CN109251244B - TCR (T cell receptor) for recognizing LMP1 antigen derived from EBV (Epstein-Barr Virus) membrane protein - Google Patents

Abstract

The present invention provides a T Cell Receptor (TCR) capable of specifically binding short peptide ILWRLGATI derived from LMP1 antigen, which antigen short peptide ILWRLGATI 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.

Description

TCR (T cell receptor) for recognizing LMP1 antigen derived from EBV (Epstein-Barr Virus) membrane protein

Technical Field

The invention relates to a TCR capable of recognizing LMP1 antigen-derived short peptide, 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. ILWRLGATI is a short peptide derived from LMP1 antigen, which is a target for the treatment of LMP1 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 ILWRLGATI-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 CAVGSNFGNEKLTF (SEQ ID NO: 12); and/or the amino acid sequence of CDR3 of the variable domain of the TCR beta chain is CSARRLGTEAFF (SEQ ID NO: 15).

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

αCDR1-DSVNN(SEQ ID NO:10)

αCDR2-IPSGT(SEQ ID NO:11)

alpha CDR3-CAVGSNFGNEKLTF (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-CSARRLGTEAFF(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 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 encoding the variable domain of the TCR α chain SEQ ID NO: 2.

in another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 6.

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 of the invention, there is provided 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 comprises Burkitt's lymphoma, Hodgkin's lymphoma, EBV positive post-transplant lymphoproliferative disorder (PTLD), nasopharyngeal carcinoma, or the like.

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, a TCR β chain amino acid 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 the molecular weight marker (marker), the middle lane is the non-reducing gel, and the rightmost lane is the reducing gel.

FIG. 7 is a BIAcore kinetic profile of binding of soluble TCRs of the invention to the ILWRLGATI-HLA A0201 complex.

FIG. 8 shows that T cells transduced with the TCR of the invention were very responsive to activation of target cells loaded with their specific short peptides, but not to target cells not loaded with the corresponding short peptides and target cells loaded with non-specific short peptides.

FIG. 9 shows that LMP1 CD8+ T cells have obvious killing effect on target cells LCL-A02/A11 loaded with LMP1PX224129-137ILWRLGATI short peptides; the killing effect on target cells LCL-A02/A11 which are not loaded with short peptides or loaded with nonspecific short peptides is not obvious.

Detailed Description

The present inventors have made extensive and intensive studies to find a TCR capable of specifically binding to LMP1 antigen short peptide ILWRLGATI (SEQ ID NO:9), which antigen short peptide ILWRLGATI 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, different individuals have different MHC, and different short peptides in one protein antigen can be presented on 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 the binding of antigen-specific TCRs to pMHC complexes, and then other cell membrane surface molecules of both T cells and APCs interact, which causes a series of subsequent cell signaling and other physiological reactions, thereby allowing T cells of different antigen specificities to exert immune effects 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 ILWRLGATI-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-DSVNN(SEQ ID NO:10)

αCDR2-IPSGT(SEQ ID NO:11)

alpha CDR3-CAVGSNFGNEKLTF (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-CSARRLGTEAFF(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 the corresponding function based on the CDR regions disclosed herein, so long as 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 a variant of 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 the 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 molecule comprises a variable domain and a constant domain, and the β chain variable domain amino acid sequence comprises 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 β chain variable domain 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-. From the literature, those skilled in the art are readily able to construct single chain TCR molecules comprising the CDRs regions of the invention. 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 the 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 the 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 molecule 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 soluble TCR molecules are required. 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 infections 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 exon 1 of TRAC × 01 and a cysteine residue of Ser57 of exon 1 of TRBC1 × 01 or TRBC2 × 01. 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 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01; or Tyr10 and TRBC1 and 01 of TRAC 01 exon 1 or Glu20 of TRBC2 and 01 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.

In addition, for stability, patent document PCT/CN2016/077680 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 the variable domain and at least part of the 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 association of two, three, four or more TCRs of the invention, such as might be produced as a tetramer using the tetrameric domain of p53, or a complex formed by association of a plurality of TCRs of the invention with 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 can also be used 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 ILWRLGATI-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 and the like (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 TCR may comprise a human variable domain and a murine constant domain. The drawback of this approach is the possibility of eliciting an immune response. Thus, there should be a regulatory regimen to immunosuppresse 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 portion thereof, which 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-

(SEQ ID NO:16)

αCDR2-

(SEQ ID NO:17)

αCDR3-

(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-

(SEQ ID NO:19)

βCDR2-

(SEQ ID NO:20)

βCDR3-

(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. More preferably, the nucleotide sequence of the nucleic acid molecule of the invention comprises SEQ ID NO. 4 and/or SEQ ID NO. 8.

