CN109575121B - T cell receptor recognizing AFP antigen short peptides - Google Patents

Abstract

The present invention provides a T Cell Receptor (TCR) capable of specifically binding short peptide FMNKFIYEI derived from AFP antigen, said antigen short peptide FMNKFIYEI being capable of forming a complex with HLA a0201 and being 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

T cell receptor recognizing AFP antigen short peptides

Technical Field

The present invention relates to TCRs capable of recognizing short peptides derived from the AFP antigen, to AFP-specific T cells obtained by transduction of such TCRs, and to their use in the prevention and treatment of AFP-related diseases.

Background

AFP (alpha Fetoprotein), also called alpha Fetoprotein, is a protein expressed during embryonic development and is the main component of embryonic serum. During development, AFP is expressed at relatively high levels in the yolk sac and liver, and is subsequently inhibited. In hepatocellular carcinoma, AFP expression is activated (Butterfield et al.J. Immunol.,2001, Apr 15; 166(8): 5300-8). AFP is degraded into small molecule polypeptides after intracellular production and binds to MHC (major histocompatibility complex) molecules to form complexes, which are presented on the cell surface. FMNKFIYEI (SEQ ID NO:9) are short peptides derived from the AFP antigen and are one of the targets for the treatment of AFP-related diseases.

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 combination 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 can exert immune effects on target cells. Accordingly, those skilled in the art have focused on isolating TCRs specific for short AFP antigen peptides and transducing the TCRs into T cells to obtain T cells specific for short AFP antigen peptides, thereby allowing them to function in cellular immunotherapy.

Disclosure of Invention

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

In a first aspect of the invention, there is provided a T Cell Receptor (TCR) capable of binding to the FMNKFIYEI-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 ATDPRTGANSKLT (SEQ ID NO: 12); and/or the amino acid sequence of CDR3 of the variable domain of the TCR beta chain is ASEGLAYEQY (SEQ ID NO: 15).

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

αCDR1-TSINN(SEQ ID NO:10)

αCDR2-IRSNERE(SEQ ID NO:11)

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

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

βCDR1-SEHNR(SEQ ID NO:13)

βCDR2-FQNEAQ(SEQ ID NO:14)

βCDR3-ASEGLAYEQY(SEQ ID NO:15)。

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

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

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

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

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

In another preferred embodiment, the TCR is soluble.

In another preferred embodiment, the TCR is single chain.

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

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

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

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

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

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

In another preferred embodiment, the cysteine residues form an artificial disulfide bond between the alpha and beta chain constant domains of the TCR.

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

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

thr45 and TRBC1 x 01 of TRAC x 01 exon 1 or Ser77 of TRBC2 x 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 x 01 of TRAC x 01 exon 1 or Glu15 of TRBC2 x 01 exon 1;

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

pro89 and TRBC1 x 01 of TRAC x 01 exon 1 or Ala19 of TRBC2 x 01 exon 1; 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 are substituted at one or more groups of sites selected from:

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

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

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 comprises a conjugate attached to the C-or N-terminus of the α chain and/or β 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 or SEQ ID NO: 33.

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

In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence encoding the TCR α chain SEQ ID NO:4 and/or a nucleic acid sequence comprising 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 is hepatocellular carcinoma.

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

Drawings

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

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

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

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

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

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

Fig. 9a and 9b are the amino acid and nucleotide sequences, respectively, of a single chain TCR β chain.

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

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

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

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

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

FIG. 15 results of functional validation of ELISPOT activation of effector cells that have not transduced the TCR of the invention.

Detailed Description

The present inventors have extensively and intensively studied to find a TCR capable of specifically binding to AFP antigen short peptide FMNKFIYEI (SEQ ID NO:9), which antigen short peptide FMNKFIYEI 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 often referred to as 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 intracellularly and then carried to the cell surface through 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 FMNKFIYEI-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-TSINN(SEQ ID NO:10)

αCDR2-IRSNERE(SEQ ID NO:11)

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

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

βCDR1-SEHNR(SEQ ID NO:13)

βCDR2-FQNEAQ(SEQ ID NO:14)

βCDR3-ASEGLAYEQY(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 identical to SEQ ID NO:5, having at least 90%, preferably 95%, more preferably 98% sequence identity.

