CN110407926B - TCR for identifying LMP1 antigen short peptide and coding sequence thereof - Google Patents

Abstract

The present invention provides a T Cell Receptor (TCR) capable of specifically binding short peptide MLWRLGATI derived from LMP1 antigen, which antigen short peptide MLWRLGATI can form a complex with HLA a0201 and be presented together to the cell surface. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules. In addition, the invention provides cells that transduce a TCR of the invention.

Description

TCR for identifying LMP1 antigen short peptide and coding sequence thereof

Technical Field

The invention relates to a TCR capable of recognizing LMP1 antigen short peptide and a coding sequence thereof, LMP1 specific T cells obtained by transducing the TCR and application of the T cells in preventing and treating LMP1 related diseases.

Background

EBV is a human herpes virus that is ubiquitous worldwide. Studies have shown that over 95% of adult humans contain antibodies to this virus, which means that they are infected by this virus at some stage. EBV is present in most infected individuals throughout life, and is generally less problematic. However, in some cases, EBV is associated with the development of several cancers, including Burkitt's lymphoma, Hodgkin's lymphoma, EBV-positive post-transplant lymphoproliferative disorder (PTLD), nasopharyngeal cancer, or the like. For example, LMP1 belongs to the latent membrane protein of EBV, which is expressed by most nasopharyngeal carcinoma cells (Raab-Trub N. Epstein-Barr virus in the pathogenesis of NPC [ J ]. Semin Cancer Biol,2002,12(6): 431-441.). LMP1 is degraded into small molecule polypeptides after intracellular production and is presented to the cell surface as a complex with MHC (major histocompatibility complex) molecules. MLWRLGATI (SEQ ID NO:9) is a short peptide derived from LMP1 antigen, which is a target for the treatment of LMP 1-related diseases. For the treatment of the above diseases, chemotherapy, radiotherapy and the like can be used, but both of them cause damages to normal cells themselves.

T cell adoptive immunotherapy is the transfer of reactive T cells specific for a target cell antigen into a patient to act on the target cell. The T Cell Receptor (TCR) is a membrane protein on the surface of T cells that recognizes a corresponding short peptide antigen on the surface of a target cell. In the immune system, the direct physical contact between T cells and Antigen Presenting Cells (APC) is initiated by the binding of antigen short peptide specific TCR and short peptide-major histocompatibility complex (pMHC complex), and then other cell membrane surface molecules of the T cells and APC interact to cause a series of subsequent cell signaling and other physiological reactions, so that T cells with different antigen specificities exert immune effects on their target cells. Therefore, those skilled in the art have focused on isolating TCRs specific for LMP1 antigen short peptides and transducing the TCRs into T cells to obtain T cells specific for LMP1 antigen short peptides, thereby making them useful in cellular immunotherapy.

Disclosure of Invention

The invention aims to provide a T cell receptor for recognizing LMP1 antigen short peptide.

In a first aspect of the invention, there is provided a T Cell Receptor (TCR) capable of binding to the MLWRLGATI-HLA A0201 complex.

In another preferred embodiment, the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, the amino acid sequence of CDR3 of the TCR alpha chain variable domain is ALNAGAGNMLT (SEQ ID NO: 12); and/or the amino acid sequence of CDR3 of the variable domain of the TCR beta chain is SASETSGSYEQF (SEQ ID NO: 15).

In another preferred embodiment, the 3 Complementarity Determining Regions (CDRs) of the TCR α chain variable domain are:

αCDR1-TRDTTYY(SEQ ID NO:10)

αCDR2-RNSFDEQN(SEQ ID NO:11)

alpha CDR3-ALNAGAGNMLT (SEQ ID NO: 12); and/or

The 3 complementarity determining regions of the TCR β chain variable domain are:

βCDR1-DFQATT(SEQ ID NO:13)

βCDR2-SNEGSKA(SEQ ID NO:14)

βCDR3-SASETSGSYEQF(SEQ ID NO:15)。

in another preferred embodiment, the TCR comprises a TCR alpha chain variable domain which is an amino acid sequence having at least 90% sequence identity to SEQ ID No. 1, and a TCR beta chain variable domain; and/or the TCR β chain variable domain is identical to SEQ ID NO:5 an amino acid sequence having at least 90% sequence identity.

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

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

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

In another preferred embodiment, the α chain amino acid sequence of the TCR is SEQ ID NO:3 and/or the beta chain amino acid sequence of the TCR is SEQ ID NO 7.

In another preferred embodiment, the TCR is soluble.

In another preferred embodiment, the TCR is single chain.

In another preferred embodiment, the TCR is formed by linking an α chain variable domain to a β chain variable domain via a peptide linker.

In another preferred embodiment, the TCR has one or more mutations in amino acid 11, 13, 19, 21, 53, 76, 89, 91, or 94 of the α chain variable region, and/or in the penultimate 3-, 5-, or 7-position of the short peptide amino acid of the α chain J gene; and/or the TCR has one or more mutations in beta chain variable region amino acid 11, 13, 19, 21, 53, 76, 89, 91, or 94 th, and/or beta chain J gene short peptide amino acid penultimate 2,4 or 6 th, wherein the amino acid position numbering is according to the position numbering listed in IMGT (international immunogenetic information system).

In another preferred embodiment, the α chain variable domain amino acid sequence of the TCR comprises SEQ ID NO 32 and/or the β chain variable domain amino acid sequence of the TCR comprises SEQ ID NO 34.

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

In another preferred embodiment, the TCR comprises (a) all or part of a TCR α chain, excluding the transmembrane domain; and (b) all or part of a TCR β 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, the cysteine residues form an artificial disulfide bond between the alpha and beta chain constant domains of the TCR.

In another preferred embodiment, the cysteine residues forming the artificial disulfide bond in the TCR are substituted at one or more groups of sites selected from the group consisting of:

thr48 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser57 of TRBC2 × 01 exon 1;

thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser77 of TRBC2 × 01 exon 1;

tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC2 x 01;

thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1;

ser15 and TRBC1 × 01 of TRAC × 01 exon 1 or Glu15 of TRBC2 × 01 exon 1;

arg53 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser54 of TRBC2 × 01 exon 1;

pro89 and TRBC1 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01; and

tyr10 and TRBC1 × 01 of exon 1 of TRAC × 01 or Glu20 of exon 1 of TRBC2 × 01.

In another preferred embodiment, the α chain amino acid sequence of the TCR is SEQ ID NO 26 and/or the β chain amino acid sequence of the TCR is SEQ ID NO 28.

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

In another preferred embodiment, the cysteine residues that form the artificial interchain disulfide bond in the TCR replace one or more groups of sites selected from the group consisting of:

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

amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01;

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

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

In another preferred embodiment, the TCR comprises an alpha chain variable domain and a beta chain variable domain and all or part of the beta chain constant domain, excluding the transmembrane domain, but which does not comprise an alpha chain constant domain, the alpha chain variable domain of the TCR forming a heterodimer with the beta chain.

In another preferred embodiment, the TCR has a conjugate attached to the C-or N-terminus of the alpha and/or beta chain.

In another preferred embodiment, the conjugate that binds to the T cell receptor is a detectable label, a therapeutic agent, a PK modifying moiety or a combination of any of these. Preferably, the therapeutic agent is an anti-CD 3 antibody.

In a second aspect of the invention, there is provided a multivalent TCR complex comprising at least two TCR molecules, and wherein at least one of the TCR molecules is a TCR according to the first aspect of the invention.

In a third aspect of the invention, there is provided a nucleic acid molecule comprising a nucleic acid sequence encoding a TCR molecule according to the first aspect of the invention, or the complement thereof.

In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO: 33.

In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO:6 or SEQ ID NO 35.

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

in a fourth aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the third aspect of the invention; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector.

In a fifth aspect of the invention, there is provided an isolated host cell comprising a vector according to the fourth aspect of the invention or a genome into which has been integrated an exogenous nucleic acid molecule according to the third aspect of the invention.

In a sixth aspect of the invention, there is provided a cell which transduces a nucleic acid molecule according to the third aspect of the invention or a vector according to the fourth aspect of the invention; preferably, the cell is a T cell or a stem cell.

In a seventh aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to the first aspect of the invention, a TCR complex according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a cell according to the sixth aspect of the invention.

In an eighth aspect, the invention provides the use of a T cell receptor according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a cell according to the sixth aspect of the invention, for the manufacture of a medicament for the treatment of a tumour or an autoimmune disease.

In a ninth aspect, the invention provides a method of treating a disease comprising administering to a subject in need thereof an amount of a T cell receptor according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a cell according to the sixth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention;

preferably, the disease is a tumor, preferably the tumor is hepatocellular carcinoma.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1a, FIG. 1b, FIG. 1c, FIG. 1d, FIG. 1e and FIG. 1f are the amino acid sequence of the TCR α chain variable domain, the nucleotide sequence of the TCR α chain variable domain, the amino acid sequence of the TCR α chain, the nucleotide sequence of the TCR α chain, the amino acid sequence of the TCR α chain with leader sequence and the nucleotide sequence of the TCR α chain with leader sequence, respectively.

Fig. 2a, fig. 2b, fig. 2c, fig. 2d, fig. 2e and fig. 2f are a TCR β chain variable domain amino acid sequence, a TCR β chain variable domain nucleotide sequence, a TCR β chain amino acid sequence, a TCR β chain nucleotide sequence with a leader sequence, and a TCR β chain nucleotide sequence with a leader sequence, respectively.

FIG. 3 is CD8 of monoclonal cells ⁺ And tetramer-PE double positive staining results.

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

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

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

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

FIGS. 8a and 8b are the amino acid and nucleotide sequences, respectively, of the variable domain of the single chain TCR α chain.

Figure 9a and figure 9b are the amino acid and nucleotide sequences, respectively, of the variable domain of the single-chain TCR β chain.

