CN110577590B - TCR capable of recognizing AFP antigen and encoding nucleic acid thereof - 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

TCR capable of recognizing AFP antigen and encoding nucleic acid thereof

Technical Field

The present invention relates to a TCR capable of recognizing a short peptide derived from an AFP antigen and the coding sequence thereof, to AFP-specific T cells obtained by transduction of the above TCR, 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) is a short peptide derived from the AFP antigen, which is a target 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 binding of antigen short peptide specific TCR and short peptide-major histocompatibility complex (pMHC complex), and then other cell membrane surface molecules of the T cells and APC interact to cause a series of subsequent cell signaling and other physiological reactions, so that T cells with different antigen specificities exert immune effects on their target cells. 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 AVMGDSNYQLI (SEQ ID NO: 12); and/or the amino acid sequence of CDR3 of the variable domain of the TCR beta chain is ASSQNLAGGPGTDTQY (SEQ ID NO: 15).

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

α CDR1-YGGTVN(SEQ ID NO:10)

α CDR2-YFSGDPLV(SEQ ID NO:11)

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

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

β CDR1-SGHVS(SEQ ID NO:13)

β CDR2-FQNEAQ(SEQ ID NO:14)

β CDR3-ASSQNLAGGPGTDTQY(SEQ ID NO:15)。

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

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

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

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

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

In another preferred embodiment, the TCR is soluble.

In another preferred embodiment, the TCR is single chain.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 has a conjugate attached to the C-or N-terminus of the alpha and/or beta chain.

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

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

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

In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence encoding the variable domain of the TCR α chain SEQ ID NO:2 or SEQ ID NO: 33.

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

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

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

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

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

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

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

In a ninth aspect 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 is CD8 of monoclonal cells ⁺ And tetramer-PE double positive staining results.

Figure 4a and figure 4b are the amino acid and nucleotide sequences, respectively, of a soluble TCR alpha chain.

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

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

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

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

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

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

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

FIG. 12 is a BIAcore kinetic profile of binding of soluble TCRs of the invention to the 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 the functional verification of the ELISPOT activation of the resulting T cell clones.

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

Detailed Description

The present inventors have 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 and may be MHC class I or II molecules. Therefore, it is specific for antigen presentation, different individuals have different MHC, and different short peptides in one protein antigen can be presented on the cell surface of respective APC. Human MHC is commonly referred to as an HLA gene or HLA complex.

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

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

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

Natural interchain disulfide bond and artificial interchain disulfide bond

A set of disulfide bonds, referred to herein as "native interchain disulfide bonds", exist between the C α and C β chains in 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 TRAC 01 and TRBC1 × 01 or TRBC2 × 01 amino acid sequences 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 is P (proline) in the order from the N-terminus to the C-terminus, and thus it may be described as Pro60 of TRBC1 × 01 or TRBC2 × 01 exon 1 in the invention, or as the 60 th amino acid of TRBC1 × 01 or TRBC2 × 01 exon 1, such as in TRBC1 × 01 or TRBC2 × 01, and the 61 th amino acid is Q (glutamine) in the order from the N-terminus to the C-terminus, and thus it may be described as TRBC1 × 01 or TRBC 6301 × 01 exon 01, and further as TRBC 858 × 8, and this may be described as TRBC 858 × 8, or TRBC 851, and this may be also be described as TRBC 8536. In the present invention, the position numbering of the amino acid sequences of the variable regions TRAV and TRBV follows the position numbering listed in IMGT. If an amino acid in TRAV, the position listed in IMGT is numbered 46, it is described herein as the 46 th amino acid of TRAV, and so on. In the present invention, the sequence position numbers of other amino acids are specifically described.

Detailed Description

TCR molecules

During antigen processing, antigens are degraded within cells and then carried to the cell surface by MHC molecules. T cell receptors are capable of recognizing peptide-MHC complexes on the surface of antigen presenting cells. Accordingly, a first aspect of the invention provides a TCR molecule capable of binding 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 a CDR having the amino acid sequence:

α CDR1-YGGTVN(SEQ ID NO:10)

α CDR2-YFSGDPLV(SEQ ID NO:11)

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

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

β CDR1-SGHVS(SEQ ID NO:13)

β CDR2-FQNEAQ(SEQ ID NO:14)

β CDR3-ASSQNLAGGPGTDTQY(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 meant to be TCR molecules comprising the above-described alpha and/or beta chain CDR region sequences and any suitable framework structure. The TCR α chain variable domain of the invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity to SEQ ID No. 1; and/or the TCR β chain variable domain of the invention is a variant of SEQ ID NO:5, preferably 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. Specifically, in one aspect the α chain of the heterodimeric TCR molecules comprises a variable domain and a constant domain, the α chain variable domain amino acid sequence comprising CDR1(SEQ ID NO: 10), CDR2(SEQ ID NO: 11) and CDR3(SEQ ID NO:12) of the above-described α chain. Preferably, the TCR molecule comprises the alpha chain variable domain amino acid sequence SEQ ID NO 1. More preferably, the amino acid sequence of the α chain variable domain of the TCR molecule is SEQ ID NO 1. In another aspect, the β chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the β chain variable domain amino acid sequence comprises CDR1(SEQ ID NO:13), CDR2(SEQ ID NO: 14), and CDR3(SEQ ID NO:15) of the above-described β chain. Preferably, the TCR molecule comprises the beta chain variable domain amino acid sequence SEQ ID NO 5. More preferably, the amino acid sequence of the β chain variable domain of the TCR molecule is SEQ ID NO 5.