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

In a preferred embodiment of the invention, the TCR molecules of the invention are single chain TCR molecules consisting of part or all of the α chain and/or part or all of the β chain. Single chain TCR molecules are described in Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-. 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 an alpha chain variable domain amino acid sequence SEQ ID NO 1. More preferably, the α chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO 1. The amino acid sequence of the variable domain of the beta chain 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 beta chain described above. Preferably, the single chain TCR molecule comprises a beta chain variable domain amino acid sequence SEQ ID NO 5. More preferably, the amino acid sequence of the β chain variable domain of the single chain TCR molecule is SEQ ID NO 5.

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

Naturally occurring TCRs are membrane proteins that are stabilized by their transmembrane regions. Like immunoglobulins (antibodies) as antigen recognition molecules, TCRs can also be developed for diagnostic and therapeutic applications, where it is desirable to obtain soluble TCR molecules. Soluble TCR molecules do not include their transmembrane regions. Soluble TCRs have a wide range of uses, not only for studying the interaction of TCRs with pmhcs, but also as diagnostic tools for detecting infection or as markers for autoimmune diseases. Similarly, soluble TCRs can be used to deliver therapeutic agents (e.g., cytotoxic or immunostimulatory compounds) to cells presenting a specific antigen, and in addition, soluble TCRs can be conjugated to other molecules (e.g., anti-CD 3 antibodies) to redirect T cells to target them to cells presenting a particular antigen. The present invention also provides soluble TCRs with specificity for AFP 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 x 01 of TRAC x 01 exon 1 or Ala19 of TRBC2 x 01 exon 1; or Tyr10 and TRBC1 x 01 of TRAC x 01 exon 1 or TRBC2 x 01 of Glu20 of exon 1. I.e., a cysteine residue, in place of any of the above-described alpha and beta chain constant domains. Deletion of the native disulfide bond can be achieved by truncating at most 50, or at most 30, or at most 15, or at most 10, or at most 8 or fewer amino acids at one or more of the C-termini of the TCR constant domains of the invention such that they do not include a cysteine residue, or by mutating a cysteine residue that forms a native disulfide 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 a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, with or without the artificial disulfide bond 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, the inventive TCR, on the other hand, also includes TCRs having mutations in their hydrophobic core region, preferably mutations that improve the stability of the inventive soluble TCR, as described in 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 from the last amino acid position of the short peptide of the alpha chain J gene (TRAJ), and/or positions 2,4,6 from the last amino acid position of the short peptide of the beta chain J gene (TRBJ), wherein the numbering of the positions of the amino acid sequences is according to the numbering of the positions listed in the International Immunogenetic information System (IMGT). The above-mentioned international system of immunogenetics information is known to the skilled person and the position numbering of the amino acid residues of the different TCRs in IMGT can be derived from this database.

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

In addition, for stability, patent document 201510260322.4 also discloses that the introduction of an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR can significantly improve the stability of the TCR. Thus, the high affinity TCRs of the invention may also contain an artificial interchain disulfide bond between the α chain variable region and the β chain constant region. Specifically, the cysteine residues that form the artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR are substituted for: amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01; amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01; amino acid 46 of TRAV and amino acid 61 of TRBC1 x 01 or TRBC2 x 01 exon 1; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01. Preferably, such a TCR may comprise (i) all or part of a TCR α chain, excluding its transmembrane domain, and (ii) all or part of a TCR β chain, excluding its transmembrane domain, wherein (i) and (ii) both comprise 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 also to generate intermediates for other multivalent TCR complexes having such applications.