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

FIG. 11 is a gel diagram of the soluble single chain TCR obtained after purification. The left lane is the molecular weight marker (marker) and the right lane is the non-reducing gel.

FIG. 12 is a BIAcore kinetic profile of binding of soluble TCRs of the invention to the MLWRLGATI-HLA A0201 complex.

FIG. 13 is a BIAcore kinetic profile of binding of soluble single chain TCRs of the invention to the MLWRLGATI-HLA A0201 complex.

FIG. 14 shows the results of functional verification of the ELISPOT activation of the resulting T cell clones.

FIG. 15 is a graphical representation of the results of functional confirmation of ELISPOT activation of effector cells transduced with the TCRs of the invention.

Detailed Description

The present inventors have made extensive and intensive studies to find a TCR capable of specifically binding to LMP1 antigen short peptide MLWRLGATI (SEQ ID NO:9), which antigen short peptide MLWRLGATI can form a complex with HLA A0201 and be presented together on the cell surface. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules. In addition, the invention provides cells that transduce a TCR of the invention.

Term(s) for

MHC molecules are proteins of the immunoglobulin superfamily, which may be MHC class I or class II molecules. Therefore, it is specific for antigen presentation, has different MHC among different individuals, and can present different short peptides of one protein antigen onto the cell surface of respective APC. Human MHC is commonly referred to as an HLA gene or HLA complex.

The T Cell Receptor (TCR), is the only receptor for a specific antigenic peptide presented on the Major Histocompatibility Complex (MHC). In the immune system, direct physical contact between T cells and Antigen Presenting Cells (APCs) is initiated by binding of antigen-specific TCRs to pMHC complexes, and then other cell membrane surface molecules of both T cells and APCs interact, which leads to a series of subsequent cell signaling and other physiological responses, thereby allowing T cells of different antigen specificities to exert an immune effect on their target cells.

TCRs are cell membrane surface glycoproteins that exist as heterodimers from either the α chain/β chain or the γ chain/δ chain. In 95% of T cells the TCR heterodimer consists of α and β chains, while 5% of T cells have TCRs consisting of γ and δ chains. Native α β heterodimeric TCRs have an α chain and a β chain, which constitute subunits of an α β heterodimeric TCR. Broadly, each of the α and β chains comprises a variable region, a linker region and a constant region, and the β chain also typically contains a short diversity region between the variable region and the linker region, but the diversity region is often considered to be part of the linker region. Each variable region comprises 3 CDRs (complementarity determining regions) CDR1, CDR2 and CDR3, which are chimeric in framework structures (framework regions). The CDR regions determine the binding of the TCR to the pMHC complex, where CDR3 is recombined from variable and connecting regions, referred to as hypervariable regions. The α and β chains of a TCR are generally regarded as having two "domains" each, namely a variable domain and a constant domain, the variable domain being made up of linked variable regions and linking regions. The sequences of TCR constant domains can be found in public databases of the international immunogenetic information system (IMGT), such as the constant domain sequence of the α chain of the TCR molecule is "TRAC 01", the constant domain sequence of the β chain of the TCR molecule is "TRBC 1 01" or "TRBC 2 01". In addition, the α and β chains of the TCR also comprise a transmembrane region and a cytoplasmic region, the cytoplasmic region being very short.

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

Natural interchain disulfide bond and artificial interchain disulfide bond

A set of disulfide bonds, referred to herein as "native interchain disulfide bonds," exist between the C α and C β chains of the membrane proximal region of native TCRs. 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 an "artificial interchain disulfide bond".

For convenience of description of the positions of disulfide bonds, the positions of the amino acid sequences of TRAC 01 and TRBC1 × 01 or TRBC2 × 01 are numbered in the order from the N-terminus to the C-terminus, such as in TRBC1 × 01 or TRBC2 × 01, and the 60 th amino acid in the order from the N-terminus to the C-terminus is P (proline), and thus in the present invention it can be described as Pro60 of TRBC1 × 01 or TRBC2 × 01 exon 1, and also as the 60 th amino acid of TRBC1 × 01 or TRBC2 × 01 exon 1, and as in 737bc 3 × 01 or TRBC2 × 01, and the 61 th amino acid in the order from the N-terminus to the C-terminus is Q (glutamine), and thus in the present invention it can be described as TRBC1 × 01 or TRBC 6301 × 01, or TRBC 8501, and similarly as TRBC 8261 or glbc 891. In the present invention, the position numbering of the amino acid sequences of the variable regions TRAV and TRBV follows the position numbering listed in IMGT. If an amino acid in TRAV, the position listed in IMGT is numbered 46, it is described herein as the 46 th amino acid of TRAV, and so on. In the present invention, the sequence position numbers of other amino acids are specifically described.

Detailed Description

TCR molecules

During antigen processing, antigens are degraded within cells and then carried to the cell surface by MHC molecules. T cell receptors are capable of recognizing peptide-MHC complexes on the surface of antigen presenting cells. Accordingly, a first aspect of the invention provides a TCR molecule capable of binding MLWRLGATI-HLA a0201 complex. Preferably, the TCR molecule is isolated or purified. The α and β chains of the TCR each have 3 Complementarity Determining Regions (CDRs).

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

αCDR1-TRDTTYY(SEQ ID NO:10)

αCDR2-RNSFDEQN(SEQ ID NO:11)

alpha CDR3-ALNAGAGNMLT (SEQ ID NO: 12); and/or

The 3 complementarity determining regions of the TCR β chain variable domain are:

βCDR1-DFQATT(SEQ ID NO:13)

βCDR2-SNEGSKA(SEQ ID NO:14)

βCDR3-SASETSGSYEQF(SEQ ID NO:15)。

chimeric TCRs can be prepared by embedding the above-described amino acid sequences of the CDR regions of the invention into any suitable framework. One skilled in the art can design or synthesize a TCR molecule with a corresponding function based on the CDR regions disclosed herein, provided that the framework structure is compatible with the CDR regions of the TCR of the invention. Thus, the TCR molecules of the invention are those which comprise the above-described α and/or β chain CDR region sequences and any suitable framework structure. The TCR α chain variable domain of the 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 TCR β chain variable domain of the invention is identical to SEQ ID NO:5, having at least 90%, preferably 95%, more preferably 98% sequence identity.

In a preferred embodiment of the invention, the TCR molecules of the invention are heterodimers consisting of α and β chains. In particular, in one aspect the α chain of the heterodimeric TCR molecules comprises a variable domain and a constant domain, the α chain variable domain amino acid sequence comprising CDR1(SEQ ID NO: 10), CDR2(SEQ ID NO: 11) and CDR3(SEQ ID NO:12) of the above-described α chain. Preferably, the TCR molecule comprises an alpha chain variable domain amino acid sequence 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. In another aspect, the β chain of the heterodimeric TCR molecules comprises a variable domain and a constant domain, and the β chain variable domain amino acid sequence comprises the CDR1(SEQ ID NO:13), CDR2(SEQ ID NO: 14), and CDR3(SEQ ID NO:15) of the above-described β chain. Preferably, the TCR molecule comprises the beta chain variable domain amino acid sequence SEQ ID NO 5. More preferably, the amino acid sequence of the variable domain of the β chain of the TCR molecule is SEQ ID No. 5.

In a preferred embodiment of the invention, the TCR molecules of the invention are single chain TCR molecules consisting of part or all of the α chain and/or part or all of the β chain. Single chain TCR molecules are described in Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-. One skilled in the art can readily construct single chain TCR molecules comprising the CDRs regions of the present invention, based on the teachings in the literature. In particular, the single chain TCR molecule comprises V α, V β and C β, preferably linked in order from N-terminus to C-terminus.

The alpha chain variable domain amino acid sequence of the single chain TCR molecule comprises the CDR1(SEQ ID NO: 10), CDR2(SEQ ID NO: 11) and CDR3(SEQ ID NO:12) of the alpha chain described above. Preferably, the single chain TCR molecule comprises an alpha chain variable domain amino acid sequence SEQ ID NO 1. More preferably, the α chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO 1. The amino acid sequence of the beta chain variable domain of the single chain TCR molecule comprises the CDR1(SEQ ID NO:13), CDR2(SEQ ID NO: 14) and CDR3(SEQ ID NO:15) of the above-described beta chain. Preferably, the single chain TCR molecule comprises a beta chain variable domain amino acid sequence SEQ ID NO 5. More preferably, the amino acid sequence of the β chain variable domain of the single chain TCR molecule is SEQ ID NO 5.

In a preferred embodiment of the invention, the constant domain of the TCR molecules of the invention is a human constant domain. The human constant domain amino acid sequences are known to those skilled in the art or can be obtained by consulting published databases of relevant books or IMGT (international immunogenetic information system). For example, the constant domain sequence of the α chain of the TCR molecules of the invention can be "TRAC 01", and the constant domain sequence of the β chain of the TCR molecules can be "TRBC 1 01" or "TRBC 2 01". Arg at position 53 of the amino acid sequence given in TRAC 01 of IMGT, here denoted: TRAC × 01 Arg53 of exon 1, and so on. Preferably, the amino acid sequence of the α chain of the TCR molecules of the invention is SEQ ID No. 3, and/or the amino acid sequence of the β chain is SEQ ID No. 7.

Naturally occurring TCRs are membrane proteins that are stabilized by their transmembrane regions. Like immunoglobulins (antibodies) as antigen recognition molecules, TCRs can also be developed for diagnostic and therapeutic applications, where it is desirable to obtain soluble TCR molecules. Soluble TCR molecules do not include their transmembrane regions. Soluble TCRs have a wide range of uses, not only for studying the interaction of TCRs with pmhcs, but also as diagnostic tools for detecting infection or as markers for autoimmune diseases. Similarly, soluble TCRs can be used to deliver therapeutic agents (e.g., cytotoxic or immunostimulatory compounds) to cells presenting a specific antigen, and in addition, soluble TCRs can be conjugated to other molecules (e.g., anti-CD 3 antibodies) to redirect T cells to target them to cells presenting a particular antigen. The present invention also obtains soluble TCRs specific for LMP1 antigen short peptides.