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

The alpha chain variable domain amino acid sequence of the single chain TCR molecule comprises the CDR1(SEQ ID NO: 10), CDR2(SEQ ID NO: 11) and CDR3(SEQ ID NO:12) of the above-described alpha chain. Preferably, the single chain TCR molecule comprises the alpha chain variable domain amino acid sequence SEQ ID NO 1. More preferably, the α chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO 1. The amino acid sequence of the beta chain variable domain of the single chain TCR molecule comprises the CDR1(SEQ ID NO:13), CDR2(SEQ ID NO: 14) and CDR3(SEQ ID NO:15) of the above-described beta chain. Preferably, the single chain TCR molecule comprises the beta chain variable domain amino acid sequence SEQ ID NO 5. More preferably, the amino acid sequence of the variable domain of the β chain 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 may be "TRAC 01", and the constant domain sequence of the β chain of the TCR molecules may be "TRBC 1 01" or "TRBC 2 01". Arg at position 53 of the amino acid sequence given in TRAC 01 of IMGT, here denoted: TRAC × 01 Arg53 of exon 1, and so on. Preferably, the amino acid sequence of the α chain of the TCR molecule of the invention is SEQ ID NO 3 and/or the amino acid sequence of the β chain is SEQ ID NO 7.

Naturally occurring TCRs are membrane proteins that are stabilized by their transmembrane regions. Like immunoglobulins (antibodies) as antigen recognition molecules, TCRs can also be developed for diagnostic and therapeutic applications, where soluble TCR molecules are required. Soluble TCR molecules do not include their transmembrane regions. Soluble TCRs have a wide range of uses, not only for studying the interaction of TCRs with pmhcs, but also as diagnostic tools for detecting infections or as markers for autoimmune diseases. Similarly, soluble TCRs can be used to deliver therapeutic agents (e.g., cytotoxic or immunostimulatory compounds) to cells presenting a specific antigen, and in addition, soluble TCRs can be conjugated to other molecules (e.g., anti-CD 3 antibodies) to redirect T cells to target them to cells presenting a particular antigen. The present invention also 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 TRAC × 01 exon 1 and a cysteine residue of Ser57 of TRBC1 × 01 or TRBC2 × 01 exon 1. Other sites for introducing cysteine residues to form disulfide bonds may also be: thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser77 of TRBC2 × 01 exon 1; tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC2 x 01; thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1; ser15 and TRBC1 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 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01; 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 both TRAC constant domain sequences and TRBC1 or TRBC2 constant domain sequences, with or without the artificial disulfide bonds introduced as described above between the constant domains. The TRAC constant domain sequence and TRBC1 or TRBC2 constant domain sequences of the TCR may be linked by the native disulfide bond present in the TCR.

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

The TCR with the mutated hydrophobic core region of the invention can be a stable soluble single chain TCR formed by connecting the variable domains of the alpha and beta chains of the TCR by a flexible peptide chain. It should be noted that the flexible peptide chain of the present invention can be any peptide chain suitable for linking the variable domains of TCR α and β chains. The single-chain soluble TCR constructed as in example 4 of the invention has an alpha chain variable domain amino acid sequence of SEQ ID NO. 32 and an 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, regarding stability, patent document 201680003540.2 also discloses that introduction of an artificial interchain disulfide bond between an α chain variable region and a β chain constant region of TCR can significantly improve the stability of 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 TRBC1 x 01 or TRBC2 x 01 exon 1. Preferably, such a TCR may comprise (i) all or part of a TCR α chain, excluding its transmembrane domain, and (ii) all or part of a TCR β chain, excluding its transmembrane domain, wherein (i) and (ii) both comprise a variable domain and at least part of a constant domain of the TCR chain, the α chain forming a heterodimer with the β chain. More preferably, such a TCR may comprise the a chain variable domain and the β chain variable domain and all or part of the β chain constant domain, excluding the transmembrane domain, but which does not comprise the a chain constant domain, the a chain variable domain of the TCR forming a heterodimer with the β chain.