The TCRs of the invention may be used alone or in covalent or other association, preferably covalently, with a conjugate. The conjugates include a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the FMNKFIYEI-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 with or coupled to 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. Therefore, there should be a regulatory scheme for immunosuppression when it is used for adoptive T cell therapy to allow for the engraftment of murine expressing T cells.

It should be understood that the amino acid names herein are expressed in terms of international single-letter or three-letter english letters, and the single-letter english letter and three-letter english letters of the amino acid names correspond to the following relationships: ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y) and Val (V).

Nucleic acid molecules

A second aspect of the invention provides a nucleic acid molecule encoding a TCR molecule of the first aspect of the invention or a 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 CDR regions of the α chain of the TCR molecule of the first aspect of the invention is as follows:

αCDR1-actagtataaacaat(SEQ ID NO:16)

αCDR2-atacgttcaaatgaaagagag(SEQ ID NO:17)

αCDR3-gctacggaccctcgaactggagccaatagtaagctgaca(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-tctgaacacaaccgc(SEQ ID NO:19)

βCDR2-ttccagaatgaagctcaa(SEQ ID NO:20)

βCDR3-gccagcgagggactagcgtacgagcagtac(SEQ ID NO:21)

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

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

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

The nucleotide sequence may be codon optimized. Different cells differ in the utilization of specific codons, and the expression level can be increased by changing the codons in the sequence according to the type of the cell. Codon usage tables for mammalian cells as well as for various other organisms are well known to those skilled in the art.

The full-length sequence of the nucleic acid molecule of the present invention or a fragment thereof can be obtained by, but not limited to, PCR amplification, recombination, or artificial synthesis. At present, DNA sequences encoding the TCRs of the invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. The DNA may be the coding strand or the non-coding strand.

Carrier

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

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

Preferably, the vector can transfer the nucleotide of the invention into a cell, e.g., a T cell, such that the cell expresses a TCR specific for the AFP 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).

AFP antigen associated diseases

The present invention also relates to a method for the treatment and/or prevention of a disease associated with AFP in a subject, comprising the step of adoptive transfer of AFP-specific T cells to the subject. The AFP-specific T cells recognize FMNKFIYEI-HLA A0201 complex.

The AFP-specific T-cells of the invention can be used to treat any AFP-related disease presenting an AFP antigen short peptide FMNKFIYEI-HLA A0201 complex. Including but not limited to tumors such as hepatocellular carcinoma.

Method of treatment

Treatment may be effected by isolating T cells from patients or volunteers suffering from a disease associated with the AFP 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 an AFP-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 itself. 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 inventive TCR can be combined with AFP antigen short peptide complex FMNKFIYEI-HLA A0201, and the cell transduced with the inventive TCR can be specifically activated and has strong killing effect on target cell.

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 Russell 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 cloned AFP antigen short peptide specific T cells

Peripheral Blood Lymphocytes (PBLs) from healthy volunteers of genotype HLA-A0201 were stimulated with synthetic short peptide FMNKFIYEI (SEQ ID NO: 9; Beijing Baisheng Gene technology, Inc.). FMNKFIYEI short peptide and HLA-A0201 with biotin label are renatured to prepare 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. Monoclonal cells were stained with tetramer and double positive clones selected are shown in FIG. 3.

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

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

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

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

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

αCDR1-TSINN(SEQ ID NO:10)

αCDR2-IRSNERE(SEQ ID NO:11)

αCDR3-ATDPRTGANSKLT(SEQ ID NO:12)

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

βCDR1-SEHNR(SEQ ID NO:13)

βCDR2-FQNEAQ(SEQ ID NO:14)

βCDR3-ASEGLAYEQY(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 comprises the following specific steps: the TCR alpha chain and the TCR beta chain are connected by overlap PCR to obtain the TCR alpha-2A-TCR beta segment. And (3) carrying out enzyme digestion and connection on the lentivirus expression vector and the TCR alpha-2A-TCR beta to obtain pLenti-TRA-2A-TRB-IRES-NGFR plasmid. As a control, a lentiviral vector pLenti-eGFP expressing eGFP was also constructed. The pseudovirus was then packaged again at 293T/17.