To obtain a soluble TCR, in one aspect, the inventive TCR may be one in which an artificial disulfide bond is introduced between residues of the constant domains of its alpha and beta chains. Cysteine residues form an artificial interchain disulfide bond between the alpha and beta chain constant domains of the TCR. Cysteine residues may be substituted for other amino acid residues at appropriate positions in native TCRs to form artificial interchain disulfide bonds. For example, a disulfide bond is formed by substituting Thr48 of TRAC × 01 exon 1 and a cysteine residue of Ser57 of TRBC1 × 01 or TRBC2 × 01 exon 1. Other sites for introducing cysteine residues to form disulfide bonds may also be: thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser77 of TRBC2 × 01 exon 1; tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC2 x 01; thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1; ser15 and TRBC1 × 01 of TRAC × 01 exon 1 or Glu15 of TRBC2 × 01 exon 1; arg53 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser54 of TRBC2 × 01 exon 1; pro89 and TRBC1 x 01 of TRAC x 01 exon 1 or Ala19 of TRBC2 x 01 exon 1; or Tyr10 and TRBC1 x 01 of TRAC x 01 exon 1 or TRBC2 x 01 of Glu20 of exon 1. I.e., a cysteine residue, in place of any of the above-described alpha and beta chain constant domains. The TCR constant domains of the invention may be truncated at one or more of their C-termini 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 as not to include a cysteine residue for the purpose of deleting the native disulphide bond, or by mutating the cysteine residue forming the native disulphide bond to another amino acid.

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

To obtain a soluble TCR, on the other hand, the inventive TCR also comprises a TCR having a mutation in its hydrophobic core region, preferably a mutation that enables an improved stability of the inventive soluble TCR, as described in the patent publication WO 2014/206304. Such TCRs may be mutated at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or positions 3,5,7 of the reciprocal amino acid position of the short peptide of the alpha chain J gene (TRAJ), and/or positions 2,4,6 of the reciprocal amino acid position of the short peptide of the beta chain J gene (TRBJ), wherein the position numbering of the amino acid sequence is according to the position numbering listed in the International immunogenetic information System (IMGT). The above-mentioned international system of immunogenetics information is known to the skilled person and the position numbering of the amino acid residues of the different TCRs in IMGT can be derived from this database.

The TCR with the mutated hydrophobic core region of the invention can be a stable soluble single chain TCR formed by connecting the variable domains of the alpha and beta chains of the TCR by a flexible peptide chain. It should be noted that the flexible peptide chain of the present invention can be any peptide chain suitable for linking the TCR α and β chain variable domains. The single-chain soluble TCR constructed in the embodiment 4 of the invention has the alpha chain variable domain amino acid sequence of SEQ ID NO. 32 and the encoded nucleotide sequence of SEQ ID NO. 33; the amino acid sequence of the beta chain variable domain is SEQ ID NO. 34, and the coded nucleotide sequence is SEQ ID NO. 35.

In addition, for stability, patent document 201510260322.4 also discloses that the introduction of an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR can significantly improve the stability of the TCR. Thus, the high affinity TCRs of the invention may also contain an artificial interchain disulfide bond between the α chain variable region and the β chain constant region. Specifically, the cysteine residues that form the artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR are substituted for: amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01; amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01; amino acid 46 of TRAV and amino acid 61 of TRBC1 x 01 or TRBC2 x 01 exon 1; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01. Preferably, such a TCR may comprise (i) all or part of a TCR α chain, excluding its transmembrane domain, and (ii) all or part of a TCR β chain, excluding its transmembrane domain, wherein (i) and (ii) both comprise a variable domain and at least part of a constant domain of the TCR chain, the α chain forming a heterodimer with the β chain. More preferably, such a TCR may comprise the a chain variable domain and the β chain variable domain and all or part of the β chain constant domain, excluding the transmembrane domain, but which does not comprise the a chain constant domain, the a chain variable domain of the TCR forming a heterodimer with the β chain.

The TCRs of the invention may also be provided in the form of multivalent complexes. Multivalent TCR complexes of the invention comprise polymers formed by binding two, three, four or more TCRs of the invention, such as might be formed by using the tetrameric domain of p53 to create a tetramer, or a complex of multiple TCRs of the invention bound to another molecule. The TCR complexes of the invention can be used to track or target cells presenting a particular antigen in vitro or in vivo, and also to generate intermediates for other multivalent TCR complexes having such applications.

The TCRs of the invention may be used alone or in covalent or other association, preferably covalently, with a conjugate. The conjugates include a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the MLWRLGATI-HLA a0201 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.

Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.

Therapeutic agents that may be associated or conjugated with the TCRs of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, Cancer metastasis reviews (Cancer metastasis) 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, Proc. Natl. Acad. Sci. USA (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, 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 11, 1234); 8. liposomes (Mamot et al, 2005, Cancer research)65, 11631); 9. nano magnetic particles; 10. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, and the like.

In addition, the TCRs of the 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 inventive TCRs may comprise a human variable domain and a murine constant domain. The drawback of this approach is the possibility of eliciting an immune response. Therefore, there should be a regulatory scheme for immunosuppression when it is used for adoptive T cell therapy to allow for the engraftment of murine expressing T cells.

It should be understood that the amino acid names herein are expressed in terms of international single-letter or three-letter english letters, and the single-letter english letter and three-letter english letters of the amino acid names correspond to the following relationships: 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) and Val (V).

Nucleic acid molecules

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

The nucleotide sequence encoding the α chain CDR regions of the TCR molecules of the first aspect of the invention is as follows:

αCDR1-acccgtgatactacttattac(SEQ ID NO:16)

αCDR2-cggaactcttttgatgagcaaaat(SEQ ID NO:17)

αCDR3-gctctgaacgccggggcaggcaacatgctcacc(SEQ ID NO:18)

the nucleotide sequence encoding the β chain CDR regions of the TCR molecules of the first aspect of the invention is as follows:

βCDR1-gactttcaggccacaact(SEQ ID NO:19)

βCDR2-tccaatgagggctccaaggcc(SEQ ID NO:20)

βCDR3-agtgctagtgagactagcgggagctatgagcagttc(SEQ ID NO:21)

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

The nucleotide sequence of the nucleic acid molecule of the invention may be single-stranded or double-stranded, the nucleic acid molecule may be RNA or DNA, and may or may not comprise an intron. Preferably, the nucleotide sequence of the nucleic acid molecule of the invention does not comprise an intron but is capable of encoding a polypeptide of the invention, e.g. the nucleotide sequence of the nucleic acid molecule of the invention encoding a TCR alpha chain variable domain of the invention comprises SEQ ID NO 2 and/or the nucleotide sequence of the nucleic acid molecule of the invention encoding a TCR beta chain variable domain of the invention comprises SEQ ID NO 6. Alternatively, the nucleotide sequence of a nucleic acid molecule of the invention encoding a TCR α chain variable domain of the invention comprises SEQ ID NO 33 and/or the nucleotide sequence of a nucleic acid molecule of the invention encoding a TCR β chain variable domain of the invention comprises SEQ ID NO 35. More preferably, the nucleotide sequence of the nucleic acid molecule of the invention comprises SEQ ID NO. 4 and/or SEQ ID NO. 8. Alternatively, the nucleotide sequence of the nucleic acid molecule of the invention is SEQ ID NO. 31.

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

The nucleotide sequence may be codon optimized. Different cells differ in the utilization of specific codons, and the expression level can be increased by changing the codons in the sequence according to the type of the cell. Codon usage tables for mammalian cells as well as for 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 be obtained by, but not limited to, PCR amplification, recombination or artificial synthesis. At present, DNA sequences encoding the TCRs of the invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. The DNA may be the coding strand or the non-coding strand.

Carrier

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

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

Preferably, the vector can transfer the nucleotide of the invention into a cell, e.g., a T cell, such that the cell expresses a TCR specific for the LMP1 antigen. Ideally, the vector should be capable of sustained high level expression in T cells.

Cells

The invention also relates to genetically engineered host cells that have been engineered with the vectors or coding sequences of the invention. The host cell comprises a vector of the invention or has integrated into its chromosome a nucleic acid molecule of the invention. The host cell is selected from: prokaryotic and eukaryotic cells, such as E.coli, yeast cells, CHO cells, and the like.

In addition, the invention also includes isolated cells, particularly T cells, that express the TCRs of the invention. The T cell may be derived from a T cell isolated from a subject, or may be part of a mixed population of cells isolated from a subject, such as a population of Peripheral Blood Lymphocytes (PBLs). For example, the cells may be isolated from Peripheral Blood Mononuclear Cells (PBMC), which may be CD4 ⁺ Helper T cell or CD8 ⁺ Cytotoxic T cells. The cell may be in CD4 ⁺ Helper T cell/CD 8 ⁺ A mixed population of cytotoxic T cells. Generally, the cells can be activated with an antibody (e.g., an anti-CD 3 or anti-CD 28 antibody) to render them more amenable to transfection, e.g., transfection with a vector comprising a nucleotide sequence encoding a TCR molecule of the invention.

Alternatively, the cell of the invention may also be or be derived from a stem cell, such as a Hematopoietic Stem Cell (HSC). Gene transfer to HSCs does not result in TCR expression on the cell surface, since the CD3 molecule is not expressed on the stem cell surface. However, when stem cells differentiate into lymphoid precursors (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 many methods suitable for T cell transfection using DNA or RNA encoding the TCR of the invention (e.g., Robbins et al, (2008) J.Immunol.180: 6116-. T cells expressing the TCRs of the invention may be used for adoptive immunotherapy. Those skilled in the art will be able to recognize many suitable methods for adoptive therapy (e.g., Rosenberg et al, (2008) Nat Rev Cancer8 (4): 299-308).