It will be appreciated that, due to the degeneracy of the genetic code, different nucleotide sequences 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, baculoviral 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 ILWRLGATI-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 ILWRLGATI-HLA A0201 complex. Including but not limited to Burkitt's lymphoma, Hodgkin's lymphoma, EBV positive post-transplant lymphoproliferative disorder (PTLD), nasopharyngeal carcinoma, or 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 disclosed by the invention can be combined with LMP1 antigen short peptide complex ILWRLGATI-HLA A0201, and cells transduced with the TCR disclosed by the invention can be specifically activated and have a 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 PRAME-specific T cells

Peripheral Blood Lymphocytes (PBLs) from healthy volunteers of genotype HLA-A02 were stimulated with synthetic short peptide (Nanjing Kingskan Biotech Co., Ltd.) ILWRLGATI (SEQ ID NO.9, named PX224 in the present invention). And (3) renaturing the short peptide and HLA-A0201 with biotin labels to prepare the pHLA haploid. These haploids were combined with streptavidin labeled with PE (BD Co.) to form PE-labeled tetramers, which were sorted for double positive anti-CD 8-APC cells. The sorted cells were expanded and subjected to secondary sorting as described above, followed by single cloning by limiting dilution.

Because the whole experiment process takes longer time, the influence factors are extremely large, the experiment is complex, the cell performance can not be predicted at all, and the success rate of obtaining the corresponding T cell monoclonal is very low even through layer-by-layer screening and strict detection. It is generally difficult to obtain TCRs with the desired activity through only a few batches of experiments.

In the invention, through intensive research and a large number of experiments of the inventor, the double-positive monoclonal cell meeting the conditions is finally obtained. Monoclonal cells were stained with tetramer and double positive clones were selected as shown in FIG. 3.

Even if a single T cell clone is successfully selected, the resulting TCR may not be satisfactory because in many cases the resulting TCR may not be successfully renatured or may have poor affinity for the corresponding epitope after renaturation, or may not bind. Further validation of binding activity is required.

Example 2 construction of TCR Gene and vector for obtaining LMP 1-specific T cell clones

Using Quick-RNA^TMMiniPrep (ZYMO research) extracted the total RNA of the PX 224-specific, HLA-A02-restricted T cell clone selected in example 1. cDNA was synthesized using the SMART RACE cDNA amplification kit from clontech, using primers designed to be conserved at the C-terminus of the human TCR gene. The sequences were cloned into the T vector (TAKARA) and sequenced. 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 after sequencing, and figure 1a, figure 1b, figure 1c and figure 1d 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 and a TCR alpha chain nucleotide sequence; fig. 2a, fig. 2b, fig. 2c and fig. 2d 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 and a TCR β chain nucleotide sequence, respectively.

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

αCDR1-DSVNN(SEQ ID NO:10)

αCDR2-IPSGT(SEQ ID NO:11)

αCDR3-CAVGSNFGNEKLTF(SEQ ID NO:12)

the beta chain comprises CDRs having the following amino acid sequences:

βCDR1-DFQATT(SEQ ID NO:13)

βCDR2-SNEGSKA(SEQ ID NO:14)

βCDR3-CSARRLGTEAFF(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: v region genes of a TCR alpha chain and a TCR beta chain are respectively connected with C regions of conserved regions of a mouse TCR alpha chain and a TCR beta chain by overlap PCR to obtain a TCR alpha-2A-TCR beta fragment. The lentivirus expression vector and TCR alpha-2A-TCR beta are connected by enzyme digestion to obtain pLenti-LMP1TRA-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 exon 1 TRAC × 01 and Ser57 of exon 1 TRBC2 × 01, respectively; the amino acid sequence and nucleotide sequence of the alpha chain are shown in FIGS. 4a and 4b, respectively, and the amino acid sequence and nucleotide sequence of the beta chain are shown in FIGS. 5a and 5b, respectively, and the introduced cysteine residues are shown in bold and underlined letters. 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 transformed into expression bacteria BL21(DE3) by chemical transformation method, and the bacteria are grown in LB culture solution and OD₆₀₀Inclusion 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 binding characterisation

BIAcore analysis

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

Binding activity of the TCR molecules obtained in examples 3 and 5 to the ILWRLGATI-HLA A0201 complex was measured using a BIAcore T200 real-time assay system. Anti-streptavidin antibody (GenScript) was added to coupling buffer (10mM sodium acetate buffer, pH 4.77), and then the antibody was passed through CM5 chip previously activated with EDC and NHS to immobilize the antibody on the chip surface, and finally the unreacted activated surface was blocked with ethanolamine hydrochloric acid solution to complete the coupling process at a coupling level of about 15,000 RU.

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

The ILWRLGATI-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 the supernatant, adding 30ml of 10-fold diluted BugBuster to resuspend the inclusion bodies, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, repeating twice, adding 30ml of 20mM Tris-HCl pH 8.0 to resuspend the inclusion bodies, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, finally dissolving the inclusion bodies by using 20mM Tris-HCl 8M urea, detecting the purity of the inclusion bodies by SDS-PAGE, and detecting the concentration by using a BCA kit.

b. Renaturation

The synthesized short peptide ILWRLGATI (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. ILWRLGATI peptide was added 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 ℃) at 25mg/L (final concentration), 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 carried out at 4 ℃ for at least 3 days until completion, and SDS-PAGE checked for success or failure of the renaturation.

c. Purification after renaturation

The renaturation buffer was replaced 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 a Mi llipore ultrafiltration tube, the biotinylated pMHC was purified by gel filtration chromatography, and HiPrep was pre-equilibrated with filtered PBS using an Akta purifier (GE general electric Co., Ltd.)^TM16/60S200HR 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 using BIAcore Evaluation software, and the kinetic profile of binding of soluble TCR molecules of the invention to ILWRLGATI-HLA A0201 complex is shown in FIG. 7. The map shows that the soluble TCR molecule obtained by the invention can be combined with ILWRLGATI-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 5 transfection of LMP1 specific T cell receptor Lentiviral packaging with Primary T cells into LMP1TCR