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

The TCRs of the invention may be used alone or in covalent or other association, preferably covalently, with a conjugate. The conjugates include a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the 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 or conjugated with the TCRs of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, Cancer metastasis reviews (Cancer metastasis) 24, 539); 2. biotoxicity (Chaudhary et al, 1989, Nature 339, 394; Epel et al, 2002, Cancer Immunology and Immunotherapy)51, 565); 3. cytokines such as IL-2 and the like (Gillies et al, 1992, Proc. Natl. Acad. Sci. USA (PNAS)89, 1428; Card et al, 2004, Cancer Immunology and Immunotherapy)53, 345; Halin et al, 2003, Cancer Research 63, 3202); 4. antibody Fc fragment (Mosquera et al, 2005, Journal Of Immunology 174, 4381); 5. antibody scFv fragments (Zhu et al, 1995, International Journal of Cancer 62,319); 6. gold nanoparticles/nanorods (Lapotko et al, 2005, Cancer communication (Cancer letters)239, 36; Huang et al, 2006, Journal of the American Chemical Society 128, 2115); 7. viral particles (Peng et al, 2004, Gene therapy)11, 1234); 8. liposomes (Mamot et al, 2005, Cancer research 65, 11631); 9. nano magnetic particles; 10. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, and the like.

In addition, the TCRs of the invention may also be hybrid TCRs comprising sequences derived from more than one species. For example, studies have shown that murine TCRs are more efficiently expressed in human T cells than human TCRs. Thus, the inventive TCR may comprise a human variable domain and a murine constant domain. The drawback of this approach is the possibility of eliciting an immune response. Thus, there should be a regulatory regimen to immunosuppresse when it is used for adoptive T cell therapy to allow for the engraftment of murine expressing T cells.

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

Nucleic acid molecules

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

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

α CDR1-tatggtggaactgttaat(SEQ ID NO:16)

α CDR2-tacttttcaggggatccactggtt(SEQ ID NO:17)

α CDR3-gccgtgatgggggatagcaactatcagttaatc(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:

β CDR1-tcgggtcatgtatcc(SEQ ID NO:19)

β CDR2-ttccagaatgaagctcaa(SEQ ID NO:20)

β CDR3-gccagcagtcaaaacctagcgggagggccaggcacagatacgcagtat(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, SEQ ID NO 17 and SEQ ID NO 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, SEQ ID NO 20 and SEQ ID NO 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 contain 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, the nucleic acid sequence encoding the TCR of the present invention may be identical to or a degenerate variant of the nucleic acid sequences shown in the figures of the present invention. As one example herein, a "degenerate variant" refers to a nucleic acid sequence that encodes a protein sequence having SEQ ID NO. 1, but differs from the sequence of SEQ ID NO. 2.

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

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

Carrier

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

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

Preferably, the vector can transfer the nucleotide of the invention into a cell, e.g., a T cell, such that the cell expresses a TCR specific for the 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 using the vectors or coding sequences of the invention. The host cell comprises the vector of the invention or has the nucleic acid molecule of the invention integrated into the chromosome. 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, for example 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: 6116-6131). T cells expressing the inventive TCR can be used in 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 adoptively transferring 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 and the like.

Method of treatment

Treatment may be effected by isolating T cells from patients or volunteers suffering from a disease associated with the 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 was able to bind to AFP antigen short peptide complex FMNKFIYEI-HLA a0201, while cells transduced with the inventive TCR were able to be specifically activated.

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 noted 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 publisher), or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

Example 1 cloning of 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; Baisheng Gene technologies, Inc., Beijing Sai). And (3) renaturing FMNKFIYEI short peptide and HLA-A0201 with biotin label to prepare pHLA haploid. These haploids combined with streptavidin labeled with PE (BD Co.) to form PE-labeled tetramers, which were sorted from anti-CD 8-APC double positive cells. The sorted cells were expanded and subjected to secondary sorting as described above, followed by single cloning by limiting dilution. Monoclonal cells were stained with tetramer and double positive clones were selected as shown in FIG. 3. The double positive clones obtained by layer-by-layer screening also need to meet the requirement of further functional test.

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

Firstly, preparing an ELISPOT flat plate, wherein the ELISPOT experiment steps are as follows: the components of the assay were added to the ELISPOT plate in the following order: 40 μ l T2 cells 5X 10 ⁵ After 40 μ l of effector cells (2000T cell clones/well) per ml of cells (i.e., 20,000T 2 cells/well), 20 μ l of specific short peptide was added to the experimental group, 20 μ l of nonspecific short peptide was added to the control group, 20 μ l of medium (test medium) was added to the blank group, and 2 duplicate 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 a specific short peptide, but showed no substantial response to T2 cells loaded with other unrelated peptides and unloaded with a 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 the amino acid sequence of the variable domain of TCR β chain and the variable domain of TCR β chain, respectivelyA domain nucleotide sequence, a TCR beta chain amino acid sequence, a TCR beta chain nucleotide sequence, a TCR beta chain amino acid sequence with a leader sequence and a TCR beta chain nucleotide sequence with a leader sequence.