Example 3 expression, refolding and purification of AFP 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 respectively transformed into the table by a chemical transformation methodReach bacterium BL21(DE3), which was grown in LB medium at OD ₆₀₀ Inclusion bodies formed after expression of the α and β chains of the TCR were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution several times at 0.6 final induction with final concentration of 0.5mM IPTG, and finally dissolved in 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT),10mM ethylenediaminetetraacetic acid (EDTA),20mM Tris (pH 8.1).

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

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

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

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

Example 5 expression, renaturation and purification of soluble Single chain TCR specific for short peptides of the AFP antigen

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

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

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

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

Example 6 binding characterization

BIAcore analysis

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

Binding activity of the TCR molecules obtained in examples 3 and 5 to the FMNKFIYEI-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 FMNKFIYEI-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 FMNKFIYEI-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 FMNKFIYEI (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. FMNKFIYEI 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 the biotinylated Complex

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.) ^TM 16/60S200HR 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 single peak elution at approximately 55 ml. The fractions containing the protein were pooled, concentrated using a Mill ipore ultrafiltration tube, protein concentration was determined by BCA (Thermo), and biotinylated pMHC molecules were stored in aliquots at-80 ℃ with the addition of the protease inhibitor cocktail (Roche).

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

Example 7 activation of T cells transducing TCRs of the invention

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

ELISPOT scheme

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

Reagent

Test medium: 10% FBS (Gibco, catalog No. 16000-

Wash buffer (PBST): 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 antibodies, streptavidin-alkaline phosphatase and BCIP/NBT solution)

Method

Target cell preparation

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

Effector cell preparation

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

Preparation of short peptide solution

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

ELISPOT

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

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

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

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

All wells were prepared in duplicate for addition.

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

The mixture was diluted with PBS containing 10% FBS at 1: streptavidin-alkaline phosphatase was diluted 100, 100 microliters of diluted streptavidin-alkaline phosphatase was added to each well and the wells were incubated for 1 hour at room temperature. The plates were then washed 2 times with 4 washes of PBS and tapped on a paper towel to remove excess wash buffer and PBS. After washing, 100 microliter of BCIP/NBT solution provided by the kit is added for development. And covering the well plate with tinfoil paper in the developing period, keeping the well plate in the dark, and standing for 5-15 minutes. 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 TCR-transduced T cells of the invention were tested for IFN- γ release in response to target cells loaded with AFP antigen short peptide FMNKFIYEI by ELISPOT assay (as described above). The number of ELSPOT spots observed in each well was plotted using a graphipad prism 6.

As shown in FIG. 15, T cells transduced with the TCR of the invention were shown to be very active against target cells loaded with their specific short peptides, whereas T cells not transduced with the TCR of the invention were shown to be essentially non-active against the corresponding target cells.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes 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> Guangdong Xiangxue accurate medical technology Limited

<120> T cell receptor recognizing AFP antigen short peptide

<130> P2017-1880

<160> 37

<170> PatentIn version 3.5

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<213> Artificial sequence (Artificial sequence)