(a) Production of lentiviruses by Rapid-mediated transient transfection of 293T/17 cells

A third generation lentiviral packaging system was used to package lentiviruses containing the gene encoding the desired TCR. 293T/17 cells were transfected with 4 plasmids (a lentiviral vector containing pLenti-LMP1TRA-2A-TRB-IRES-NGFR as described In example 2, and 3 plasmids containing other components necessary for the construction of infectious but non-replicating lentiviral particles) using rapid-mediated transient transfection (Express-In-mediated transfection (Open Biosystems)).

For transfection, cells were seeded at day 0 on a 15 cm petri dish at 1.7X 10⁷293T/17 cells, which were distributed evenly on the culture dish with a degree of confluence slightly higher than 50%. On day 1, plasmids were transfected, pLenti-LMP1TRA-2A-TRB-IRES-NGFR and pLenti-eGFP pseudoviruses were packaged, and the above expression plasmids were mixed with packaging plasmids pMDLg/pRRE, pRSV-REV and pMD.2G in a 15 cm diameter plate in the following amounts: 22.5 microgram: 15 microgram: 7.5 micrograms. The ratio of the transfection reagent PEI-MAX to the plasmid was 2:1 and the amount used was 114.75. mu.g per dish. The specific operation is as follows: adding the expression plasmid and the packaging plasmid into 1800 microliters of OPTI-MEM (Gibbo, catalog No. 31985-₂And (5) culturing. 5-7 hours of transfection, the transfection medium was removed and replaced with DMEM (Gibbo, Cat. C11995500bt) complete medium containing 10% fetal bovine serum at 37 deg.C/5% CO₂And (5) culturing. Culture supernatants containing packaged lentiviruses were collected on days 3 and 4. To harvest the packaged lentivirus, the collected culture supernatant was centrifuged at 3000g for 15min to remove cell debris, filtered through a 0.45 micron filter (Merck Millipore, catalog number SLGP033RB) and finally concentrated using a 50KD cut-off concentration tube (Merck Millipore), catalog number UFC905096, to remove most of the supernatant, and finally concentrated to 1ml, aliquots were frozen at-80 ℃. Pseudovirus samples were taken for virus titer determination, procedures were referenced to p24ELISA (Clontech, cat # 632200) kit instructions. As a control, a pseudovirus transformed with pLenti-eGFP was also included.

(b) Transduction of primary T cells with lentiviruses containing LMP 1-specific T cell receptor genes

CD8 isolated from blood of healthy volunteers⁺T cells, then transduced with the packaged lentivirus. These cells were counted in 48-well plates in a 1X 10 format in 1640 (Gibbo, Cat. No. C11875500bt) medium containing 30IU/ml IL-2 with 10% FBS (Gibbo, Cat. No. C10010500BT)⁶Cells/ml (0.5 ml/well) were incubated with pre-washed anti-CD 3/CD28 antibody-coated beads (T cell amplicons, life technologies, cat No. 11452D) overnight for stimulation, cells: bead 3: 1.

after overnight stimulation, the concentrated lentivirus of PRAME specific T cell receptor gene was added at an MOI of 10 according to the virus titer measured with the p24ELISA kit and centrifuged at 32 ℃ and 900g for 1 hour. After infection, the lentivirus infection solution was removed and the cells were resuspended in 1640 medium containing 10% FBS containing 30IU/ml IL-2 at 37 ℃/5% CO₂The cells were cultured for 3 days. Cells were counted 3 days after transduction and diluted to 0.5X 10⁶Individual cells/ml. The cells were counted every two days, replaced or added with fresh medium containing 30IU/ml IL-2, maintaining the cells at 0.5X 10⁶-1×10⁶Individual cells/ml. Cells were analyzed by flow cytometry starting on day 3 and starting on day 5 for functional assays (e.g., ELISPOT for IFN- γ release and non-radioactive cytotoxicity assays). Freezing storage of aliquots of cells, at least 4X 10, from day 10 or as the cells slow down division and become smaller in size⁶Individual cell/tube (1X 10)⁷Individual cells/ml, 90% FBS/10% DMSO).

Example 6 cell activation functional validation

ELISPOT scheme

The following assay was performed to demonstrate activation of TCR-transduced T cells in response specifically 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-

Washing buffer solution: 0.01M PBS/0.05% Tween 20

PBS (Gibbo Co., catalog number C10010500BT)

PVDF ELISPOT 96 well plate (Merck Millipore, Cat. No. 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 of this example were Epstein-Barr virus (EBV) transformed immortalized Lymphoblastoid Cell Lines (LCLs). B95-8 cells were induced to produce EBV-containing culture supernatants by phorbol myristate acetate (TPA), centrifuged at 4 deg.C/600 g for 10min to remove impurities, filtered through 0.22 μm filter, and aliquoted for-70 deg.C storage. From Peripheral Blood Lymphocytes (PBLs) of healthy volunteers of the genotype HLA-A11/A02/A24 (both homozygote and heterozygote), 10ml of 2X 10-concentration blood was taken⁷One ml PBL suspension in 25 cm square culture flask, adding cyclosporine at 37 deg.C/CO₂Incubate for 1 hour in incubator, thaw one EBV portion quickly, add to the cells at 1/10 dilution, shake gently and place the flask upright at 37 deg.C/CO₂Culturing in an incubator. After 12 days of culture, the culture was continued by adding 10ml of a medium, and after about 30 days, the culture was further expanded and subjected to flow assay, in which CD19 was present⁺CD23^hiCD58⁺Is an immortalized Lymphoblastoid Cell Line (LCL). The ELISPOT test uses HLA-A02LCL as target cells.