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

α CDR1-YGGTVN(SEQ ID NO:10)

α CDR2-YFSGDPLV(SEQ ID NO:11)

α CDR3-AVMGDSNYQLI(SEQ ID NO:12)

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

β CDR1-SGHVS(SEQ ID NO:13)

β CDR2-FQNEAQ(SEQ ID NO:14)

β CDR3-ASSQNLAGGPGTDTQY(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: and connecting the full-length genes of the TCR alpha chain and the TCR beta chain by overlap PCR to obtain the TCR alpha-2A-TCR beta fragment. 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 soluble TCR specific for short peptide AFP antigen

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 the nucleotide sequence of the alpha chain are respectively shown in figure 4a and figure 4b, and the amino acid sequence and the nucleotide sequence of the beta chain are respectively shown in figure 5a and figure 5 b. The above-mentioned gene sequences of interest for the TCR alpha and beta chains were synthesized and inserted into the expression vector pET28a + (Novagene) by standard methods described in Molecular Cloning A Laboratory Manual (third edition, Sambrook and Russell), and the upstream and downstream Cloning sites were NcoI and NotI, respectively. The insert was confirmed by sequencing without error.

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

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

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

According to the disclosure of 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 (linker) 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 its beta-chain variable domain are shown in FIG. 9a and FIG. 9b, respectively; the amino acid sequence and the nucleotide sequence of the linker sequence are shown in FIG. 10a and FIG. 10b, respectively.

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, spread on LB plates containing kanamycin, cultured at 37 ℃ for overnight inversion, positive clones were selected for PCR screening, positive recombinants were sequenced, and after the sequence was determined to be correct, 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 using Bugbuster Master Mix (Merck), the inclusion bodies were recovered by centrifugation at 6000rpm for 15min, washed with Bugbuster (Merck) to remove cell debris and membrane components, and the inclusion bodies were collected by centrifugation at 6000rpm for 15 min. The inclusion bodies were dissolved in buffer (20mM Tris-HCl pH 8.0,8M urea), the insoluble material was removed by high speed centrifugation, the supernatant was quantitated by BCA method and split charged, and stored at-80 ℃ for further use.

To 5mg of solubilized single-chain TCR inclusion body protein, 2.5mL of buffer (6M Gua-HCl, 50mM Tris-HCl pH 8.1, 100mM NaCl, 10mM EDTA) was added, DTT was added to a final concentration of 10mM, and treatment was performed 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 of E.coli bacterial liquid for inducing expression of heavy chain or light chain, centrifuging at 8000g at 4 ℃ for 10min, washing the bacterial cells once with 10ml of PBS, then resuspending the bacterial cells by vigorous shaking with 5ml of BugBuster Master Mix Extraction Reagents (Merck), carrying out rotary incubation at room temperature for 20min, centrifuging at 6000g at 4 ℃ for 15min, discarding supernatant, and collecting inclusion bodies.

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

b. Renaturation

The synthesized short peptide 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 on 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 Millipore ultrafiltration tubes while displacing the buffer to 20mM Tris pH 8.0, followed by addition of biotinylation reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50. mu. M D-Biotin, 100. mu.g/ml BirA enzyme (GST-BirA), incubation of the mixture overnight at room temperature, and SDS-PAGE to determine the completion of biotinylation.

e. Purification of biotinylated complexes

The biotinylated pMHC molecules were concentrated to 1ml using Millipore ultrafiltration tubes, the biotinylated pMHC was purified by gel filtration chromatography, and HiPrep was pre-equilibrated with filtered PBS using Akta purifier (GE general electric Co., Ltd.) ^TM 16/60S200HR column (GE general electric) was loaded with 1ml of concentrated biotinylated pMHC molecules and then eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules appeared as a unimodal elution at approximately 55 ml. The fractions containing the protein were pooled, concentrated using Millipore ultrafiltration tubes, protein concentration was determined by BCA (Thermo), and biotinylated pMHC molecules were stored in aliquots at-80 ℃ by 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 (Gibbo, catalog number 16000-

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

PBS (Gibbo Co., catalog number C10010500BT)

PVDF ELISPOT 96-well plate (Merck Millipore, Cat. No. MSIPS4510)

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

Method

Target cell preparation

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

Effector cell preparation

The effector cells (T cells) of this experiment were CD8 expressing the TCR specific for the 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 in the test medium at 2 × the desired final concentration. Negative control effector cells were treated as well.

Preparation of short peptide solution

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

ELISPOT

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

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

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

100 microliter of effector cells (1 x 10) ⁴ Individual control effector cells/well and 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, then 3 times with wash buffer, and tapped on a paper towel to remove residual wash buffer. Then, the mixture was mixed with PBS containing 10% FBS at a ratio of 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.

PBS containing 10% FBS was used at 1: streptavidin-alkaline phosphatase was diluted 100, 100 microliters of diluted streptavidin-alkaline phosphatase was added to each well and the wells were incubated for 1 hour at room temperature. The plates were then washed 2 times with 4 washes of PBS and tapped on a paper towel to remove excess wash buffer and PBS. After washing, 100 microliter of BCIP/NBT solution provided by the kit is added for development. And covering the well plate with tin foil paper in dark during development, and standing for 5-15 minutes. During this period, spots on the developing well plate were routinely detected, and the optimal time for terminating the reaction was determined. The BCIP/NBT solution was removed and the well plate was rinsed with double-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).