LMP1 antigen-related diseases

The present invention also relates to a method of treating and/or preventing a disease associated with LMP1 in a subject comprising the step of adoptively transferring LMP1 specific T cells to the subject. The LMP 1-specific T cells recognized MLWRLGATI-HLA A0201 complex.

The LMP1 specific T cells of the invention can be used for treating any LMP1 related diseases presenting LMP1 antigen short peptide MLWRLGATI-HLA A0201 complex. Including but not limited to tumors such as nasopharyngeal carcinoma, burkitt's lymphoma, hodgkin's lymphoma, and the like.

Method of treatment

Treatment may be effected by isolating T cells from patients or volunteers suffering from a disease associated with the LMP1 antigen and introducing the TCR of the invention into such T cells, followed by reinfusion of these genetically engineered cells into the patient. Accordingly, the present invention provides a method of treating a LMP 1-related disease comprising infusing into a patient an isolated T cell expressing a TCR of the invention, preferably the T cell is derived from the patient himself. Generally, this involves (1) isolating T cells from the 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 the genetically modified T cells into the patient. The number of cells isolated, transfected and transfused can be determined by a physician.

The main advantages of the invention are:

(1) the TCR can be combined with LMP1 antigen short peptide complex MLWRLGATI-HLA A0201, and cells transduced with the TCR can be specifically activated and have strong killing effect on target cells.

The following specific examples further illustrate the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example as described in Sambrook and Russel et al, Molecular Cloning: A Laboratory Manual (third edition) (2001) CSHL Press, or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

Example 1 cloning of LMP1 antigen short peptide specific T cells

Peripheral Blood Lymphocytes (PBLs) from healthy volunteers of genotype HLA-A0201 were stimulated with synthetic short peptide MLWRLGATI (SEQ ID NO.: 9; Baisheng Gene technologies, Inc., Beijing Sai). MLWRLGATI short peptide and HLA-A0201 with biotin label are renatured to prepare pHLA haploid. These haploids combined with streptavidin labeled with PE (BD Co.) to form PE-labeled tetramers, which were sorted from anti-CD 8-APC double positive cells. The sorted cells were expanded and subjected to secondary sorting as described above, followed by single cloning by limiting dilution. Monoclonal cells were stained with tetramer and double positive clones were selected as shown in FIG. 3. The double positive clones obtained by screening layer by layer also need to meet the requirement of further function test.

The function and specificity of the T cell clone were further tested by ELISPOT assay. Methods for detecting cell function using the ELISPOT assay are well known to those skilled in the art. The effector cells used in the IFN-gamma ELISPOT experiment in this example were T cell clones obtained in the present invention, the target cells were T2 cells loaded with the short peptide of the present invention, and the control group were T2 cells loaded with other short peptides and T2 cells not loaded with any short peptide.

Firstly, preparing an ELISPOT plate, wherein the ELISPOT experiment steps are as follows: the components of the assay were added to the ELISPOT plate in the following order: 40 μ l T2 cells 5X 10 ⁵ After 40. mu.l of effector cells (2000T cell clones/well) per ml of cells (i.e.20,000T 2 cells/well), 20. mu.l of specific short peptide was added to the experimental group, 20. mu.l of nonspecific short peptide was added to the control group, 20. mu.l of medium (test medium) was added to the blank group, and 2 replicate wells were set. Then incubated overnight (37 ℃, 5% CO) ₂ ). The plates were then washed and subjected to secondary detection and color development, the plates were dried for 1 hour, and spots formed on the membrane were counted using an immuno-spot plate READER (ELISPOT READER system; AID Co.). As shown in FIG. 14, the obtained T cell clone specific to a specific antigen showed a specific response to T2 cells loaded with the short peptide of the present invention, but showed no substantial response to T2 cells loaded with other unrelated peptides and unloaded with the short peptide.

Example 2 construction of TCR Gene and vector for obtaining LMP1 antigen short peptide specific T cell clone

Using Quick-RNA ^TM MiniPrep (ZYMO research) extracted the total RNA of the antigen short peptide MLWRLGATI-specific HLA-A0201-restricted T cell clone selected in example 1. cDNA was synthesized using the SMART RACE cDNA amplification kit from clontech, using primers designed to preserve the C-terminal region of the human TCR gene. The sequences were cloned into the T vector (TAKARA) and sequenced. It should be noted that the sequence is a complementary sequence, not including introns. The alpha chain and beta chain sequence structures of the TCR expressed by the double positive clone are respectively shown in figure 1 and figure 2 by sequencing, and figure 1a, figure 1b, figure 1c, figure 1d, figure 1e and figure 1f are respectively a TCR alpha chain variable domain amino acid sequence, a TCR alpha chain variable domain nucleotide sequence, a TCR alpha chain amino acid sequence, a TCR alpha chain nucleotide sequence, a TCR alpha chain amino acid sequence with a leader sequence and a TCR alpha chain nucleotide sequence with the leader sequence; fig. 2a, fig. 2b, fig. 2c, fig. 2d, fig. 2e and fig. 2f are a TCR β 0 chain variable domain amino acid sequence, a TCR β 1 chain variable domain nucleotide sequence, a TCR β 2 chain amino acid sequence, a TCR β 3 chain nucleotide sequence, a TCR β chain amino acid sequence with a leader sequence and a TCR β chain nucleotide sequence with a leader sequence, respectively.

The alpha chain was identified to comprise CDRs with the following amino acid sequences:

αCDR1-TRDTTYY(SEQ ID NO:10)

αCDR2-RNSFDEQN(SEQ ID NO:11)

αCDR3-ALNAGAGNMLT(SEQ ID NO:12)

the beta chain comprises CDRs having the amino acid sequences:

βCDR1-DFQATT(SEQ ID NO:13)

βCDR2-SNEGSKA(SEQ ID NO:14)

βCDR3-SASETSGSYEQF(SEQ ID NO:15)。

the full length genes for the TCR α and β chains were cloned into the lentiviral expression vector pllenti (addendum) by overlap (overlap) PCR, respectively. The method specifically comprises the following steps: the TCR alpha chain and the TCR beta chain are connected by overlap PCR to obtain the TCR alpha-2A-TCR beta segment. And (3) carrying out enzyme digestion and connection on the lentivirus expression vector and the TCR alpha-2A-TCR beta to obtain pLenti-TRA-2A-TRB-IRES-NGFR plasmid. As a control, a lentiviral vector pLenti-eGFP expressing eGFP was also constructed. The pseudovirus was then packaged again at 293T/17.

Example 3 expression, refolding and purification of LMP1 antigen short peptide specific soluble TCR

To obtain soluble TCR molecules, the α and β chains of the TCR molecules of the invention may comprise only the variable and part of the constant domains thereof, respectively, and a cysteine residue has been introduced into the constant domains of the α and β chains, respectively, to form artificial interchain disulfide bonds, at the positions Thr48 of TRAC × 01 exon 1 and Ser57 of TRBC2 × 01 exon 1, respectively; the amino acid sequence and the nucleotide sequence of the alpha chain are respectively shown in figure 4a and figure 4b, and the amino acid sequence and the nucleotide sequence of the beta chain are respectively shown in figure 5a and figure 5 b. The above-mentioned gene sequences of interest for the TCR alpha and beta chains were synthesized and inserted into the expression vector pET28a + (Novagene) by standard methods described in Molecular Cloning A Laboratory Manual (third edition, Sambrook and Russell), and the upstream and downstream Cloning sites were NcoI and NotI, respectively. The insert was confirmed by sequencing without error.

The expression vectors of TCR alpha and beta chains are respectively transformed into expression bacteria BL21(DE3) by a chemical transformation method, and the bacteria grow by LB culture solution and OD ₆₀₀ Inclusion bodies formed after expression of the α and β chains of the TCR were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution several times at 0.6 final induction with final concentration of 0.5mM IPTG, and finally dissolved in 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT),10mM ethylenediaminetetraacetic acid (EDTA),20mM Tris (pH 8.1).

The TCR α and β chains after lysis were separated by 1: 1 was rapidly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1),3.7mM cystamine,6.6mM β -mercaptamine (4 ℃) to a final concentration of 60 mg/mL. After mixing, the solution was dialyzed against 10 volumes of deionized water (4 ℃ C.) and after 12 hours, the deionized water was changed to a buffer (20mM Tris, pH 8.0) and dialysis was continued at 4 ℃ for 12 hours. The solution after completion of dialysis was filtered through a 0.45. mu.M filter and then purified by an anion exchange column (HiTrap Q HP,5ml, GE Healthcare). The TCR eluted with peaks containing successfully renatured α and β dimers was confirmed by SDS-PAGE gel. The TCR was subsequently further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was greater than 90% as determined by SDS-PAGE and the concentration was determined by BCA. The SDS-PAGE gel of the soluble TCR of the invention is shown in FIG. 6.

Example 4 Generation of soluble Single chain TCR specific for short peptides of LMP1 antigen

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

The target gene was digested simultaneously with Nco I and Not I, and ligated with pET28a vector digested simultaneously with Nco I and Not I. The ligation product was transformed into e.coli DH5 α, coated with LB plates containing kanamycin, inverted cultured overnight at 37 ℃, positive clones were selected for PCR screening, positive recombinants were sequenced, and after the correct sequence was determined, recombinant plasmids were extracted and transformed into e.coli BL21(DE3) for expression.