Effector cell preparation

The effector cells (T cells) of this assay were CD8 expressing LMP 1-specific TCR analyzed by flow cytometry in example 3⁺T cells and CD8 of the same volunteer⁺T served as negative control effector cells. T cells were stimulated with anti-CD 3/CD28 coated beads (T cell amplicons, life technologies), transduced with a lentivirus carrying the LMP1 specific TCR gene (according to example 3), expanded in 1640 medium containing 10% FBS with 30IU/ml IL-2 until 9-12 days after transduction, then the cells were placed in test medium and washed by centrifugation at 300g for 10min at RT. Then theCells were resuspended in the assay medium at 2 × the desired final concentration. Negative control effector cells were treated as well.

ELISPOT

The well plate was prepared as follows according to the manufacturer's instructions: 10ml of sterile PBS per plate 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 plate, and any residual wash buffer was removed by flicking and tapping the ELISPOT well plate on paper.

LMP1 CD8⁺T cells (TCR-transduced T cells, effector cells, of the invention, also designated "IL 14CD 8" in the invention⁺TCELL”)、CD8⁺T cells (negative control effector cells) and LCL-A02/A11 (target cells) were prepared as described in example 3, and the corresponding short peptides were added to the corresponding experimental groups, where PX224 was LMP1PX224129-137ILWRLGATI short peptide, and the remainder was non-LMP 1 TCR-specific binding short peptide.

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

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

50 microliters of effector cells (1000 LMP1TCR positive T cells).

20 microliter 10^-5Mol/l LMP1PX224129-137 ILWRLGATI/other short peptide solution (final concentration is 10%^-6Moles/liter).

All wells were made in triplicate for addition.

The plates were then incubated overnight (37 ℃/5% CO2) for the next day, the medium was discarded, the plates were washed 2 times with double distilled water and 3 times with wash buffer, and tapped on a paper towel to remove residual wash buffer. Primary antibody was then detected by dilution with PBS containing 10% FBS and added to each well at 100. mu.l/well. The well plate was incubated at room temperature for 2 hours, washed 3 times with wash buffer and the well plate was tapped on a paper towel 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 plate was then washed 3 times with wash buffer and 2 times with PBS, and the excess wash buffer and PBS was removed by tapping the plate on a paper towel. 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. Spots on the developing plate were routinely detected during this period to determine the optimum time for terminating the reaction. The BCIP/NBT solution was removed and the well plate was rinsed with double-distilled water to stop the development reaction, spun-dried, then the bottom of the well plate was removed, the well plate was dried at room temperature until each well was completely dried, and then the spots formed in the bottom film of the well plate were counted using an immune spot plate counter (CTL, cell Technology Limited).

Results

The LMP1TCR transduced T cells were tested for IFN- γ release by ELISPOT assay (described above) in response to LMP1PX224129-137ILWRLGATI short peptide-loaded target cells and non-specific short peptide-loaded target cells. The number of ELSPOT spots observed in each well was plotted using a graphipad prism 6.

The results of the experiment are shown in FIG. 8, LMP1 CD8 alone⁺Addition of the corresponding short peptides by T cells (effector cells) or LCL cells (target cells) releases little IFN-. gamma..

LMP1 CD8⁺T cells (effector cells) were able to react with PX 224-supplemented LCL-A02/A11 cells to release more IFN- γ.

LMP1 CD8⁺T cells (effector cells) released little IFN-. gamma.from LCL-A02/A11 cells supplemented with other short peptides.

CD8⁺T cells (negative control effector cells) released very little IFN-. gamma.from LCL-A02/A11 cells supplemented with PX 224.

In conclusion, T cells transduced with the TCR of the invention have a good activation response to target cells loaded with the specific short peptide, and no activation response to target cells not loaded with the corresponding short peptide and target cells loaded with non-specific short peptide.

Example 7 verification of cell killing function

This test is a colorimetric substitution test for the 51Cr release cytotoxicity test, and quantitatively determines Lactate Dehydrogenase (LDH) released after cell lysis. LDH released in the medium was detected using a 30 min coupled enzymatic reaction in which LDH converted a tetrazolium salt (INT) to red formazan (formazan). The amount of red product produced is proportional to the number of cells lysed. 490nm visible absorbance data can be collected using a standard 96-well plate reader.

Material

CytoTox

Non-radioactive cytotoxicity assays (Promega, G1780) contained a substrate mixture, assay buffer, lysis solution and stop buffer.

Test medium: 5% FBS (heat-inactivated, Gibbo, Cat. No. 16000-.