As a result, the

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 (effector cells) transduced with the TCR of the invention were shown to be very responsive to activation of target cells loaded with their specific short peptides, whereas T cells (effector cells) transduced with other TCRs were shown to be essentially non-responsive to activation of 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> a TCR recognizing AFP antigen and nucleic acid encoding the same

<130> P2018-0966

<160> 37

<170> PatentIn version 3.5

<210> 1

<211> 114

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 1

Ala Gln Ser Val Ser Gln His Asn His His Val Ile Leu Ser Glu Ala

1 5 10 15

Ala Ser Leu Glu Leu Gly Cys Asn Tyr Ser Tyr Gly Gly Thr Val Asn

20 25 30

Leu Phe Trp Tyr Val Gln Tyr Pro Gly Gln His Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Ile Lys Ser Lys Phe Ser Phe Asn Leu Arg Lys Pro Ser

65 70 75 80

Val Gln Trp Ser Asp Thr Ala Glu Tyr Phe Cys Ala Val Met Gly Asp

85 90 95

Ser Asn Tyr Gln Leu Ile Trp Gly Ala Gly Thr Lys Leu Ile Ile Lys

100 105 110

Pro Asp

<210> 2

<211> 342

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

gcccagtctg tgagccagca taaccaccac gtaattctct ctgaagcagc ctcactggag 60

ttgggatgca actattccta tggtggaact gttaatctct tctggtatgt ccagtaccct 120

ggtcaacacc ttcagcttct cctcaagtac ttttcagggg atccactggt taaaggcatc 180

aagggctttg aggctgaatt tataaagagt aaattctcct ttaatctgag gaaaccctct 240

gtgcagtgga gtgacacagc tgagtacttc tgtgccgtga tgggggatag caactatcag 300

ttaatctggg gcgctgggac caagctaatt ataaagccag at 342

<210> 3

<211> 254

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 3

Ala Gln Ser Val Ser Gln His Asn His His Val Ile Leu Ser Glu Ala

1 5 10 15

Ala Ser Leu Glu Leu Gly Cys Asn Tyr Ser Tyr Gly Gly Thr Val Asn

20 25 30

Leu Phe Trp Tyr Val Gln Tyr Pro Gly Gln His Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Ile Lys Ser Lys Phe Ser Phe Asn Leu Arg Lys Pro Ser

65 70 75 80

Val Gln Trp Ser Asp Thr Ala Glu Tyr Phe Cys Ala Val Met Gly Asp

85 90 95

Ser Asn Tyr Gln Leu Ile Trp Gly Ala Gly Thr Lys Leu Ile Ile Lys

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

gcccagtctg tgagccagca taaccaccac gtaattctct ctgaagcagc ctcactggag 60

ttgggatgca actattccta tggtggaact gttaatctct tctggtatgt ccagtaccct 120

ggtcaacacc ttcagcttct cctcaagtac ttttcagggg atccactggt taaaggcatc 180

aagggctttg aggctgaatt tataaagagt aaattctcct ttaatctgag gaaaccctct 240

gtgcagtgga gtgacacagc tgagtacttc tgtgccgtga tgggggatag caactatcag 300

ttaatctggg gcgctgggac caagctaatt ataaagccag 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> 118

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 5

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Arg Leu Thr Val Leu

115

<210> 6

<211> 354

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

ggtgctggag tctcccagtc ccctaggtac aaagtcgcaa agagaggaca ggatgtagct 60

ctcaggtgtg atccaatttc gggtcatgta tccctttttt ggtaccaaca ggccctgggg 120

caggggccag agtttctgac ttatttccag aatgaagctc aactagacaa atcggggctg 180

cccagtgatc gcttctttgc agaaaggcct gagggatccg tctccactct gaagatccag 240

cgcacacagc aggaggactc cgccgtgtat ctctgtgcca gcagtcaaaa cctagcggga 300

gggccaggca cagatacgca gtattttggc ccaggcaccc ggctgacagt gctc 354

<210> 7

<211> 297

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 7

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Met Val Lys Arg Lys Asp Ser Arg Gly