<400> 2

agtcaacagg gagaagagga tcctcaggcc ttgagcatcc aggagggtga aaatgccacc 60

atgaactgca gttacaaaac tagtataaac aatttacagt ggtatagaca aaattcaggt 120

agaggccttg tccacctaat tttaatacgt tcaaatgaaa gagagaaaca cagtggaaga 180

ttaagagtca cgcttgacac ttccaagaaa agcagttcct tgttgatcac ggcttcccgg 240

gcagcagaca ctgcttctta cttctgtgct acggaccctc gaactggagc caatagtaag 300

ctgacatttg gaaaaggaat aactctgagt gttagaccag at 342

<210> 3

<211> 254

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 3

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250

<210> 4

<211> 762

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

agtcaacagg gagaagagga tcctcaggcc ttgagcatcc aggagggtga aaatgccacc 60

atgaactgca gttacaaaac tagtataaac aatttacagt ggtatagaca aaattcaggt 120

agaggccttg tccacctaat tttaatacgt tcaaatgaaa gagagaaaca cagtggaaga 180

ttaagagtca cgcttgacac ttccaagaaa agcagttcct tgttgatcac ggcttcccgg 240

gcagcagaca ctgcttctta cttctgtgct acggaccctc gaactggagc caatagtaag 300

ctgacatttg gaaaaggaat aactctgagt gttagaccag atatccagaa ccctgaccct 360

gccgtgtacc agctgagaga ctctaaatcc agtgacaagt ctgtctgcct attcaccgat 420

tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 480

actgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 540

aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 600

ttccccagcc cagaaagttc ctgtgatgtc aagctggtcg agaaaagctt tgaaacagat 660

acgaacctaa actttcaaaa cctgtcagtg attgggttcc gaatcctcct cctgaaagtg 720

gccgggttta atctgctcat gacgctgcgg ctgtggtcca gc 762

<210> 5

<211> 112

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 5

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

1 5 10 15

Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu

20 25 30

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

35 40 45

Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg

50 55 60

Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln

65 70 75 80

Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Glu Gly

85 90 95

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

100 105 110

<210> 6

<211> 336

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

gatactggag tctcccagga ccccagacac aagatcacaa agaggggaca gaatgtaact 60

ttcaggtgtg atccaatttc tgaacacaac cgcctttatt ggtaccgaca gaccctgggg 120

cagggcccag agtttctgac ttacttccag aatgaagctc aactagaaaa atcaaggctg 180

ctcagtgatc ggttctctgc agagaggcct aagggatctt tctccacctt ggagatccag 240

cgcacagagc agggggactc ggccatgtat ctctgtgcca gcgagggact agcgtacgag 300

cagtacttcg ggccgggcac caggctcacg gtcaca 336

<210> 7

<211> 291

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 7

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

1 5 10 15

Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu

20 25 30

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

35 40 45

Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg

50 55 60

Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln

65 70 75 80

Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Glu Gly

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Ser Arg Gly

290

<210> 8

<211> 873

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

gatactggag tctcccagga ccccagacac aagatcacaa agaggggaca gaatgtaact 60

ttcaggtgtg atccaatttc tgaacacaac cgcctttatt ggtaccgaca gaccctgggg 120

cagggcccag agtttctgac ttacttccag aatgaagctc aactagaaaa atcaaggctg 180

ctcagtgatc ggttctctgc agagaggcct aagggatctt tctccacctt ggagatccag 240

cgcacagagc agggggactc ggccatgtat ctctgtgcca gcgagggact agcgtacgag 300

cagtacttcg ggccgggcac caggctcacg gtcacagagg acctgaaaaa cgtgttccca 360

cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 420