Microwell round bottom 96 well tissue culture plates (Nunc, Cat. No. 163320)

96-well immunoplate Maxisorb (Nunc, Cat. No. 442404)

Method

Target cell preparation

The target cell LCL used in this assay was prepared as described above for the ELISPOT protocol. Target cells were prepared in assay medium: the concentration of the target cells is adjusted to 3X 10⁵One/ml, 50. mu.l/well to obtain 1.5X 10⁴Individual cells/well.

Effector cell preparation

The effector cells (T cells) of this assay were CD8 expressing LMP 1-specific TCR analyzed by flow cytometry in example 3⁺T cells. Effector cell to target cell ratio 10: 1,5: 1,2.5: 1,1.25:1 (diluted to 3X 10 if 10: 1)⁶One/ml, 50. mu.l/well to obtain 1.5X 10⁵Is smallCells/well).

Preparation of short peptide solution

LMP1PX224129-137ILWRLGATI (or other nonspecific short peptide) short peptide is diluted to 10% with 5% FBS-containing phenol red-free RPMI1640 medium^-5Working solution in the range of 10 final concentration after addition to the experimental group^-6M。

(a) Detection of killing capability of effector cells by loading target cells with short peptides at different concentrations

Preparation of the test

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

50ul of target cells (prepared as described above) were added to each well

50ul of effector cells (prepared as described above) were added to each well

-12ul of the short peptide solution was added to each well

8ul culture supplement wells (20 ul medium was directly supplemented to the experimental group without short peptide load).

A control group was prepared as follows:

experimental group without short peptide loading: contains 50ul effector cells and 50ul target cells.

Effector cells release spontaneously: there were only 50ul of effector cells.

Target cells release: there are only 50ul of target cells.

Maximum release of target cells: there are only 50ul of target cells.

Reagent medium control: only 120ul of medium was present.

All wells were made in triplicate with a final volume of 120ul (insufficient media make-up).

Incubate at 37 ℃ for 24 hours. Before collecting the supernatants from all wells, the target cells maximum release control wells were placed on the cells at-70 ℃ for approximately 30 minutes and thawed at 37 ℃ for 15 minutes to allow total lysis of the target cells.

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

Calculation results

The absorbance values of the medium background were subtracted from all the absorbance values of the experimental, target cell spontaneous release and effector cell spontaneous release groups.

The corrected values obtained above were substituted into the following formula to calculate the percent cytotoxicity resulting from each effect-to-target ratio.

% cytotoxicity 100 × (experiment-effector cell spontaneous-target cell spontaneous)/(target cell maximal-target cell spontaneous)

Results

LMP1TCR transduced T cells were tested for LDH release in response to LMP1PX224129-137ILWRLGATI short peptide-loaded target cells by non-radioactive cytotoxicity assays (described above). The absorbance of 490nm visible light in each well was plotted using graphpad prism 6.

The statistical result of the experimental data is shown in FIG. 9, the concentration of the short peptide is 10 when the LMP1PX224129-137ILWRLGATI is loaded^-6The M effective target ratio is 10: 1 and 5:1, LMP1 CD8+ T cells have obvious killing effect on target cells LCL-A02/A11; the killing effect on target cells LCL-A02/A11 which are not loaded with short peptides or loaded with nonspecific short peptides is not obvious. Homologous CD8⁺The T cells have no obvious killing effect on LCL-A02/A11 loaded with LMP1PX224129-137ILWRLGATI short peptides.

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 and 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 present invention as defined by the appended claims.

Sequence listing

<110> Guangzhou biomedical and health research institute of Chinese academy of sciences