Example 5 expression, renaturation and purification of a soluble Single chain TCR specific for the LMP1 antigen short peptide

The BL21(DE3) colony containing the recombinant plasmid pET28 a-template strand prepared in example 4 was inoculated in its entirety into LB medium containing kanamycin, cultured at 37 ℃ to OD600 of 0.6 to 0.8, IPTG was added to a final concentration of 0.5mM, and the culture was continued at 37 ℃ for 4 hours. The cell pellet was harvested by centrifugation at 5000rpm for 15min, the cell pellet was lysed using Bugbuster Master Mix (Merck), the inclusion bodies were recovered by centrifugation at 6000rpm for 15min, washed with Bugbuster (Merck) to remove cell debris and membrane components, and the inclusion bodies were collected by centrifugation at 6000rpm for 15 min. The inclusion bodies were dissolved in buffer (20mM Tris-HCl pH 8.0,8M urea), the insoluble material was removed by high speed centrifugation, the supernatant was quantitated by BCA method and split charged, and stored at-80 ℃ for further use.

To 5mg of solubilized single-chain TCR inclusion body protein, 2.5mL of buffer (6M Gua-HCl, 50mM Tris-HCl pH 8.1, 100mM NaCl, 10mM EDTA) was added, DTT was added to a final concentration of 10mM, and treatment was carried out at 37 ℃ for 30 min. The treated single-chain TCR was added dropwise to 125mL of renaturation buffer (100mM Tris-HCl pH 8.1,0.4M L-arginine, 5M urea, 2mM EDTA,6.5mM beta-mercaptoethylamine, 1.87mM Cystamine) with a syringe, stirred at 4 ℃ for 10min, and then the renaturation solution was filled into a cellulose membrane dialysis bag with a cut-off of 4kDa, and the bag was placed in 1L of precooled water and stirred slowly at 4 ℃ overnight. After 17 hours, the dialysate was changed to 1L of pre-chilled buffer (20mM Tris-HCl pH 8.0), dialysis was continued for 8h at 4 ℃ and then the dialysate was changed to the same fresh buffer and dialysis was continued overnight. After 17 hours, the sample was filtered through a 0.45 μ M filter, vacuum degassed, passed through an anion exchange column (HiTrap Q HP, GE Healthcare), protein purified by a linear gradient of 0-1M NaCl in 20mM Tris-HCl pH 8.0, the fractions collected were subjected to SDS-PAGE analysis, fractions containing single-chain TCR concentrated and then further purified by a gel filtration column (Superdex 7510/300, GE Healthcare), the target fraction was also subjected to SDS-PAGE analysis.

The eluted fractions for BIAcore analysis were further tested for purity by gel filtration. The conditions are as follows: the chromatographic column Agilent Bio SEC-3(300A, phi 7.8X 300mM) and the mobile phase are 150mM phosphate buffer solution, the flow rate is 0.5mL/min, the column temperature is 25 ℃, and the ultraviolet detection wavelength is 214 nm.

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

Example 6 binding characterization

BIAcore analysis

This example demonstrates that soluble TCR molecules of the invention are capable of specifically binding to the MLWRLGATI-HLA a0201 complex.

Binding activity of the TCR molecules obtained in examples 3 and 5 to the MLWRLGATI-HLA A0201 complex was measured using a BIAcore T200 real-time assay system. The coupling process was completed by adding an anti-streptavidin antibody (GenScript) to a coupling buffer (10mM sodium acetate buffer, pH 4.77), then flowing the antibody through a CM5 chip previously activated with EDC and NHS to immobilize the antibody on the chip surface, and finally blocking the unreacted activated surface with ethanolamine hydrochloride solution to complete the coupling at a level of about 15,000 RU.

Low concentration of streptavidin was flowed over the surface of the antibody-coated chip, then MLWRLGATI-HLA A0201 complex was flowed through the detection channel, the other channel served as a reference channel, and 0.05mM biotin was flowed through the chip at a flow rate of 10. mu.L/min for 2min, blocking the remaining binding sites of streptavidin.

The MLWRLGATI-HLA A0201 complex is prepared as follows:

a. purification of

Collecting 100ml E.col i bacterial liquid for inducing expression of heavy chain or light chain, centrifuging at 4 ℃ for 10min at 8000g, washing the thallus once with 10ml PBS, then resuspending the thallus with 5ml BugBuster Master Mix Extraction Reagents (Merck) by vigorous shaking, rotatably incubating at room temperature for 20min, centrifuging at 4 ℃ for 15min at 6000g, discarding supernatant, and collecting inclusion body.

Resuspending the inclusion bodies in 5ml of BugBuster Master Mix, and rotary incubating at room temperature for 5 min; adding 30ml of 10-fold diluted BugBuster, uniformly mixing, and centrifuging at 4 ℃ at 6000g for 15 min; discarding supernatant, adding 30ml of 10-fold diluted BugBuster to resuspend the inclusion bodies, mixing, centrifuging at 4 ℃ for 15min, repeating twice, adding 30ml of 20mM Tris-HCl pH 8.0 to resuspend the inclusion bodies, mixing, centrifuging at 4 ℃ for 15min at 6000g, finally dissolving the inclusion bodies by 20mM Tris-HCl 8M urea, detecting the purity of the inclusion bodies by SDS-PAGE, and detecting the concentration by a BCA kit.

b. Renaturation

The synthesized short peptide MLWRLGATI (Beijing Baisheng Gene technology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg/ml. Inclusion of light and heavy chains was solubilized with 8M Urea, 20mM Tris pH 8.0, 10mM DTT and further denatured by addition of 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA prior to renaturation. MLWRLGATI peptide was added at 25mg/L (final concentration) to a renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidative glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4 ℃), followed by the addition of 20mg/L of light chain and 90mg/L of heavy chain in sequence (final concentration, heavy chain was added in three portions, 8 h/time), and renaturation was performed at 4 ℃ for at least 3 days until completion, and SDS-PAGE was checked for success or failure of renaturation.

c. Purification after renaturation

The renaturation buffer was exchanged by dialysis against 10 volumes of 20mM Tris pH 8.0, at least twice to reduce the ionic strength of the solution sufficiently. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and then loaded onto a HiTrap Q HP (GE general electric) anion exchange column (5ml bed volume). The protein was eluted using an Akta purifier (GE general electric) with a 0-400mM NaCl linear gradient prepared in 20mM Tris pH 8.0, pMHC was eluted at about 250mM NaCl, the peak fractions were collected, and the purity was checked by SDS-PAGE.

d. Biotinylation of the compound

The purified pMHC molecules were concentrated using a Mill ipore ultrafiltration tube while replacing the buffer with 20mM Tris pH 8.0, followed by addition of biotinylation reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50. mu. M D-Biotin, 100. mu.g/ml BirA enzyme (GST-BirA), incubation of the mixture overnight at room temperature, and SDS-PAGE to determine the completion of biotinylation.

e. Purification of biotinylated complexes

The biotinylated pMHC molecules were concentrated to 1ml using Millipore ultrafiltration tubes, the biotinylated pMHC was purified by gel filtration chromatography, and HiPrep was pre-equilibrated with filtered PBS using Akta purifier (GE general electric Co., Ltd.) ^TM 16/60S 200HR column (GE general electric) was loaded with 1ml of concentrated biotinylated pMHC molecules and then eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules appeared as a unimodal elution at approximately 55 ml. The fractions containing the protein were pooled, concentrated using a Mill ipore ultrafiltration tube, protein concentration was determined by BCA (Thermo), and biotinylated pMHC molecules were stored in aliquots at-80 ℃ with the addition of the protease inhibitor cocktail (Roche).

Kinetic parameters were calculated by BIAcore Evaluation software, and kinetic profiles of the soluble TCR molecules of the invention and the binding of the soluble single-chain TCR molecules constructed by the invention to MLWRLGATI-HLA A0201 complex were obtained as shown in FIGS. 12 and 13, respectively. The maps show that both soluble TCR molecules and soluble single-chain TCR molecules obtained by the invention can be combined with MLWRLGATI-HLA A0201 complex. Meanwhile, the method is used for detecting the binding activity of the soluble TCR molecule and the short peptides of other unrelated antigens and the HLA complex, and the result shows that the TCR molecule is not bound with other unrelated antigens.

Example 7 activation of T cells transduced with a TCR of the invention

Constructing a lentivirus vector containing the TCR target gene, transducing T cells, and carrying out an ELISPOT functional verification test.

ELISPOT scheme

The following experiments were performed to demonstrate the specific activation response of the TCR-transduced T cells of the invention to target cells. IFN-. gamma.production as measured by the ELISPOT assay was used as a readout for T cell activation.

Reagent

Test medium: 10% FBS (Gibbo, catalog number 16000-

Wash buffer (PBST): 0.01M PBS/0.05% Tween 20

PBS (Gibbo Co., catalog number C10010500BT)

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

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

Method

Target cell preparation

The target cells used in this experiment were T2 cells loaded with a specific short peptide. Target cells were prepared in experimental media: the concentration of the target cells is adjusted to 2.0X 10 ⁵ One/ml, 100. mu.l/well to obtain 2.0X 10 ⁴ Individual cells/well.

Effector cell preparation

The effector cells (T cells) of this experiment were CD8 expressing the TCR specific for the LMP1 antigen short peptide of the invention ⁺ T cells, and CD8 of the same volunteer not transfected with the TCR of the invention ⁺ T was used as a control group. Use of anti-CD 3/CD28 packageT cells were stimulated by beads (T cell amplicons, life technologies), transduced with lentiviruses carrying the LMP1 antigen short peptide specific TCR gene, expanded in 1640 medium containing 10% FBS with 50IU/ml IL-2 and 10ng/ml IL-7 until 9-12 days post transduction, then placed in assay medium and washed by centrifugation at 300g for 10min at RT. The cells were then resuspended in the test medium at 2 × the desired final concentration. Negative control effector cells were treated as well.

