CN110577591B - T cell receptor for identifying AFP antigen short peptide and its coding sequence - Google Patents

Abstract

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

Description

T cell receptor for identifying AFP antigen short peptide and its coding sequence

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 (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 an 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. Therefore, those skilled in the art have focused on the isolation of TCRs specific for short AFP antigen peptides and the transduction of the TCRs into T cells to obtain T cells specific for short AFP antigen peptides, thereby enabling 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 AVETSYDKVI (SEQ ID NO: 12); and/or the amino acid sequence of CDR3 of the variable domain of the TCR beta chain is ASSYGAGGPLDTQY (SEQ ID NO: 15).

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

αCDR1-VGISA (SEQ ID NO:10)

αCDR2-LSSGK (SEQ ID NO:11)

α CDR3-AVETSYDKVI (SEQ ID NO: 12); and/or

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

βCDR1-SGHVS (SEQ ID NO:13)

βCDR2-FNYEAQ (SEQ ID NO:14)

βCDR3-ASSYGAGGPLDTQY (SEQ ID NO:15)。

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

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

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

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

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

In another preferred embodiment, the TCR is soluble.

In another preferred embodiment, the TCR is single-chain.

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

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

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

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

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

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

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

In another preferred embodiment, the cysteine residues that form the artificial disulfide bond in the TCR are substituted at one or more groups of positions selected from:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Drawings

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Detailed Description

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

Term(s) for

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

The T Cell Receptor (TCR), is the only receptor for a specific antigenic peptide presented on the Major Histocompatibility Complex (MHC). In the immune system, direct physical contact between T cells and Antigen Presenting Cells (APCs) is initiated by 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 a TCR 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 linking 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), e.g. 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 "TRBC1 01" or "TRBC2 01". In addition, the α and β chains of the TCR also comprise a transmembrane region and a cytoplasmic region, the cytoplasmic region being very short.

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

Natural interchain disulfide bond and artificial interchain disulfide bond

A set of disulfide bonds, referred to herein as "native interchain disulfide bonds," exist between the C α and C β chains of the membrane proximal region of native TCRs. In the present invention, the artificially introduced interchain covalent disulfide bond whose position is different from that of the natural interchain disulfide bond is referred to as an "artificial interchain disulfide bond".

For convenience of description of the positions of disulfide bonds, the positions of TRAC 01 and TRBC1 × 01 or TRBC2 × 01 amino acid sequences are numbered in order from N-terminus to C-terminus, for example, in TRBC1 × 01 or TRBC2 × 01, the 60 th amino acid in order from N-terminus to C-terminus is P (proline), and thus, in the present invention, it may be described as TRBC1 × 01 or TRBC2 × 01 exon 1 Pro60, and also as TRBC1 × 01 or TRBC2 × 01 exon 160, for example, in TRBC1 × 01 or TRBC2 × 01, and the 61 st amino acid in order from N-terminus to C-terminus is Q (glutamine), and thus, in the present invention, it may be described as TRBC1 × 01 or TRBC2 × 01 exon 1 Gln61, and also as TRBC1 or TRBC2 × 01, and so on, and further, it may be described as TRBC1 × 01 or TRBC2 × 01. In the present invention, the position numbering of the amino acid sequences of the variable regions TRAV and TRBV follows the position numbering listed in IMGT. If an amino acid in TRAV, the position listed in IMGT is numbered 46, it is described herein as the 46 th amino acid of TRAV, and so on. In the present invention, the sequence position numbers of other amino acids are specifically described.

Detailed Description

TCR molecules

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

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

αCDR1-VGISA (SEQ ID NO:10)

αCDR2-LSSGK (SEQ ID NO:11)

α CDR3-AVETSYDKVI (SEQ ID NO: 12); and/or

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

βCDR1-SGHVS (SEQ ID NO:13)

βCDR2-FNYEAQ (SEQ ID NO:14)

βCDR3-ASSYGAGGPLDTQY (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, having at least 90%, preferably 95%, more preferably 98% sequence identity.