<120> a TCR recognizing LMP1 antigen derived from EBV membrane protein

<130> P2017-1290

<160> 29

<170> PatentIn version 3.5

<210> 1

<211> 110

<212> PRT

<213> artificial sequence

<220>

<223> TCR alpha chain variable Domain

<400> 1

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

1 5 10 15

Ala Asn Ser Thr Leu Arg Cys Asn Phe Ser Asp Ser Val Asn Asn Leu

20 25 30

Gln Trp Phe His Gln Asn Pro Trp Gly Gln Leu Ile Asn Leu Phe Tyr

35 40 45

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

50 55 60

Ala Thr Glu Arg Tyr Ser Leu Leu Tyr Ile Ser Ser Ser Gln Thr Thr

65 70 75 80

Asp Ser Gly Val Tyr Phe Cys Ala Val Gly Ser Asn Phe Gly Asn Glu

85 90 95

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

100 105 110

<210> 2

<211> 330

<212> DNA

<213> artificial sequence

<220>

<223> TCR alpha chain variable Domain

<400> 2

ggaatacaag tggagcagag tcctccagac ctgattctcc aggagggagc caattccacg 60

ctgcggtgca atttttctga ctctgtgaac aatttgcagt ggtttcatca aaacccttgg 120

ggacagctca tcaacctgtt ttacattccc tcagggacaa aacagaatgg aagattaagc 180

gccacgactg tcgctacgga acgctacagc ttattgtaca tttcctcttc ccagaccaca 240

gactcaggcg tttatttctg tgctgtggga tctaactttg gaaatgagaa attaaccttt 300

gggactggaa caagactcac catcataccc 330

<210> 3

<211> 251

<212> PRT

<213> artificial sequence

<220>

<223> TCR alpha chain

<400> 3

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

1 5 10 15

Ala Asn Ser Thr Leu Arg Cys Asn Phe Ser Asp Ser Val Asn Asn Leu

20 25 30

Gln Trp Phe His Gln Asn Pro Trp Gly Gln Leu Ile Asn Leu Phe Tyr

35 40 45

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

50 55 60

Ala Thr Glu Arg Tyr Ser Leu Leu Tyr Ile Ser Ser Ser Gln Thr Thr

65 70 75 80

Asp Ser Gly Val Tyr Phe Cys Ala Val Gly Ser Asn Phe Gly Asn Glu

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

245 250

<210> 4

<211> 753

<212> DNA

<213> artificial sequence

<220>

<223> TCR alpha chain

<400> 4

ggaatacaag tggagcagag tcctccagac ctgattctcc aggagggagc caattccacg 60

ctgcggtgca atttttctga ctctgtgaac aatttgcagt ggtttcatca aaacccttgg 120

ggacagctca tcaacctgtt ttacattccc tcagggacaa aacagaatgg aagattaagc 180

gccacgactg tcgctacgga acgctacagc ttattgtaca tttcctcttc ccagaccaca 240

gactcaggcg tttatttctg tgctgtggga tctaactttg gaaatgagaa attaaccttt 300

gggactggaa caagactcac catcataccc aatatccaga accctgaccc tgccgtgtac 360

cagctgagag actctaaatc cagtgacaag tctgtctgcc tattcaccga ttttgattct 420

caaacaaatg tgtcacaaag taaggattct gatgtgtata tcacagacaa aactgtgcta 480

gacatgaggt ctatggactt caagagcaac agtgctgtgg cctggagcaa caaatctgac 540

tttgcatgtg caaacgcctt caacaacagc attattccag aagacacctt cttccccagc 600

ccagaaagtt cctgtgatgt caagctggtc gagaaaagct ttgaaacaga tacgaaccta 660

aactttcaaa acctgtcagt gattgggttc cgaatcctcc tcctgaaagt ggccgggttt 720

aatctgctca tgacgctgcg gctgtggtcc agc 753

<210> 5

<211> 114

<212> PRT

<213> artificial sequence

<220>

<223> TCR beta chain variable domain

<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

Arg Arg Leu Gly Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr

100 105 110

Val Val

<210> 6

<211> 342

<212> DNA

<213> artificial sequence

<220>

<223> TCR beta chain variable domain

<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 gcagtgctcg gaggctcggc 300

actgaagctt tctttggaca aggcaccaga ctcacagttg ta 342

<210> 7

<211> 291

<212> PRT

<213> artificial sequence

<220>

<223> TCR beta chain

<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

Arg Arg Leu Gly Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Lys Asp Phe

290

<210> 8

<211> 873

<212> DNA

<213> artificial sequence

<220>

<223> TCR beta chain

<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 gcagtgctcg gaggctcggc 300

actgaagctt tctttggaca aggcaccaga ctcacagttg tagaggacct gaacaaggtg 360

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 420

gccacactgg tgtgcctggc cacaggcttc ttccccgacc acgtggagct gagctggtgg 480

gtgaatggga aggaggtgca cagtggggtc agcacggacc cgcagcccct caaggagcag 540

cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 600

tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 660

gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 720

ggtagagcag actgtggctt tacctcggtg tcctaccagc aaggggtcct gtctgccacc 780

atcctctatg agatcctgct agggaaggcc accctgtatg ctgtgctggt cagcgccctt 840

gtgttgatgg ccatggtcaa gagaaaggat ttc 873

<210> 9

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> antigen short peptide

<400> 9

Ile Leu Trp Arg Leu Gly Ala Thr Ile

1 5

<210> 10

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> α CDR1

<400> 10

Asp Ser Val Asn Asn

1 5

<210> 11

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> α CDR2

<400> 11

Ile Pro Ser Gly Thr

1 5

<210> 12

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> α CDR3

<400> 12

Cys Ala Val Gly Ser Asn Phe Gly Asn Glu Lys Leu Thr Phe

1 5 10

<210> 13

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> β CDR1

<400> 13

Asp Phe Gln Ala Thr Thr

1 5

<210> 14

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> β CDR2

<400> 14

Ser Asn Glu Gly Ser Lys Ala

1 5

<210> 15

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> β CDR3

<400> 15

Cys Ser Ala Arg Arg Leu Gly Thr Glu Ala Phe Phe

1 5 10

<210> 16

<211> 15

<212> DNA

<213> artificial sequence

<220>

<223> α CDR1

<400> 16

gactctgtga acaat 15

<210> 17

<211> 15

<212> DNA

<213> artificial sequence

<220>

<223> α CDR2

<400> 17

attccctcag ggaca 15

<210> 18

<211> 42

<212> DNA

<213> artificial sequence

<220>

<223> α CDR3

<400> 18

tgtgctgtgg gatctaactt tggaaatgag aaattaacct tt 42

<210> 19

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> β CDR1

<400> 19

gactttcagg ccacaact 18

<210> 20

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> β CDR2

<400> 20

tccaatgagg gctccaaggc c 21

<210> 21

<211> 36

<212> DNA

<213> artificial sequence

<220>

<223> β CDR3

<400> 21

tgcagtgctc ggaggctcgg cactgaagct ttcttt 36

<210> 22

<211> 271

<212> PRT

<213> artificial sequence

<220>

<223> TCR alpha chain having leader sequence

<400> 22

Met Lys Arg Ile Leu Gly Ala Leu Leu Gly Leu Leu Ser Ala Gln Val

1 5 10 15

Cys Cys Val Arg Gly Ile Gln Val Glu Gln Ser Pro Pro Asp Leu Ile

20 25 30

Leu Gln Glu Gly Ala Asn Ser Thr Leu Arg Cys Asn Phe Ser Asp Ser

35 40 45

Val Asn Asn Leu Gln Trp Phe His Gln Asn Pro Trp Gly Gln Leu Ile

50 55 60

Asn Leu Phe Tyr Ile Pro Ser Gly Thr Lys Gln Asn Gly Arg Leu Ser

65 70 75 80

Ala Thr Thr Val Ala Thr Glu Arg Tyr Ser Leu Leu Tyr Ile Ser Ser

85 90 95

Ser Gln Thr Thr Asp Ser Gly Val Tyr Phe Cys Ala Val Gly Ser Asn

100 105 110

Phe Gly Asn Glu Lys Leu Thr Phe Gly Thr Gly Thr Arg Leu Thr Ile

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

<210> 23

<211> 813

<212> DNA

<213> artificial sequence

<220>

<223> TCR alpha chain having leader sequence

<400> 23

atgaagagga tattgggagc tctgctgggg ctcttgagtg cccaggtttg ctgtgtgaga 60

ggaatacaag tggagcagag tcctccagac ctgattctcc aggagggagc caattccacg 120

ctgcggtgca atttttctga ctctgtgaac aatttgcagt ggtttcatca aaacccttgg 180

ggacagctca tcaacctgtt ttacattccc tcagggacaa aacagaatgg aagattaagc 240

gccacgactg tcgctacgga acgctacagc ttattgtaca tttcctcttc ccagaccaca 300

gactcaggcg tttatttctg tgctgtggga tctaactttg gaaatgagaa attaaccttt 360

gggactggaa caagactcac catcataccc aatatccaga accctgaccc tgccgtgtac 420

cagctgagag actctaaatc cagtgacaag tctgtctgcc tattcaccga ttttgattct 480

caaacaaatg tgtcacaaag taaggattct gatgtgtata tcacagacaa aactgtgcta 540

gacatgaggt ctatggactt caagagcaac agtgctgtgg cctggagcaa caaatctgac 600

tttgcatgtg caaacgcctt caacaacagc attattccag aagacacctt cttccccagc 660

ccagaaagtt cctgtgatgt caagctggtc gagaaaagct ttgaaacaga tacgaaccta 720

aactttcaaa acctgtcagt gattgggttc cgaatcctcc tcctgaaagt ggccgggttt 780

aatctgctca tgacgctgcg gctgtggtcc agc 813

<210> 24

<211> 305

<212> PRT

<213> artificial sequence

<220>

<223> TCR beta chain having leader 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 Arg Arg

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Phe

305

<210> 25

<211> 915

<212> DNA

<213> artificial sequence

<220>

<223> TCR beta chain having leader 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 cggaggctcg gcactgaagc tttctttgga 360