Preparation of short peptide solution

The corresponding short peptide was added to the corresponding target cell (T2) experiment group to give a final concentration of 1. mu.g/ml of short peptide in ELISPOT well plates.

ELISPOT

The well plate was prepared as follows according to the manufacturer's instructions: 10ml of sterile PBS per plate was added at 1: anti-human IFN-. gamma.capture antibody was diluted at 200, and 100. mu.l of the diluted capture antibody was aliquoted into each well. The plates were incubated overnight at 4 ℃. After incubation, the well plates were washed to remove excess capture antibody. 100 μ l/well of RPMI1640 medium containing 10% FBS was added and the well plates were incubated at room temperature for 2 hours to close the well plates. The media was then washed from the well plates, and any residual wash buffer was removed by flicking and tapping the ELISPOT well plates on paper.

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

100 microliter of target cells 2 x 10 ⁵ Cells/ml (total of about 2 x 10 was obtained) ⁴ Individual target cells/well).

100 microliter of effector cells (1 x 10) ⁴ Individual control effector cells/well and LMP1TCR positive T cells/well).

All wells were prepared in duplicate for addition.

The well plates were then incubated overnight (37 deg.C/5% CO) ₂ ) The next day, the medium was discarded, the well plate was washed 2 times with double distilled water and 3 times with wash buffer, and tapped on a paper towel to remove residual wash buffer. Then the cells were washed with 10% FBS in PBS 1: the detection antibody was diluted at 200 and added to each well at 100. mu.l/well. Incubate well plates at room temperature for 2 hours, wash 3 times with wash buffer, tap wells on paper towelThe plate was washed to remove excess wash buffer.

PBS containing 10% FBS was used at 1: streptavidin-alkaline phosphatase was diluted 100, 100 microliters of diluted streptavidin-alkaline phosphatase was added to each well and the wells were incubated for 1 hour at room temperature. The plates were then washed 2 times with 4 washes of PBS and tapped on a paper towel to remove excess wash buffer and PBS. After washing, 100 microliter of BCIP/NBT solution provided by the kit is added for development. And covering the well plate with tinfoil paper in the developing period, keeping the well plate in the dark, and standing for 5-15 minutes. During this period, spots on the developing well plate were routinely detected, and the optimal time for terminating the reaction was determined. The BCIP/NBT solution was removed and the well plate was rinsed with double steam to stop the development reaction, spun dry, then the bottom of the well plate was removed, the well plate was dried at room temperature until each well was completely dried, and the spots formed in the bottom membrane of the well plate were counted using an immunopot plate counter (CTL, Cellular Technology Limited).

Results

The TCR transduced T cells of the invention were tested for IFN- γ release in response to target cells loaded with LMP1 antigen short peptide MLWRLGATI by ELISPOT assay (described above). The number of ELSPOT spots observed in each well was plotted using a graphipad prism 6.

As shown in FIG. 15, T cells transduced with the TCR of the invention were shown to be very active against target cells loaded with their specific short peptides, whereas T cells transduced with other TCRs were shown to be essentially non-active against the corresponding target cells.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.

Sequence listing

<110> Guangdong Xiangxue accurate medical technology Limited

<120> TCR for identifying LMP1 antigen short peptide and coding sequence thereof

<130> P2018-0692

<160> 37

<170> PatentIn version 3.5

<210> 1

<211> 115

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 1

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

1 5 10 15

Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg Asp Thr Thr Tyr

20 25 30

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

35 40 45

Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile Ser Gly Arg Tyr

50 55 60

Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn Phe Thr Ile Thr

65 70 75 80

Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys Ala Leu Asn Ala

85 90 95

Gly Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val

100 105 110

Lys Pro His

115

<210> 2

<211> 345

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

gctcagaagg taactcaagc gcagactgaa atttctgtgg tggagaagga ggatgtgacc 60

ttggactgtg tgtatgaaac ccgtgatact acttattact tattctggta caagcaacca 120

ccaagtggag aattggtttt ccttattcgt cggaactctt ttgatgagca aaatgaaata 180

agtggtcggt attcttggaa cttccagaaa tccaccagtt ccttcaactt caccatcaca 240

gcctcacaag tcgtggactc agcagtatac ttctgtgctc tgaacgccgg ggcaggcaac 300

atgctcacct ttggaggggg aacaaggtta atggtcaaac cccat 345

<210> 3

<211> 255

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 3

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

1 5 10 15

Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg Asp Thr Thr Tyr

20 25 30

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

35 40 45

Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile Ser Gly Arg Tyr

50 55 60

Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn Phe Thr Ile Thr

65 70 75 80

Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys Ala Leu Asn Ala

85 90 95

Gly Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

<210> 4

<211> 765

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

gctcagaagg taactcaagc gcagactgaa atttctgtgg tggagaagga ggatgtgacc 60

ttggactgtg tgtatgaaac ccgtgatact acttattact tattctggta caagcaacca 120

ccaagtggag aattggtttt ccttattcgt cggaactctt ttgatgagca aaatgaaata 180

agtggtcggt attcttggaa cttccagaaa tccaccagtt ccttcaactt caccatcaca 240

gcctcacaag tcgtggactc agcagtatac ttctgtgctc tgaacgccgg ggcaggcaac 300

atgctcacct ttggaggggg aacaaggtta atggtcaaac cccatatcca gaaccctgac 360

cctgccgtgt accagctgag agactctaaa tccagtgaca agtctgtctg cctattcacc 420

gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 480

aaaactgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 540

aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 600

ttcttcccca gcccagaaag ttcctgtgat gtcaagctgg tcgagaaaag ctttgaaaca 660

gatacgaacc taaactttca aaacctgtca gtgattgggt tccgaatcct cctcctgaaa 720

gtggccgggt ttaatctgct catgacgctg cggctgtggt ccagc 765

<210> 5

<211> 116

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 5

Gly Ala Val Val Ser Gln His Pro Ser Trp Val Ile Cys Lys Ser Gly

1 5 10 15

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

20 25 30

Met Phe Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala

35 40 45

Thr Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys

50 55 60

Asp Lys Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr

65 70 75 80

Val Thr Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala

85 90 95

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

100 105 110

Leu Thr Val Leu

115

<210> 6

<211> 348

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

ggtgctgtcg tctctcaaca tccgagctgg gttatctgta agagtggaac ctctgtgaag 60

atcgagtgcc gttccctgga ctttcaggcc acaactatgt tttggtatcg tcagttcccg 120

aaacagagtc tcatgctgat ggcaacttcc aatgagggct ccaaggccac atacgagcaa 180

ggcgtcgaga aggacaagtt tctcatcaac catgcaagcc tgaccttgtc cactctgaca 240

gtgaccagtg cccatcctga agacagcagc ttctacatct gcagtgctag tgagactagc 300

gggagctatg agcagttctt cgggccaggg acacggctca ccgtgcta 348

<210> 7

<211> 295

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 7

Gly Ala Val Val Ser Gln His Pro Ser Trp Val Ile Cys Lys Ser Gly

1 5 10 15

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

20 25 30

Met Phe Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala

35 40 45

Thr Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys

50 55 60

Asp Lys Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr

65 70 75 80

Val Thr Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Lys Arg Lys Asp Ser Arg Gly

290 295

<210> 8

<211> 885

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

ggtgctgtcg tctctcaaca tccgagctgg gttatctgta agagtggaac ctctgtgaag 60

atcgagtgcc gttccctgga ctttcaggcc acaactatgt tttggtatcg tcagttcccg 120

aaacagagtc tcatgctgat ggcaacttcc aatgagggct ccaaggccac atacgagcaa 180

ggcgtcgaga aggacaagtt tctcatcaac catgcaagcc tgaccttgtc cactctgaca 240

gtgaccagtg cccatcctga agacagcagc ttctacatct gcagtgctag tgagactagc 300

gggagctatg agcagttctt cgggccaggg acacggctca ccgtgctaga ggacctgaaa 360

aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 420

caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 480

tggtgggtga atgggaagga ggtgcacagt ggggtcagca cagacccgca gcccctcaag 540

gagcagcccg ccctcaatga ctccagatac tgcctgagca gccgcctgag ggtctcggcc 600

accttctggc agaacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660

gagaatgacg agtggaccca ggatagggcc aaacctgtca cccagatcgt cagcgccgag 720

gcctggggta gagcagactg tggcttcacc tccgagtctt accagcaagg ggtcctgtct 780

gccaccatcc tctatgagat cttgctaggg aaggccacct tgtatgccgt gctggtcagt 840

gccctcgtgc tgatggccat ggtcaagaga aaggattcca gaggc 885

<210> 9

<211> 9

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 9

Met Leu Trp Arg Leu Gly Ala Thr Ile

1 5

<210> 10

<211> 7

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 10

Thr Arg Asp Thr Thr Tyr Tyr

1 5

<210> 11

<211> 8

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 11

Arg Asn Ser Phe Asp Glu Gln Asn

1 5

<210> 12

<211> 11

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 12

Ala Leu Asn Ala Gly Ala Gly Asn Met Leu Thr

1 5 10

<210> 13

<211> 6

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 13

Asp Phe Gln Ala Thr Thr

1 5

<210> 14

<211> 7

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 14

Ser Asn Glu Gly Ser Lys Ala

1 5

<210> 15

<211> 12

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 15

Ser Ala Ser Glu Thr Ser Gly Ser Tyr Glu Gln Phe

1 5 10

<210> 16

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 16

acccgtgata ctacttatta c 21

<210> 17

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 17

cggaactctt ttgatgagca aaat 24

<210> 18

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 18

gctctgaacg ccggggcagg caacatgctc acc 33

<210> 19

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 19

gactttcagg ccacaact 18

<210> 20

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 20

tccaatgagg gctccaaggc c 21

<210> 21

<211> 36

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 21

agtgctagtg agactagcgg gagctatgag cagttc 36

<210> 22

<211> 275

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 22

Met Leu Thr Ala Ser Leu Leu Arg Ala Val Ile Ala Ser Ile Cys Val

1 5 10 15

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

20 25 30

Val Val Glu Lys Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg

35 40 45

Asp Thr Thr Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Gly Glu

50 55 60

Leu Val Phe Leu Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile

65 70 75 80

Ser Gly Arg Tyr Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn

85 90 95

Phe Thr Ile Thr Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys

100 105 110

Ala Leu Asn Ala Gly Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Trp Ser Ser