ctggtgtgcc tggccacagg cttctacccc gaccacgtgg agctgagctg gtgggtgaat 480

gggaaggagg tgcacagtgg ggtcagcaca gacccgcagc ccctcaagga gcagcccgcc 540

ctcaatgact ccagatactg cctgagcagc cgcctgaggg tctcggccac cttctggcag 600

aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 660

tggacccagg atagggccaa acctgtcacc cagatcgtca gcgccgaggc ctggggtaga 720

gcagactgtg gcttcacctc cgagtcttac cagcaagggg tcctgtctgc caccatcctc 780

tatgagatct tgctagggaa ggccaccttg tatgccgtgc tggtcagtgc cctcgtgctg 840

atggccatgg tcaagagaaa ggattccaga ggc 873

<210> 9

<211> 9

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 9

Phe Met Asn Lys Phe Ile Tyr Glu Ile

1 5

<210> 10

<211> 5

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 10

Thr Ser Ile Asn Asn

1 5

<210> 11

<211> 7

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 11

Ile Arg Ser Asn Glu Arg Glu

1 5

<210> 12

<211> 13

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 12

Ala Thr Asp Pro Arg Thr Gly Ala Asn Ser Lys Leu Thr

1 5 10

<210> 13

<211> 5

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 13

Ser Glu His Asn Arg

1 5

<210> 14

<211> 6

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 14

Phe Gln Asn Glu Ala Gln

1 5

<210> 15

<211> 10

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 15

Ala Ser Glu Gly Leu Ala Tyr Glu Gln Tyr

1 5 10

<210> 16

<211> 15

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 16

actagtataa acaat 15

<210> 17

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 17

atacgttcaa atgaaagaga g 21

<210> 18

<211> 39

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 18

gctacggacc ctcgaactgg agccaatagt aagctgaca 39

<210> 19

<211> 15

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 19

tctgaacaca accgc 15

<210> 20

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 20

ttccagaatg aagctcaa 18

<210> 21

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 21

gccagcgagg gactagcgta cgagcagtac 30

<210> 22

<211> 274

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 22

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Ser Ser

<210> 23

<211> 822

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 23

atggaaactc tcctgggagt gtctttggtg attctatggc ttcaactggc tagggtgaac 60

agtcaacagg gagaagagga tcctcaggcc ttgagcatcc aggagggtga aaatgccacc 120

atgaactgca gttacaaaac tagtataaac aatttacagt ggtatagaca aaattcaggt 180

agaggccttg tccacctaat tttaatacgt tcaaatgaaa gagagaaaca cagtggaaga 240

ttaagagtca cgcttgacac ttccaagaaa agcagttcct tgttgatcac ggcttcccgg 300

gcagcagaca ctgcttctta cttctgtgct acggaccctc gaactggagc caatagtaag 360

ctgacatttg gaaaaggaat aactctgagt gttagaccag atatccagaa ccctgaccct 420

gccgtgtacc agctgagaga ctctaaatcc agtgacaagt ctgtctgcct attcaccgat 480

tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 540

actgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 600

aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 660

ttccccagcc cagaaagttc ctgtgatgtc aagctggtcg agaaaagctt tgaaacagat 720

acgaacctaa actttcaaaa cctgtcagtg attgggttcc gaatcctcct cctgaaagtg 780

gccgggttta atctgctcat gacgctgcgg ctgtggtcca gc 822

<210> 24

<211> 310

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 24

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu

65 70 75 80

Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu

85 90 95

Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Arg Lys Asp Ser Arg Gly

305 310

<210> 25

<211> 930

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 25

atgggcacca gcctcctctg ctggatggcc ctgtgtctcc tgggggcaga tcacgcagat 60

