

1 5 10 15

Ala Gly Ala Cys Cys Cys Cys Ala Ala Ala Ala Thr Thr Cys Cys Gly

20 25 30

Cys Ala Thr Cys Cys Cys Thr Gly Ala Ala Gly Ala Thr Ala Gly Gly Ala

35 40 45

Cys Ala Gly Ala Gly Cys Ala Thr Gly Ala Cys Ala Cys Thr Gly Cys

50 55 60

Ala Gly Thr Gly Thr Ala Cys Cys Cys Ala Gly Gly Ala Thr Ala Thr

65 70 75 80

Gly Ala Ala Cys Cys Ala Thr Ala Ala Cys Thr Ala Cys Ala Thr Gly

85 90 95

Thr Ala Cys Thr Gly Gly Thr Ala Thr Cys Gly Ala Cys Ala Ala Gly

100 105 110

Ala Cys Cys Cys Ala Gly Gly Cys Ala Thr Gly Gly Gly Gly Cys Thr

115 120 125

Gly Ala Ala Gly Cys Thr Gly Ala Thr Thr Thr Ala Thr Thr Ala Thr

130 135 140

Thr Cys Ala Gly Thr Thr Gly Gly Thr Gly Cys Thr Gly Gly Thr Ala

145 150 155 160

Thr Cys Ala Cys Thr Gly Ala Thr Ala Ala Ala Gly Gly Ala Gly Ala

165 170 175

Ala Gly Thr Cys Cys Cys Gly Ala Ala Thr Gly Gly Cys Thr Ala Cys

180 185 190

Ala Ala Cys Gly Thr Cys Thr Cys Cys Ala Gly Ala Thr Cys Ala Ala

195 200 205

Cys Cys Ala Cys Ala Gly Ala Gly Gly Ala Thr Thr Thr Cys Cys Cys

210 215 220

Gly Cys Thr Cys Ala Gly Gly Cys Thr Gly Gly Ala Gly Thr Thr Gly

225 230 235 240

Gly Cys Thr Gly Cys Thr Cys Cys Cys Thr Cys Cys Cys Ala Gly Ala

245 250 255

Cys Ala Thr Cys Thr Gly Thr Gly Thr Ala Cys Thr Thr Cys Thr Gly

260 265 270

Thr Gly Cys Cys Ala Gly Cys Ala Gly Cys Cys Thr Thr Ala Gly Cys

275 280 285

Gly Gly Gly Ala Gly Gly Thr Thr Gly Gly Gly Thr Gly Ala Gly Cys

290 295 300

Ala Gly Thr Thr Cys Thr Thr Cys Gly Gly Gly Cys Cys Ala Gly Gly

305 310 315 320

Gly Ala Cys Ala Cys Gly Gly Cys Thr Cys Ala Cys Cys Gly Thr Gly

325 330 335

Cys Thr Ala

<210> 9

<211> 759

<212> DNA

<213> Artificial sequence

<400> 9

aaacaggagg tgacgcagat tcctgcagct ctgagtgtcc cagaaggaga aaacttggtt 60

ctcaactgca gtttcactga tagcgctatt tacaacctcc agtggtttag gcaggaccct 120

gggaaaggtc tcacatctct gttgcttatt cagtcaagtc agagagagca aacaagtgga 180

agacttaatg cctcgctgga taaatcatca ggacgtagta ctttatacat tgcagcttct 240

cagcctggtg actcagccac ctacctctgt gctgctacat caggaggaag ctacatacct 300

acatttggaa gaggaaccag ccttattgtt catccgtata tccagaaccc tgaccctgcc 360

gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 420

gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 480

gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540

tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600

cccagcccag aaagttcctg tgatgtcaag ctggtcgaga aaagctttga aacagatacg 660

aacctaaact ttcaaaacct gtcagtgatt gggttccgaa tcctcctcct gaaagtggcc 720

gggtttaatc tgctcatgac gctgcggctg tggtccagc 759

<210> 10

<211> 6

<212> PRT

<213> Artificial sequence

<400> 10

Asp Ser Ala Ile Tyr Asn

1 5

<210> 11

<211> 7

<212> PRT

<213> Artificial sequence

<400> 11

Ile Gln Ser Ser Gln Arg Glu

1 5

<210> 12

<211> 11

<212> PRT

<213> Artificial sequence

<400> 12

Ala Ala Thr Ser Gly Gly Ser Tyr Ile Pro Thr

1 5 10

<210> 13

<211> 5

<212> PRT

<213> Artificial sequence

<400> 13

Met Asn His Asn Tyr

1 5

<210> 14

<211> 6

<212> PRT

<213> Artificial sequence

<400> 14

Ser Val Gly Ala Gly Ile

1 5

<210> 15

<211> 12

<212> PRT