275

<210> 23

<211> 825

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 23

atgctgactg ccagcctgtt gagggcagtc atagcctcca tctgtgttgt atccagcatg 60

gctcagaagg taactcaagc gcagactgaa atttctgtgg tggagaagga ggatgtgacc 120

ttggactgtg tgtatgaaac ccgtgatact acttattact tattctggta caagcaacca 180

ccaagtggag aattggtttt ccttattcgt cggaactctt ttgatgagca aaatgaaata 240

agtggtcggt attcttggaa cttccagaaa tccaccagtt ccttcaactt caccatcaca 300

gcctcacaag tcgtggactc agcagtatac ttctgtgctc tgaacgccgg ggcaggcaac 360

atgctcacct ttggaggggg aacaaggtta atggtcaaac cccatatcca gaaccctgac 420

cctgccgtgt accagctgag agactctaaa tccagtgaca agtctgtctg cctattcacc 480

gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 540

aaaactgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 600

aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 660

ttcttcccca gcccagaaag ttcctgtgat gtcaagctgg tcgagaaaag ctttgaaaca 720

gatacgaacc taaactttca aaacctgtca gtgattgggt tccgaatcct cctcctgaaa 780

gtggccgggt ttaatctgct catgacgctg cggctgtggt ccagc 825

<210> 24

<211> 309

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 24

Met Leu Leu Leu Leu Leu Leu Leu Gly Pro Gly Ser Gly Leu Gly Ala

1 5 10 15

Val Val Ser Gln His Pro Ser Trp Val Ile Cys Lys Ser Gly Thr Ser

20 25 30

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

35 40 45

Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala Thr Ser

50 55 60

Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys Asp Lys

65 70 75 80

Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr Val Thr

85 90 95

Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Ser Glu

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Lys Asp Ser Arg Gly

305

<210> 25

<211> 927

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 25

atgctgctgc ttctgctgct tctggggcca ggctccgggc ttggtgctgt cgtctctcaa 60

catccgagct gggttatctg taagagtgga acctctgtga agatcgagtg ccgttccctg 120

gactttcagg ccacaactat gttttggtat cgtcagttcc cgaaacagag tctcatgctg 180

atggcaactt ccaatgaggg ctccaaggcc acatacgagc aaggcgtcga gaaggacaag 240

tttctcatca accatgcaag cctgaccttg tccactctga cagtgaccag tgcccatcct 300

gaagacagca gcttctacat ctgcagtgct agtgagacta gcgggagcta tgagcagttc 360

ttcgggccag ggacacggct caccgtgcta gaggacctga aaaacgtgtt cccacccgag 420

gtcgctgtgt ttgagccatc agaagcagag atctcccaca cccaaaaggc cacactggtg 480

tgcctggcca caggcttcta ccccgaccac gtggagctga gctggtgggt gaatgggaag 540

gaggtgcaca gtggggtcag cacagacccg cagcccctca aggagcagcc cgccctcaat 600

gactccagat actgcctgag cagccgcctg agggtctcgg ccaccttctg gcagaacccc 660

cgcaaccact tccgctgtca agtccagttc tacgggctct cggagaatga cgagtggacc 720

caggataggg ccaaacctgt cacccagatc gtcagcgccg aggcctgggg tagagcagac 780

tgtggcttca cctccgagtc ttaccagcaa ggggtcctgt ctgccaccat cctctatgag 840

atcttgctag ggaaggccac cttgtatgcc gtgctggtca gtgccctcgt gctgatggcc 900

atggtcaaga gaaaggattc cagaggc 927

<210> 26

<211> 208

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 26

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

1 5 10 15

Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg Asp Thr Thr Tyr

20 25 30

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

35 40 45

Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile Ser Gly Arg Tyr

50 55 60

Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn Phe Thr Ile Thr

65 70 75 80

Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys Ala Leu Asn Ala

85 90 95

Gly Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

<210> 27

<211> 624

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 27

gcgcagaaag tgacccaagc gcagactgaa atttctgtgg tggagaagga ggatgtgacc 60

ttggactgtg tgtatgaaac ccgtgatact acttattact tattctggta caagcaacca 120

ccaagtggag aattggtttt ccttattcgt cggaactctt ttgatgagca aaatgaaata 180

agtggtcggt attcttggaa cttccagaaa tccaccagtt ccttcaactt caccatcaca 240

gcctcacaag tcgtggactc agcagtatac ttctgtgctc tgaacgccgg ggcaggcaac 300

atgctcacct ttggaggggg aacaaggtta atggtcaaac cccatatcca gaaccctgac 360

cctgccgtgt accagctgag agactctaag tcgagtgaca agtctgtctg cctattcacc 420

gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 480

aaatgtgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 540

aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 600

ttcttcccca gcccagaaag ttcc 624

<210> 28

<211> 246

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 28

Gly Ala Val Val Ser Gln His Pro Ser Trp Val Ile Cys Lys Ser Gly

1 5 10 15

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

20 25 30

Met Phe Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala

35 40 45

Thr Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys

50 55 60

Asp Lys Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr

65 70 75 80

Val Thr Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Ala Trp Gly Arg Ala Asp