actggagtct cccaggaccc cagacacaag atcacaaaga ggggacagaa tgtaactttc 120

aggtgtgatc caatttctga acacaaccgc ctttattggt accgacagac cctggggcag 180

ggcccagagt ttctgactta cttccagaat gaagctcaac tagaaaaatc aaggctgctc 240

agtgatcggt tctctgcaga gaggcctaag ggatctttct ccaccttgga gatccagcgc 300

acagagcagg gggactcggc catgtatctc tgtgccagcg agggactagc gtacgagcag 360

tacttcgggc cgggcaccag gctcacggtc acagaggacc tgaaaaacgt gttcccaccc 420

gaggtcgctg tgtttgagcc atcagaagca gagatctccc acacccaaaa ggccacactg 480

gtgtgcctgg ccacaggctt ctaccccgac cacgtggagc tgagctggtg ggtgaatggg 540

aaggaggtgc acagtggggt cagcacagac ccgcagcccc tcaaggagca gcccgccctc 600

aatgactcca gatactgcct gagcagccgc ctgagggtct cggccacctt ctggcagaac 660

ccccgcaacc acttccgctg tcaagtccag ttctacgggc tctcggagaa tgacgagtgg 720

acccaggata gggccaaacc tgtcacccag atcgtcagcg ccgaggcctg gggtagagca 780

gactgtggct tcacctccga gtcttaccag caaggggtcc tgtctgccac catcctctat 840

gagatcttgc tagggaaggc caccttgtat gccgtgctgg tcagtgccct cgtgctgatg 900

gccatggtca agagaaagga ttccagaggc 930

<210> 26

<211> 208

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 26

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

<210> 27

<211> 624

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 27

atgagccagc agggcgaaga agatcctcag gccttgagca tccaggaggg tgaaaatgcc 60

accatgaact gcagttacaa aactagtata aacaatttac agtggtatag acaaaattca 120

ggtagaggcc ttgtccacct aattttaata cgttcaaatg aaagagagaa acacagtgga 180

agattaagag tcacgcttga cacttccaag aaaagcagtt ccttgttgat cacggcttcc 240

cgggcagcag acactgcttc ttacttctgt gctacggacc ctcgaactgg agccaatagt 300

aagctgacat ttggaaaagg aataactctg agtgttagac cagatatcca gaaccctgac 360

cctgccgtgt accagctgag agactctaag tcgagtgaca agtctgtctg cctattcacc 420

gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 480

aaatgtgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 540

aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 600

ttcttcccca gcccagaaag ttcc 624

<210> 28

<211> 243

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 28

Met Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg

1 5 10 15

Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg

20 25 30

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

35 40 45

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

50 55 60

Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile

65 70 75 80

Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Glu

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Arg Ala Asp

<210> 29

<211> 729

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 29

atggataccg gcgtgagcca ggaccccaga cacaagatca caaagagggg acagaatgta 60

actttcaggt gtgatccaat ttctgaacac aaccgccttt attggtaccg acagaccctg 120

gggcagggcc cagagtttct gacttacttc cagaatgaag ctcaactaga aaaatcaagg 180

ctgctcagtg atcggttctc tgcagagagg cctaagggat ctttctccac cttggagatc 240

cagcgcacag agcaggggga ctcggccatg tatctctgtg ccagcgaggg actagcgtac 300

gagcagtact tcgggccggg caccaggctc acggtcacag aggacctgaa aaacgtgttc 360

ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 420

acactggtgt gcctggccac cggtttctac cccgaccacg tggagctgag ctggtgggtg 480

aatgggaagg aggtgcacag tggggtctgc acagacccgc agcccctcaa ggagcagccc 540

gccctcaatg actccagata cgctctgagc agccgcctga gggtctcggc caccttctgg 600

caggaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 660

gagtggaccc aggatagggc caaacccgtc acccagatcg tcagcgccga ggcctggggt 720

agagcagac 729

<210> 30

<211> 250

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 30

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Arg Pro Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser

115 120 125

Glu Gly Gly Gly Ser Glu Gly Gly Thr Gly Asp Thr Gly Val Ser Gln

130 135 140

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

145 150 155 160

Cys Asp Pro Ile Ser Glu His Asn Arg Leu Tyr Trp Tyr Arg Gln Thr

165 170 175

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

180 185 190

Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg Phe Ser Ala Glu Arg Pro

195 200 205

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

210 215 220

Ser Ala Met Tyr Leu Cys Ala Ser Glu Gly Leu Ala Tyr Glu Gln Tyr

225 230 235 240

Phe Gly Pro Gly Thr Arg Leu Thr Val Thr

245 250

<210> 31

<211> 750

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 31

ggttctcaac aaggtgaaga agacccgcag gcgctgagca tccaagaggg cgaaaacgtg 60

accattaact gcagctacaa gaccagcatc aacaacctgc agtggtatcg tcaaaacagc 120

ggtcgtggcc tggttcacct gatcctgatt cgtagcaacg agcgtgaaaa acacagcggt 180

cgtctgcgtg tgaccctgga caccagcaag aaaagcagca gcctggagat taccgacgtt 240

cgtccgagcg ataccgcgag ctatttttgc gcgaccgatc cgcgtaccgg tgcgaacagc 300

aagctgacct ttggtaaagg tattaccctg agcgtgcgtc cgggtggcgg tagcgagggc 360

ggtggcagcg aaggtggcgg tagcgagggc ggtggcagcg aaggtggcac cggtgacacc 420

ggcgttagcc aggatccgcg tcacctgagc gtgaagcgtg gtcaaaacgt taccctgcgt 480

tgcgatccga ttagcgagca caaccgtctg tactggtatc gtcagacccc gggtcaaggc 540

ccggaattcc tgacctactt tcagaacgag gcgcaactgg aaaagagccg tctgctgagc 600

gaccgtttca gcgcggaacg tccgaaaggt agctttagca ccctggagat ccagcgtgtg 660

gaaccgggtg atagcgcgat gtatctgtgc gcgagcgagg gcctggcgta cgaacaatat 720

ttcggtccgg gcacccgtct gaccgttacc 750

<210> 32

<211> 114

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 32

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Arg Pro

<210> 33

<211> 342

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 33

ggttctcaac aaggtgaaga agacccgcag gcgctgagca tccaagaggg cgaaaacgtg 60

accattaact gcagctacaa gaccagcatc aacaacctgc agtggtatcg tcaaaacagc 120

ggtcgtggcc tggttcacct gatcctgatt cgtagcaacg agcgtgaaaa acacagcggt 180

cgtctgcgtg tgaccctgga caccagcaag aaaagcagca gcctggagat taccgacgtt 240

cgtccgagcg ataccgcgag ctatttttgc gcgaccgatc cgcgtaccgg tgcgaacagc 300

aagctgacct ttggtaaagg tattaccctg agcgtgcgtc cg 342

<210> 34

<211> 112

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 34

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

1 5 10 15

Gln Asn Val Thr Leu Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu

20 25 30

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

35 40 45

Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg

50 55 60

Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln

65 70 75 80

Arg Val Glu Pro Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Glu Gly

85 90 95

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

100 105 110

<210> 35

<211> 336

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 35

gacaccggcg ttagccagga tccgcgtcac ctgagcgtga agcgtggtca aaacgttacc 60

ctgcgttgcg atccgattag cgagcacaac cgtctgtact ggtatcgtca gaccccgggt 120

caaggcccgg aattcctgac ctactttcag aacgaggcgc aactggaaaa gagccgtctg 180

ctgagcgacc gtttcagcgc ggaacgtccg aaaggtagct ttagcaccct ggagatccag 240

cgtgtggaac cgggtgatag cgcgatgtat ctgtgcgcga gcgagggcct ggcgtacgaa 300

caatatttcg gtccgggcac ccgtctgacc gttacc 336

<210> 36

<211> 24

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 36

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

1 5 10 15

Gly Gly Ser Glu Gly Gly Thr Gly

20

<210> 37

<211> 72

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 37

ggtggcggta gcgagggcgg tggcagcgaa ggtggcggta gcgagggcgg tggcagcgaa 60

ggtggcaccg gt 72

Claims (37)

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

αCDR1-TSINN(SEQ ID NO:10)

αCDR2-IRSNERE(SEQ ID NO:11)

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

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

βCDR1-SEHNR(SEQ ID NO:13)

βCDR2-FQNEAQ(SEQ ID NO:14)

βCDR3-ASEGLAYEQY(SEQ ID NO:15)。

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

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

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

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

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

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

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

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

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

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

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

13. A TCR as claimed in claim 12 which has the amino acid sequence SEQ ID No. 30.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

37. Use of a TCR as claimed in any of claims 1 to 24, or a TCR complex as claimed in claim 25 or a cell as claimed in claim 34, for the preparation of a medicament for the treatment of an AFP-associated tumour or a cell presenting an AFP antigen short peptide FMNKFIYEI-HLA a0201 complex, said tumour being hepatocellular carcinoma.

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