<213> Artificial sequence

<400> 15

Ala Ser Ser Leu Ser Gly Arg Leu Gly Glu Gln Phe

1 5 10

<210> 16

<211> 816

<212> DNA

<213> Artificial sequence

<400> 16

atggagaccc tcttgggcct gcttatcctt tggctgcagc tgcaatgggt gagcagcaaa 60

caggaggtga cgcagattcc tgcagctctg agtgtcccag aaggagaaaa cttggttctc 120

aactgcagtt tcactgatag cgctatttac aacctccagt ggtttaggca ggaccctggg 180

aaaggtctca catctctgtt gcttattcag tcaagtcaga gagagcaaac aagtggaaga 240

cttaatgcct cgctggataa atcatcagga cgtagtactt tatacattgc agcttctcag 300

cctggtgact cagccaccta cctctgtgct gctacatcag gaggaagcta catacctaca 360

tttggaagag gaaccagcct tattgttcat ccgtatatcc agaaccctga ccctgccgtg 420

taccagctga gagactctaa atccagtgac aagtctgtct gcctattcac cgattttgat 480

tctcaaacaa atgtgtcaca aagtaaggat tctgatgtgt atatcacaga caaaactgtg 540

ctagacatga ggtctatgga cttcaagagc aacagtgctg tggcctggag caacaaatct 600

gactttgcat gtgcaaacgc cttcaacaac agcattattc cagaagacac cttcttcccc 660

agcccagaaa gttcctgtga tgtcaagctg gtcgagaaaa gctttgaaac agatacgaac 720

ctaaactttc aaaacctgtc agtgattggg ttccgaatcc tcctcctgaa agtggccggg 780

tttaatctgc tcatgacgct gcggctgtgg tccagc 816

<210> 17

<211> 876

<212> DNA

<213> Artificial sequence

<400> 17

aatgctggtg tcactcagac cccaaaattc cgcatcctga agataggaca gagcatgaca 60

ctgcagtgta cccaggatat gaaccataac tacatgtact ggtatcgaca agacccaggc 120

atggggctga agctgattta ttattcagtt ggtgctggta tcactgataa aggagaagtc 180

ccgaatggct acaacgtctc cagatcaacc acagaggatt tcccgctcag gctggagttg 240

gctgctccct cccagacatc tgtgtacttc tgtgccagca gccttagcgg gaggttgggt 300

gagcagttct tcgggccagg gacacggctc accgtgctag aggacctgaa aaacgtgttc 360

ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 420

acactggtgt gcctggccac aggcttctac cccgaccacg tggagctgag ctggtgggtg 480

aatgggaagg aggtgcacag tggggtcagc acagacccgc agcccctcaa ggagcagccc 540

gccctcaatg actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg 600

cagaaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 660

gagtggaccc aggatagggc caaacctgtc acccagatcg tcagcgccga ggcctggggt 720

agagcagact gtggcttcac ctccgagtct taccagcaag gggtcctgtc tgccaccatc 780

ctctatgaga tcttgctagg gaaggccacc ttgtatgccg tgctggtcag tgccctcgtg 840

ctgatggcca tggtcaagag aaaggattcc agaggc 876

<210> 18

<211> 933

<212> DNA

<213> Artificial sequence

<400> 18

atgagcatca gcctcctgtg ctgtgcagcc tttcctctcc tgtgggcagg tccagtgaat 60

gctggtgtca ctcagacccc aaaattccgc atcctgaaga taggacagag catgacactg 120

cagtgtaccc aggatatgaa ccataactac atgtactggt atcgacaaga cccaggcatg 180

gggctgaagc tgatttatta ttcagttggt gctggtatca ctgataaagg agaagtcccg 240

aatggctaca acgtctccag atcaaccaca gaggatttcc cgctcaggct ggagttggct 300

gctccctccc agacatctgt gtacttctgt gccagcagcc ttagcgggag gttgggtgag 360

cagttcttcg ggccagggac acggctcacc gtgctagagg acctgaaaaa cgtgttccca 420

cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 480

ctggtgtgcc tggccacagg cttctacccc gaccacgtgg agctgagctg gtgggtgaat 540

gggaaggagg tgcacagtgg ggtcagcaca gacccgcagc ccctcaagga gcagcccgcc 600

ctcaatgact ccagatactg cctgagcagc cgcctgaggg tctcggccac cttctggcag 660

aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 720

tggacccagg atagggccaa acctgtcacc cagatcgtca gcgccgaggc ctggggtaga 780

gcagactgtg gcttcacctc cgagtcttac cagcaagggg tcctgtctgc caccatcctc 840

tatgagatct tgctagggaa ggccaccttg tatgccgtgc tggtcagtgc cctcgtgctg 900

atggccatgg tcaagagaaa ggattccaga ggc 933

<210> 19

<211> 18

<212> DNA

<213> Artificial sequence

<400> 19

gatagcgcta tttacaac 18

<210> 20

<211> 21

<212> DNA

<213> Artificial sequence

<400> 20

attcagtcaa gtcagagaga g 21

<210> 21

<211> 33

<212> DNA

<213> Artificial sequence

<400> 21

gctgctacat caggaggaag ctacatacct aca 33

<210> 22

<211> 15

<212> DNA

<213> Artificial sequence

<400> 22

atgaaccata actac 15

<210> 23

<211> 18

<212> DNA

<213> Artificial sequence

<400> 23

tcagttggtg ctggtatc 18

<210> 24

<211> 36

<212> DNA

<213> Artificial sequence

<400> 24

gccagcagcc ttagcgggag gttgggtgag cagttc 36

<210> 25

<211> 207

<212> PRT

<213> Artificial sequence

<400> 25

Met Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val Pro Glu

1 5 10 15

Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala Ile Tyr

20 25 30

Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr Ser Leu

35 40 45

Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg Leu Asn

50 55 60

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

65 70 75 80

Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Ala Thr Ser Gly

85 90 95

Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

<210> 26

<211> 621

<212> DNA

<213> Artificial sequence

<400> 26

atgaaacagg aagttaccca gattcctgca gctctgagtg tcccagaagg agaaaacttg 60

gttctcaact gcagtttcac tgatagcgct atttacaacc tccagtggtt taggcaggac 120

cctgggaaag gtctcacatc tctgttgctt attcagtcaa gtcagagaga gcaaacaagt 180

ggaagactta atgcctcgct ggataaatca tcaggacgta gtactttata cattgcagct 240

tctcagcctg gtgactcagc cacctacctc tgtgctgcta catcaggagg aagctacata 300

cctacatttg gaagaggaac cagccttatt gttcatccgt atatccagaa ccctgaccct 360

gccgtgtacc agctgagaga ctctaagtcg agtgacaagt ctgtctgcct attcaccgat 420

tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 480

tgtgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 540

aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 600

ttccccagcc cagaaagttc c 621

<210> 27

<211> 244

<212> PRT

<213> Artificial sequence

<400> 27

Met Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Arg Ile Leu Lys Ile

1 5 10 15

Gly Gln Ser Met Thr Leu Gln Cys Thr Gln Asp Met Asn His Asn Tyr

20 25 30

Met Tyr Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Lys Leu Ile Tyr

35 40 45

Tyr Ser Val Gly Ala Gly Ile Thr Asp Lys Gly Glu Val Pro Asn Gly

50 55 60

Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Glu

65 70 75 80

Leu Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Ser Leu

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly Arg Ala Asp

<210> 28

<211> 732

<212> DNA

<213> Artificial sequence

<400> 28

atgaacgcgg gcgtgaccca gaccccaaaa ttccgcatcc tgaagatagg acagagcatg 60

acactgcagt gtacccagga tatgaaccat aactacatgt actggtatcg acaagaccca 120