caaggcacca gactcacagt tgtagaggac ctgaacaagg tgttcccacc cgaggtcgct 420

gtgtttgagc catcagaagc agagatctcc cacacccaaa aggccacact ggtgtgcctg 480

gccacaggct tcttccccga ccacgtggag ctgagctggt gggtgaatgg gaaggaggtg 540

cacagtgggg tcagcacgga cccgcagccc ctcaaggagc agcccgccct caatgactcc 600

agatactgcc tgagcagccg cctgagggtc tcggccacct tctggcagaa cccccgcaac 660

cacttccgct gtcaagtcca gttctacggg ctctcggaga atgacgagtg gacccaggat 720

agggccaaac ccgtcaccca gatcgtcagc gccgaggcct ggggtagagc agactgtggc 780

tttacctcgg tgtcctacca gcaaggggtc ctgtctgcca ccatcctcta tgagatcctg 840

ctagggaagg ccaccctgta tgctgtgctg gtcagcgccc ttgtgttgat ggccatggtc 900

aagagaaagg atttc 915

<210> 26

<211> 196

<212> PRT

<213> artificial sequence

<220>

<223> soluble TCR alpha chain

<400> 26

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

1 5 10 15

Ala Asn Ser Thr Leu Arg Cys Asn Phe Ser Asp Ser Val Asn Asn Leu

20 25 30

Gln Trp Phe His Gln Asn Pro Trp Gly Gln Leu Ile Asn Leu Phe Tyr

35 40 45

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

50 55 60

Ala Thr Glu Arg Tyr Ser Leu Leu Tyr Ile Ser Ser Ser Gln Thr Thr

65 70 75 80

Asp Ser Gly Val Tyr Phe Cys Ala Val Gly Ser Asn Phe Gly Asn Glu

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Pro Glu Asp Thr

195

<210> 27

<211> 588

<212> DNA

<213> artificial sequence

<220>

<223> soluble TCR alpha chain

<400> 27

ggtattcagg ttgaacagag tcctccagac ctgattctcc aggagggagc caattccacg 60

ctgcggtgca atttttctga ctctgtgaac aatttgcagt ggtttcatca aaacccttgg 120

ggacagctca tcaacctgtt ttacattccc tcagggacaa aacagaatgg aagattaagc 180

gccacgactg tcgctacgga acgctacagc ttattgtaca tttcctcttc ccagaccaca 240

gactcaggcg tttatttctg tgctgtggga tctaactttg gaaatgagaa attaaccttt 300

gggactggaa caagactcac catcataccc tatatccaga atccggaccc ggccgtttat 360

cagctgcgtg atagcaaaag cagcgataaa agcgtgtgcc tgttcaccga ttttgatagc 420

cagaccaacg tgagccagag caaagatagc gatgtgtaca tcaccgataa atgcgtgctg 480

gatatgcgca gcatggattt caaaagcaat agcgcggttg cgtggagcaa caaaagcgat 540

tttgcgtgcg cgaacgcgtt taacaacagc atcatcccgg aagatacg 588

<210> 28

<211> 244

<212> PRT

<213> artificial sequence

<220>

<223> soluble TCR beta chain

<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

Arg Arg Leu Gly Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly Arg Ala Asp

<210> 29

<211> 732

<212> DNA

<213> artificial sequence

<220>

<223> soluble TCR beta chain

<400> 29

ggtgcagttg tttctcagca 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 gcagtgctcg gaggctcggc 300

actgaagctt tctttggaca aggcaccaga ctcacagttg tagaagatct gaaaaatgtg 360

tttccgccgg aagtcgcggt gttcgaaccg tcggaagccg aaattagcca tacccagaaa 420

gcaacgctgg tgtgcctggc taccggcttt tatccggatc atgtggaact gtcctggtgg 480

gttaacggca aagaagtgca ctcaggtgtt tgtacggatc cgcagccgct gaaagaacaa 540

ccggcactga atgactcgcg ttatgctctg agttcccgtc tgcgcgttag cgccaccttc 600

tggcaggatc cgcgtaacca ctttcgctgt caggtccaat tctacggcct gtccgaaaat 660

gatgaatgga cccaggaccg tgcaaaaccg gtcacgcaaa tcgtgtcagc agaagcttgg 720

ggtcgtgcag at 732

Claims (27)

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

α CDR1- DSVNN (SEQ ID NO: 10)

α CDR2- IPSGT (SEQ ID NO: 11)

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

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

β CDR1- DFQATT (SEQ ID NO: 13)

β CDR2- SNEGSKA (SEQ ID NO: 14)

β CDR3- CSARRLGTEAFF (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; 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.

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

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

5. 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.

6. A TCR as claimed in claim 1 wherein 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.

7. A TCR as claimed in claim 1 which is formed by the α chain variable domain linked to the β chain variable domain by a peptide linker sequence.

8. A TCR as claimed in claim 1 which 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,

(a) and (b) each comprises a functional variable domain and further comprises at least a portion of the TCR chain constant domain.

9. A TCR as claimed in claim 1 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.

10. A TCR as claimed in claim 1 which comprises an artificial interchain disulphide bond between the α chain variable region and the β chain constant region of the TCR.

11. A TCR as claimed in claim 1 which comprises an α chain variable domain and a β chain variable domain and all or part of the β chain constant domain, excluding the transmembrane domain, but which does not comprise an α chain constant domain, the α chain variable domain of the TCR forming a heterodimer with the β chain.

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

13. A TCR as claimed in claim 12 wherein the conjugate which binds to the T cell receptor is a detectable label, a therapeutic agent, a PK modifying moiety or a combination thereof.

14. A TCR as claimed in claim 13 wherein the therapeutic agent is an anti-CD 3 antibody.

15. 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 14.

16. A nucleic acid molecule comprising a nucleic acid sequence encoding a TCR according to any one of claims 1 to 14 or the complement thereof.

17. The nucleic acid molecule of claim 16, wherein said nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 2.

18. the nucleic acid molecule of claim 16 or 17, wherein said nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 6.

19. the nucleic acid molecule of claim 16, wherein the nucleic acid molecule comprises 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.

20. a vector comprising the nucleic acid molecule of any one of claims 16-19.

21. The vector of claim 20, wherein said vector is a viral vector.

22. The vector of claim 20, wherein said vector is a lentiviral vector.

23. An isolated host cell comprising the vector or chromosome of any one of claims 20-22 and the exogenous nucleic acid molecule of any one of claims 16-19 integrated therein.

24. A cell transduced with the nucleic acid molecule of any one of claims 16 to 19 or the vector of claim 20.

25. The cell of claim 24, wherein the cell is a T cell or a stem cell.

26. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to any one of claims 1 to 14, a TCR complex according to claim 15, a nucleic acid molecule according to any one of claims 16 to 19, a vector according to any one of claims 20 to 22, or a cell according to claim 24 or 25.

27. Use of a TCR as claimed in any one of claims 1 to 14 or a TCR complex as claimed in claim 15, a nucleic acid molecule as claimed in any one of claims 16 to 19, a vector as claimed in claims 20 to 22 or a cell as claimed in claim 24 or 25 in the manufacture of a medicament for the treatment of a tumour.

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