245

<210> 29

<211> 738

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 29

ggtgcagttg ttagccaaca tccgagctgg gttatctgta agagtggaac ctctgtgaag 60

atcgagtgcc gttccctgga ctttcaggcc acaactatgt tttggtatcg tcagttcccg 120

aaacagagtc tcatgctgat ggcaacttcc aatgagggct ccaaggccac atacgagcaa 180

ggcgtcgaga aggacaagtt tctcatcaac catgcaagcc tgaccttgtc cactctgaca 240

gtgaccagtg cccatcctga agacagcagc ttctacatct gcagtgctag tgagactagc 300

gggagctatg agcagttctt cgggccaggg acacggctca ccgtgctaga ggacctgaaa 360

aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 420

caaaaggcca cactggtgtg cctggccacc ggtttctacc ccgaccacgt ggagctgagc 480

tggtgggtga atgggaagga ggtgcacagt ggggtctgca cagacccgca gcccctcaag 540

gagcagcccg ccctcaatga ctccagatac gctctgagca gccgcctgag ggtctcggcc 600

accttctggc aggacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660

gagaatgacg agtggaccca ggatagggcc aaacccgtca cccagatcgt cagcgccgag 720

gcctggggta gagcagac 738

<210> 30

<211> 254

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 30

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

1 5 10 15

Glu Asp Val Thr Ile Asp Cys Val Tyr Glu Thr Arg Asp Thr Thr Tyr

20 25 30

Tyr Leu Phe Trp Tyr Arg Gln Asp Pro Gly Gly Glu Leu Val Phe Leu

35 40 45

Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile Ser Gly Arg Tyr

50 55 60

Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn Phe Thr Ile Thr

65 70 75 80

Ala Val Gln Pro Val Asp Ser Ala Val Tyr Phe Cys Ala Leu Asn Ala

85 90 95

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

100 105 110

Lys Pro Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser

115 120 125

Glu Gly Gly Gly Ser Glu Gly Gly Thr Gly Gly Ala Val Val Ser Gln

130 135 140

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

145 150 155 160

Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr Met Phe Trp Tyr Arg Gln

165 170 175

Asp Pro Gly Gln Ser Leu Met Leu Met Ala Thr Ser Asn Glu Gly Ser

180 185 190

Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys Asp Arg Phe Leu Ile Asn

195 200 205

His Ala Ser Leu Thr Leu Ser Thr Leu Thr Ile Thr Ser Val His Pro

210 215 220

Glu Asp Ser Ser Phe Tyr Phe Cys Ser Ala Ser Glu Thr Ser Gly Ser

225 230 235 240

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

245 250

<210> 31

<211> 762

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 31

gctcaaaaag ttactcaggc ccagaccgaa ctgagcgttc cggaaggcga agatgttacc 60

attgattgtg tgtatgaaac ccgcgatacc acctattatc tgttttggta tcgtcaggat 120

ccgggcggcg aactggtttt tctgattcgc cgtaatagtt ttgatgaaca gaatgaaatc 180

agcggtcgtt atagctggaa ttttcagaaa agtaccagca gctttaattt taccattacc 240

gcagttcagc cggtggatag tgccgtttat ttttgtgccc tgaatgcagg cgccggtaat 300

atgctgacct ttggtggcgg cacccgcctg agtgtgaaac cgggtggtgg cagcgaaggt 360

ggcggtagtg aaggcggcgg cagtgaaggt ggtggctcag aaggcggcac cggtggtgca 420

gtggtgagcc agcatccgag tcatctgagc gttaaaagcg gcaccagtgt taaactggaa 480

tgtcgtagcc tggattttca ggcaaccacc atgttttggt atcgccagga tccgggtcag 540

agtctgatgc tgatggcaac cagtaatgaa ggtagtaaag caacctatga acagggtgtt 600

gaaaaagatc gctttctgat taatcatgcc agtctgaccc tgagcaccct gaccattacc 660

agtgttcatc cggaagatag cagtttttat ttttgcagtg caagcgaaac cagcggtagt 720

tatgaacagt ttttcggtcc gggcacccgc ttaaccgtgg at 762

<210> 32

<211> 114

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 32

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

1 5 10 15

Glu Asp Val Thr Ile Asp Cys Val Tyr Glu Thr Arg Asp Thr Thr Tyr

20 25 30

Tyr Leu Phe Trp Tyr Arg Gln Asp Pro Gly Gly Glu Leu Val Phe Leu

35 40 45

Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile Ser Gly Arg Tyr

50 55 60

Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn Phe Thr Ile Thr

65 70 75 80

Ala Val Gln Pro Val Asp Ser Ala Val Tyr Phe Cys Ala Leu Asn Ala

85 90 95

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

100 105 110

Lys Pro

<210> 33

<211> 342

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 33

gctcaaaaag ttactcaggc ccagaccgaa ctgagcgttc cggaaggcga agatgttacc 60

attgattgtg tgtatgaaac ccgcgatacc acctattatc tgttttggta tcgtcaggat 120

ccgggcggcg aactggtttt tctgattcgc cgtaatagtt ttgatgaaca gaatgaaatc 180

agcggtcgtt atagctggaa ttttcagaaa agtaccagca gctttaattt taccattacc 240

gcagttcagc cggtggatag tgccgtttat ttttgtgccc tgaatgcagg cgccggtaat 300

atgctgacct ttggtggcgg cacccgcctg agtgtgaaac cg 342

<210> 34

<211> 116

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 34

Gly Ala Val Val Ser Gln His Pro Ser His Leu Ser Val Lys Ser Gly

1 5 10 15

Thr Ser Val Lys Leu Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr

20 25 30

Met Phe Trp Tyr Arg Gln Asp Pro Gly Gln Ser Leu Met Leu Met Ala

35 40 45

Thr Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys

50 55 60

Asp Arg Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr

65 70 75 80

Ile Thr Ser Val His Pro Glu Asp Ser Ser Phe Tyr Phe Cys Ser Ala

85 90 95

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

100 105 110

Leu Thr Val Asp

115

<210> 35

<211> 348

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 35

ggtgcagtgg tgagccagca tccgagtcat ctgagcgtta aaagcggcac cagtgttaaa 60

ctggaatgtc gtagcctgga ttttcaggca accaccatgt tttggtatcg ccaggatccg 120

ggtcagagtc tgatgctgat ggcaaccagt aatgaaggta gtaaagcaac ctatgaacag 180

ggtgttgaaa aagatcgctt tctgattaat catgccagtc tgaccctgag caccctgacc 240

attaccagtg ttcatccgga agatagcagt ttttattttt gcagtgcaag cgaaaccagc 300

ggtagttatg aacagttttt cggtccgggc acccgcttaa ccgtggat 348

<210> 36

<211> 24

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 36

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

1 5 10 15

Gly Gly Ser Glu Gly Gly Thr Gly

20

<210> 37

<211> 72

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 37

ggtggtggca gcgaaggtgg cggtagtgaa ggcggcggca gtgaaggtgg tggctcagaa 60

ggcggcaccg gt 72

Claims (37)

1. A T Cell Receptor (TCR), wherein the TCR is capable of binding to the MLWRLGATI-HLA a0201 complex; and, the TCR comprises a TCR α chain variable domain and a TCR β chain variable domain, wherein the 3 Complementarity Determining Regions (CDRs) of the TCR α chain variable domain are:

αCDR1-TRDTTYY(SEQ ID NO:10)

αCDR2-RNSFDEQN(SEQ ID NO:11)

alpha CDR3-ALNAGAGNMLT (SEQ ID NO: 12); and

the 3 complementarity determining regions of the TCR β chain variable domain are:

βCDR1-DFQATT(SEQ ID NO:13)

βCDR2-SNEGSKA(SEQ ID NO:14)

βCDR3-SASETSGSYEQF(SEQ ID NO:15)。

2. a TCR as claimed in claim 1 which comprises a TCR α chain variable domain which is an amino acid sequence having at least 90% sequence identity to SEQ ID No. 1 and a TCR β chain variable domain.

3. A TCR as claimed in claim 1 wherein the TCR β chain variable domain is substantially identical to SEQ ID NO:5 an amino acid sequence having at least 90% sequence identity.

4. A TCR as claimed in claim 1 which comprises the α chain variable domain amino acid sequence SEQ ID NO 1.

5. A TCR as claimed in claim 1 which comprises the β chain variable domain amino acid sequence SEQ ID NO 5.

6. A TCR as claimed in claim 1 which is an α β heterodimer comprising a TCR α chain constant region TRAC 01 and a TCR β chain constant region TRBC1 01 or TRBC2 01.

7. A TCR as claimed in claim 6 wherein the α chain amino acid sequence of the TCR is SEQ ID NO:3 and the beta chain amino acid sequence of the TCR are SEQ ID NO 7.

8. A TCR as claimed in any one of claims 1 to 5 which is soluble.

9. A TCR as claimed in claim 8 which is single chain.

10. A TCR as claimed in claim 9 which is formed by the α chain variable domain linked to the β chain variable domain by a peptide linker sequence.

11. A TCR as claimed in claim 10 which has one or more mutations in amino acid 11, 13, 19, 21, 53, 76, 89, 91, or 94 of the variable domain of the α chain, and/or in the penultimate 3,5 or 7 amino acid position of the short peptide of the α chain J gene; and/or the TCR has one or more mutations in beta chain variable domain amino acid position 11, 13, 19, 21, 53, 76, 89, 91, or 94, and/or beta chain J gene short peptide amino acid penultimate 2,4 or 6, wherein the amino acid position numbering is according to the position numbering listed in IMGT (international immunogenetics information system).

12. A TCR as claimed in claim 11 in which the α chain variable domain amino acid sequence of the TCR comprises SEQ ID No. 32 and/or the β chain variable domain amino acid sequence of the TCR comprises SEQ ID No. 34.

13. A TCR as claimed in claim 12 wherein the amino acid sequence of the TCR is SEQ ID No. 30.

14. A TCR as claimed in claim 8 which comprises (a) all or part of the TCR α chain, excluding the transmembrane domain; and (b) all or part of a TCR β chain, excluding the transmembrane domain;

and (a) and (b) each comprise a functional variable domain.

15. A TCR as claimed in claim 14 wherein (a) and (b) each further comprise at least a portion of the constant domain of the TCR chain.

16. A TCR as claimed in claim 15 in which the cysteine residues form an artificial disulphide bond between the α and β chain constant domains of the TCR.

17. A TCR as claimed in claim 16 wherein the cysteine residues which form the artificial disulphide bond in the TCR are substituted at one or more groups selected from:

thr48 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser57 of TRBC2 × 01 exon 1;

thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser77 of TRBC2 × 01 exon 1;

tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC2 x 01;

thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1;

ser15 and TRBC1 × 01 of TRAC × 01 exon 1 or Glu15 of TRBC2 × 01 exon 1;

arg53 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser54 of TRBC2 × 01 exon 1;

pro89 and TRBC1 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01; and

tyr10 of TRAC × 01 exon 1 and TRBC1 × 01 or TRBC2 × 01 of Glu20 of exon 1.

18. A TCR as claimed in claim 17 in which the α chain amino acid sequence of the TCR is SEQ ID No. 26 and/or the β chain amino acid sequence of the TCR is SEQ ID No. 28.

19. A TCR as claimed in claim 15 which comprises an artificial interchain disulphide bond between the α chain variable domain and the β chain constant region of the TCR.

20. A TCR as claimed in claim 19 wherein the cysteine residues which form the artificial interchain disulphide bond in the TCR are substituted at one or more groups of sites selected from:

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

amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01;

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

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

21. A TCR as claimed in claim 19 which comprises the α chain variable domain and the β chain variable domain and all or part of the β chain constant domain, excluding the transmembrane domain, but which does not comprise the α chain constant domain, the α chain variable domain of the TCR forming a heterodimer with the β chain.

22. A TCR as claimed in claim 1 wherein a conjugate is attached to the C-or N-terminus of the α and/or β chain of the TCR.

23. A TCR as claimed in claim 22 wherein the conjugate to which the TCR is bound is a detectable label, a therapeutic agent, a PK modifying moiety or a combination thereof.

24. A TCR as claimed in claim 23 wherein the therapeutic agent is an anti-CD 3 antibody.

25. A multivalent TCR complex comprising at least two TCR molecules, and wherein at least one TCR molecule is a TCR as claimed in any one of claims 1 to 24.

26. A nucleic acid molecule comprising a nucleic acid sequence encoding a TCR according to any one of claims 1 to 24, or the complement thereof.

27. The nucleic acid molecule of claim 26, wherein said nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO: 33.

28. The nucleic acid molecule of claim 26 or 27, wherein said nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:6 or SEQ ID NO 35.

29. The nucleic acid molecule of claim 26, comprising the nucleotide sequence encoding a TCR α chain of SEQ ID NO:4 and/or comprises the nucleotide sequence encoding the TCR β chain SEQ ID NO: 8.

30. a vector comprising the nucleic acid molecule of any one of claims 26-29.

31. The vector of claim 30, wherein said vector is a viral vector.

32. The vector of claim 30, wherein said vector is a lentiviral vector.

33. An isolated host cell comprising the vector of claim 30 or a nucleic acid molecule of any one of claims 26-29 integrated into the chromosome.

34. A cell transduced with the nucleic acid molecule of any one of claims 26 to 29 or the vector of claim 30.

35. The cell of claim 34, wherein the cell is a T cell or a stem cell.

36. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to any one of claims 1 to 24, a TCR complex according to claim 25 or a cell according to claim 34.

37. Use of a TCR as claimed in any one of claims 1 to 24, or a TCR complex as claimed in claim 25 or a cell as claimed in claim 34, in the preparation of a medicament for the treatment of nasopharyngeal carcinoma, burkitt's lymphoma, and/or hodgkin's lymphoma.

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