ggcatggggc tgaagctgat ttattattca gttggtgctg gtatcactga taaaggagaa 180

gtcccgaatg gctacaacgt ctccagatca accacagagg atttcccgct caggctggag 240

ttggctgctc cctcccagac atctgtgtac ttctgtgcca gcagccttag cgggaggttg 300

ggtgagcagt tcttcgggcc agggacacgg ctcaccgtgc tagaggacct gaaaaacgtg 360

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 420

gccacactgg tgtgcctggc caccggtttc taccccgacc acgtggagct gagctggtgg 480

gtgaatggga aggaggtgca cagtggggtc tgcacagacc cgcagcccct caaggagcag 540

cccgccctca atgactccag atacgctctg agcagccgcc tgagggtctc ggccaccttc 600

tggcaggacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 660

gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 720

ggtagagcag ac 732

<210> 29

<211> 9

<212> PRT

<213> Artificial sequence

<400> 29

Glu Val Leu Val Asp Leu Phe Leu Lys

1 5

Claims (11)

1. a T cell receptor (TCR) is characterized in that, described TCR can be combined with EVLVDLFLK-HLA-A*1101 complex, described TCR comprises TCRα chain variable domain and TCRβ chain variable domain, described The three complementarity determining regions of the TCRα chain variable domain are: αCDR1-DSAIYN (SEQ ID NO: 10) αCDR2-IQSSQRE (SEQ ID NO: 11) αCDR3-AATSGGSYIPT (SEQ ID NO: 12); and The three complementarity determining regions of the TCRβ chain variable domain are: βCDR1-MNHNY (SEQ ID NO: 13) βCDR2-SVGAGI (SEQ ID NO: 14) βCDR3-ASSLSGRLGEQF (SEQ ID NO: 15). 2. The T cell receptor of claim 1, wherein the TCR comprises an alpha chain variable domain amino acid sequence of SEQ ID NO: 1. 3. The T cell receptor of claim 1, wherein the TCR comprises a beta chain variable domain amino acid sequence of SEQ ID NO:2. 4. The T cell receptor of claim 1, wherein the alpha chain amino acid sequence of the TCR is SEQ ID NO: 3, 5, 25; and the beta chain amino acid sequence of the TCR is SEQ ID NO: 4, 6, 27. 5. 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 of claim 1 . 6. A nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding the TCR molecule of claim 1 or a complementary sequence thereof. 7 . A vector, characterized in that, the vector contains the nucleic acid molecule of claim 6 . 8 . An isolated host cell, wherein the host cell contains the vector of claim 7 or the nucleic acid molecule of claim 6 integrated into the genome. 9 . A cell, characterized in that the cell is transduced with the nucleic acid molecule of claim 6 or the vector of claim 7 . 10. a pharmaceutical composition is characterized in that, described composition contains pharmaceutically acceptable carrier and TCR described in claim 1, TCR complex described in claim 5, nucleic acid molecule described in claim 6 , the vector of claim 7 , or the cell of claim 9 . 11. The T cell receptor of claim 1, or the TCR complex of claim 5, the nucleic acid molecule of claim 6, the vector of claim 7, or the cell of claim 9. The use is characterized in that it is used for preparing a medicament for treating tumors or autoimmune diseases.

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