290 295

<210> 8

<211> 891

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

ggtgctggag tctcccagtc ccctaggtac aaagtcgcaa agagaggaca ggatgtagct 60

ctcaggtgtg atccaatttc gggtcatgta tccctttttt ggtaccaaca ggccctgggg 120

caggggccag agtttctgac ttatttccag aatgaagctc aactagacaa atcggggctg 180

cccagtgatc gcttctttgc agaaaggcct gagggatccg tctccactct gaagatccag 240

cgcacacagc aggaggactc cgccgtgtat ctctgtgcca gcagtcaaaa cctagcggga 300

gggccaggca cagatacgca gtattttggc ccaggcaccc ggctgacagt gctcgaggac 360

ctgaaaaacg tgttcccacc cgaggtcgct gtgtttgagc catcagaagc agagatctcc 420

cacacccaaa aggccacact ggtgtgcctg gccacaggct tctaccccga ccacgtggag 480

ctgagctggt gggtgaatgg gaaggaggtg cacagtgggg tcagcacaga cccgcagccc 540

ctcaaggagc agcccgccct caatgactcc agatactgcc tgagcagccg cctgagggtc 600

tcggccacct tctggcagaa cccccgcaac cacttccgct gtcaagtcca gttctacggg 660

ctctcggaga atgacgagtg gacccaggat agggccaaac ctgtcaccca gatcgtcagc 720

gccgaggcct ggggtagagc agactgtggc ttcacctccg agtcttacca gcaaggggtc 780

ctgtctgcca ccatcctcta tgagatcttg ctagggaagg ccaccttgta tgccgtgctg 840

gtcagtgccc tcgtgctgat ggccatggtc aagagaaagg attccagagg c 891

<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> 6

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 10

Tyr Gly Gly Thr Val Asn

1 5

<210> 11

<211> 8

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 11

Tyr Phe Ser Gly Asp Pro Leu Val

1 5

<210> 12

<211> 11

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 12

Ala Val Met Gly Asp Ser Asn Tyr Gln Leu Ile

1 5 10

<210> 13

<211> 5

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 13

Ser Gly His Val Ser

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> 16

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 15

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

1 5 10 15

<210> 16

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 16

tatggtggaa ctgttaat 18

<210> 17

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 17

tacttttcag gggatccact ggtt 24

<210> 18

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 18

gccgtgatgg gggatagcaa ctatcagtta atc 33

<210> 19

<211> 15

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 19

tcgggtcatg tatcc 15

<210> 20

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 20

ttccagaatg aagctcaa 18

<210> 21

<211> 48

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 21

gccagcagtc aaaacctagc gggagggcca ggcacagata cgcagtat 48

<210> 22

<211> 273

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 22

Met Leu Leu Leu Leu Ile Pro Val Leu Gly Met Ile Phe Ala Leu Arg

1 5 10 15

Asp Ala Arg Ala Gln Ser Val Ser Gln His Asn His His Val Ile Leu

20 25 30

Ser Glu Ala Ala Ser Leu Glu Leu Gly Cys Asn Tyr Ser Tyr Gly Gly

35 40 45

Thr Val Asn Leu Phe Trp Tyr Val Gln Tyr Pro Gly Gln His Leu Gln

50 55 60

Leu Leu Leu Lys Tyr Phe Ser Gly Asp Pro Leu Val Lys Gly Ile Lys

65 70 75 80

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

85 90 95

Lys Pro Ser Val Gln Trp Ser Asp Thr Ala Glu Tyr Phe Cys Ala Val

100 105 110

Met Gly Asp Ser Asn Tyr Gln Leu Ile Trp Gly Ala Gly Thr Lys Leu

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Ser

<210> 23

<211> 819

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 23

atgctcctgt tgctcatacc agtgctgggg atgatttttg ccctgagaga tgccagagcc 60

cagtctgtga gccagcataa ccaccacgta attctctctg aagcagcctc actggagttg 120

ggatgcaact attcctatgg tggaactgtt aatctcttct ggtatgtcca gtaccctggt 180

caacaccttc agcttctcct caagtacttt tcaggggatc cactggttaa aggcatcaag 240

ggctttgagg ctgaatttat aaagagtaaa ttctccttta atctgaggaa accctctgtg 300

cagtggagtg acacagctga gtacttctgt gccgtgatgg gggatagcaa ctatcagtta 360

atctggggcg ctgggaccaa gctaattata aagccagata tccagaaccc tgaccctgcc 420

gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 480

gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 540

gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 600

tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 660

cccagcccag aaagttcctg tgatgtcaag ctggtcgaga aaagctttga aacagatacg 720

aacctaaact ttcaaaacct gtcagtgatt gggttccgaa tcctcctcct gaaagtggcc 780

gggtttaatc tgctcatgac gctgcggctg tggtccagc 819

<210> 24

<211> 316

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 24

Met Gly Thr Arg Leu Leu Cys Trp Val Val Leu Gly Phe Leu Gly Thr

1 5 10 15

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

20 25 30

Lys Arg Gly Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His

35 40 45

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

50 55 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro

65 70 75 80

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

85 90 95

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

100 105 110

Ser Ser Gln Asn Leu Ala Gly Gly Pro Gly Thr Asp Thr Gln Tyr Phe

115 120 125

Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe

130 135 140

Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His

145 150 155 160

Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp

165 170 175

His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly

180 185 190

Val Ser Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp

195 200 205

Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp

210 215 220

Gln Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu

225 230 235 240

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

245 250 255

Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315

<210> 25

<211> 948

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 25

atgggcacca ggctcctctg ctgggtggtc ctgggtttcc tagggacaga tcacacaggt 60

gctggagtct cccagtcccc taggtacaaa gtcgcaaaga gaggacagga tgtagctctc 120

aggtgtgatc caatttcggg tcatgtatcc cttttttggt accaacaggc cctggggcag 180

gggccagagt ttctgactta tttccagaat gaagctcaac tagacaaatc ggggctgccc 240

agtgatcgct tctttgcaga aaggcctgag ggatccgtct ccactctgaa gatccagcgc 300

acacagcagg aggactccgc cgtgtatctc tgtgccagca gtcaaaacct agcgggaggg 360

ccaggcacag atacgcagta ttttggccca ggcacccggc tgacagtgct cgaggacctg 420

aaaaacgtgt tcccacccga ggtcgctgtg tttgagccat cagaagcaga gatctcccac 480

acccaaaagg ccacactggt gtgcctggcc acaggcttct accccgacca cgtggagctg 540

agctggtggg tgaatgggaa ggaggtgcac agtggggtca gcacagaccc gcagcccctc 600

aaggagcagc ccgccctcaa tgactccaga tactgcctga gcagccgcct gagggtctcg 660

gccaccttct ggcagaaccc ccgcaaccac ttccgctgtc aagtccagtt ctacgggctc 720

tcggagaatg acgagtggac ccaggatagg gccaaacctg tcacccagat cgtcagcgcc 780

gaggcctggg gtagagcaga ctgtggcttc acctccgagt cttaccagca aggggtcctg 840

tctgccacca tcctctatga gatcttgcta gggaaggcca ccttgtatgc cgtgctggtc 900

agtgccctcg tgctgatggc catggtcaag agaaaggatt ccagaggc 948

<210> 26

<211> 207

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 26

Ala Gln Ser Val Ser Gln His Asn His His Val Ile Leu Ser Glu Ala

1 5 10 15

Ala Ser Leu Glu Leu Gly Cys Asn Tyr Ser Tyr Gly Gly Thr Val Asn

20 25 30

Leu Phe Trp Tyr Val Gln Tyr Pro Gly Gln His Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Ile Lys Ser Lys Phe Ser Phe Asn Leu Arg Lys Pro Ser

65 70 75 80

Val Gln Trp Ser Asp Thr Ala Glu Tyr Phe Cys Ala Val Met Gly Asp

85 90 95

Ser Asn Tyr Gln Leu Ile Trp Gly Ala Gly Thr Lys Leu Ile Ile Lys

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

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

165 170 175

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

180 185 190

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

195 200 205

<210> 27

<211> 621

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 27

gcgcagagcg tgagccagca taaccaccac gtaattctct ctgaagcagc ctcactggag 60

ttgggatgca actattccta tggtggaact gttaatctct tctggtatgt ccagtaccct 120

ggtcaacacc ttcagcttct cctcaagtac ttttcagggg atccactggt taaaggcatc 180

aagggctttg aggctgaatt tataaagagt aaattctcct ttaatctgag gaaaccctct 240

gtgcagtgga gtgacacagc tgagtacttc tgtgccgtga tgggggatag caactatcag 300

ttaatctggg gcgctgggac caagctaatt ataaagccag atatccagaa ccctgaccct 360

gccgtgtacc agctgagaga ctctaagtcg agtgacaagt ctgtctgcct attcaccgat 420

tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 480

tgtgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 540

aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 600

ttccccagcc cagaaagttc c 621

<210> 28

<211> 248

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 28

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Ala Glu Ala Trp Gly Arg Ala Asp

245

<210> 29

<211> 744

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 29

ggtgcaggtg ttagccagtc ccctaggtac aaagtcgcaa agagaggaca ggatgtagct 60

ctcaggtgtg atccaatttc gggtcatgta tccctttttt ggtaccaaca ggccctgggg 120

caggggccag agtttctgac ttatttccag aatgaagctc aactagacaa atcggggctg 180

cccagtgatc gcttctttgc agaaaggcct gagggatccg tctccactct gaagatccag 240

cgcacacagc aggaggactc cgccgtgtat ctctgtgcca gcagtcaaaa cctagcggga 300

gggccaggca cagatacgca gtattttggc ccaggcaccc ggctgacagt gctcgaggac 360

ctgaaaaacg tgttcccacc cgaggtcgct gtgtttgagc catcagaagc agagatctcc 420

cacacccaaa aggccacact ggtgtgcctg gccaccggtt tctaccccga ccacgtggag 480

ctgagctggt gggtgaatgg gaaggaggtg cacagtgggg tctgcacaga cccgcagccc 540

ctcaaggagc agcccgccct caatgactcc agatacgctc tgagcagccg cctgagggtc 600

tcggccacct tctggcagga cccccgcaac cacttccgct gtcaagtcca gttctacggg 660

ctctcggaga atgacgagtg gacccaggat agggccaaac ccgtcaccca gatcgtcagc 720

gccgaggcct ggggtagagc agac 744

<210> 30

<211> 255

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 30

Ala Gln Ser Val Ser Gln His Asn His His Leu Asn Val Ser Glu Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Ala Glu Phe Ile Lys Ser Lys Phe Ser Phe Asn Leu Arg Lys Pro Ser

65 70 75 80

Val Gln Pro Ser Asp Thr Ala Glu Tyr Phe Cys Ala Val Met Gly Asp

85 90 95

Ser Asn Tyr Gln Leu Ile Trp Gly Ala Gly Thr Lys Leu Ser Val Lys

100 105 110

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

115 120 125

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

130 135 140

Pro Arg Tyr Leu Ser Val Lys Arg Gly Gln Asp Val Ala Leu Arg Cys

145 150 155 160

Asp Pro Ile Ser Gly His Val Ser Leu Phe Trp Tyr Arg Gln Asp Pro

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

<210> 31

<211> 765

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 31

gctcaatctg ttagccagca taatcatcat ctgaatgtta gcgaaggcgc cagtgttgaa 60

attggttgta attatagcta cggtggtacc gtgaatctgt tttggtatcg ccaggatccg 120

ggtcagcatc tgcagctgct gctgaaatat tttagcggcg atccgctggt gaaaggcatt 180

aagggctttg aagcagaatt cattaagagc aaattcagct ttaacctgcg caaaccgagc 240

gtgcagccga gtgataccgc agaatatttt tgtgccgtta tgggtgacag caattatcag 300

ctgatttggg gtgcaggtac caaactgagt gttaaaccgg gtggcggtag tgaaggcggt 360

ggtagtgaag gtggcggtag cgaaggcggt ggcagtgaag gtggtaccgg cggcgccggt 420

gtgagccaaa gtccgcgtta tctgagtgtt aagcgcggtc aggatgttgc actgcgctgt 480

gatccgatta gcggccatgt tagcctgttt tggtaccgcc aggatcctgg tcagggtccg 540

gaatttctga cctattttca gaatgaagca cagctggata aaagtggtct gccgagcgat 600

cgtttctttg cagaacgccc ggaaggtagt gtgagcaccc tgaaaattca gcgtgttcag 660

ccggaagata gcgccgttta tttttgtgcg agcagccaga atctggcagg cggtccgggt 720

accgataccc agtattttgg tccgggcacc cgtctgaccg ttgat 765

<210> 32

<211> 113

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 32

Ala Gln Ser Val Ser Gln His Asn His His Leu Asn Val Ser Glu Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Ala Glu Phe Ile Lys Ser Lys Phe Ser Phe Asn Leu Arg Lys Pro Ser

65 70 75 80

Val Gln Pro Ser Asp Thr Ala Glu Tyr Phe Cys Ala Val Met Gly Asp

85 90 95

Ser Asn Tyr Gln Leu Ile Trp Gly Ala Gly Thr Lys Leu Ser Val Lys

100 105 110

Pro

<210> 33

<211> 339

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 33

gctcaatctg ttagccagca taatcatcat ctgaatgtta gcgaaggcgc cagtgttgaa 60

attggttgta attatagcta cggtggtacc gtgaatctgt tttggtatcg ccaggatccg 120

ggtcagcatc tgcagctgct gctgaaatat tttagcggcg atccgctggt gaaaggcatt 180

aagggctttg aagcagaatt cattaagagc aaattcagct ttaacctgcg caaaccgagc 240

gtgcagccga gtgataccgc agaatatttt tgtgccgtta tgggtgacag caattatcag 300

ctgatttggg gtgcaggtac caaactgagt gttaaaccg 339

<210> 34

<211> 118

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 34

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Arg Leu Thr Val Asp

115

<210> 35

<211> 354

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 35

ggcgccggtg tgagccaaag tccgcgttat ctgagtgtta agcgcggtca ggatgttgca 60

ctgcgctgtg atccgattag cggccatgtt agcctgtttt ggtaccgcca ggatcctggt 120

cagggtccgg aatttctgac ctattttcag aatgaagcac agctggataa aagtggtctg 180

ccgagcgatc gtttctttgc agaacgcccg gaaggtagtg tgagcaccct gaaaattcag 240

cgtgttcagc cggaagatag cgccgtttat ttttgtgcga gcagccagaa tctggcaggc 300

ggtccgggta ccgataccca gtattttggt ccgggcaccc gtctgaccgt tgat 354

<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 gtgaaggcgg tggtagtgaa ggtggcggta gcgaaggcgg tggcagtgaa 60

ggtggtaccg gc 72

Claims (37)

1. A T Cell Receptor (TCR), wherein the TCR is capable of binding to the FMNKFIYEI-HLAA0201 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-YGGTVN(SEQ ID NO:10)

αCDR2-YFSGDPLV(SEQ ID NO:11)

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

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

βCDR1-SGHVS(SEQ ID NO:13)

βCDR2-FQNEAQ(SEQ ID NO:14)

βCDR3-ASSQNLAGGPGTDTQY(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 beta chain amino acid sequence of the TCR are SEQ ID NO 7.

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

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

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

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

12. A TCR as claimed in claim 11 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 the 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 a portion of the constant domain of the TCR chain.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

22. A TCR as claimed in claim 1 wherein a conjugate is attached to the C-or N-terminus of the α and/or β chains 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 of any one of claims 1-24, 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 26-29.

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 in the preparation of a medicament for the treatment of an AFP-associated tumour presenting an AFP antigen short peptide FMNKFIYEI-HLA a0201 complex, said tumour being hepatocellular carcinoma.

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