In a preferred embodiment of the invention, the TCR molecules of the invention are heterodimers consisting of α and β chains. In particular, in one aspect the α chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, the α chain variable domain amino acid sequence comprising CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the above-described α chain. Preferably, the TCR molecule comprises an alpha chain variable domain amino acid sequence SEQ ID NO 1. More preferably, the amino acid sequence of the α chain variable domain of the TCR molecule is SEQ ID NO 1. In another aspect, the β chain of the heterodimeric TCR 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 91,12654-12658. 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 CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the alpha chain described above. Preferably, the single chain TCR molecule comprises an alpha chain variable domain amino acid sequence SEQ ID NO 1. More preferably, the α chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO 1. The amino acid sequence of the variable domain of the beta chain of the single-chain TCR molecule comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 14) and CDR3 (SEQ ID NO: 15) of the beta chain. Preferably, the single chain TCR molecule comprises the beta chain variable domain amino acid sequence SEQ ID NO 5. More preferably, the amino acid sequence of the β chain variable domain of the single chain TCR molecule is SEQ ID NO 5.

In a preferred embodiment of the invention, the constant domain of the TCR molecules of the invention is a human constant domain. The human constant domain amino acid sequences are known to those skilled in the art or can be obtained by consulting published databases of relevant books or IMGT (international immunogenetic information system). For example, the constant domain sequence of the α chain of the TCR molecules of the invention can be "TRAC 01", and the constant domain sequence of the β chain of the TCR molecules can be "TRBC1 01" or "TRBC2 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 invention also provides soluble TCRs specific for short AFP antigen peptides.

To obtain a soluble TCR, in one aspect, the inventive TCR may be one in which an artificial disulfide bond is introduced between residues of the constant domains of its alpha and beta chains. Cysteine residues form an artificial interchain disulfide bond between the alpha and beta chain constant domains of the TCR. Cysteine residues may be substituted for other amino acid residues at appropriate positions in native TCRs to form artificial interchain disulfide bonds. For example, a disulfide bond is formed by substituting Thr48 of exon 1 of TRAC × 01 and a cysteine residue of Ser57 of exon 1 of TRBC1 × 01 or TRBC2 × 01. Other sites for introducing cysteine residues to form disulfide bonds may also be: thr45 of TRAC × 01 exon 1 and TRBC1 × 01 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 of TRAC × 01 exon 1 and TRBC1 × 01 or Asp59 of TRBC2 × 01 exon 1; ser15 of TRAC × 01 exon 1 and TRBC1 × 01 or TRBC2 × 01 exon 1 Glu15; arg53 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser54 of TRBC2 × 01 exon 1; pro89 of exon 1 TRAC × 01 and TRBC1 × 01 or TRBC2 × 01 of Ala19 of exon 1; or Tyr10 of exon 1 of TRAC × 01 and TRBC1 × 01 or TRBC2 × 01, glu20 of exon 1. I.e., a cysteine residue, in place of any of the above-described alpha and beta chain constant domains. The TCR constant domains of the invention may be truncated at one or more of their C-termini by up to 50, or up to 30, or up to 15, or up to 10, or up to 8 or fewer amino acids, so as not to include a cysteine residue for the purpose of deleting the native disulphide bond, or by mutating the cysteine residue forming the native disulphide bond to another amino acid.

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

To obtain a soluble TCR, the inventive TCRs, on the other hand, also include TCRs having mutations in their hydrophobic core region, preferably mutations that improve the stability of the inventive soluble TCRs, as described in patent publication No. 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 alpha chain J gene (TRAJ) short peptide amino acid position reciprocal 3,5,7, and/or beta chain J gene (TRBJ) short peptide amino acid position reciprocal 2,4,6, wherein the position numbering of the amino acid sequences is according to the position numbering listed in the International immunogenetic information System (IMGT). The skilled person is aware of the above international immunogenetic information system and can derive from this database the position numbering of the amino acid residues of different TCRs in IMGT.

The TCR with the mutated hydrophobic core region of the present invention may be a stable soluble single chain TCR consisting of a flexible peptide chain connecting the variable domains of the α and β chains of the TCR. It should be noted that the flexible peptide chain of the present invention can be any peptide chain suitable for linking the TCR α and β chain variable domains. The single-chain soluble TCR constructed 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, for stability, patent document 201680003540.2 also discloses that introduction of an artificial interchain disulfide bond between the α chain variable region and the 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 exon 1 of TRBC1 x 01 or TRBC2 x 01; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01. Preferably, such a TCR may comprise (i) all or part of a TCR α chain, excluding its transmembrane domain, and (ii) all or part of a TCR β chain, excluding its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least part of the constant domain of the TCR chain, the α chain forming a heterodimer with the β chain. More preferably, such a TCR may comprise the a chain variable domain and the β chain variable domain and all or part of the β chain constant domain, excluding the transmembrane domain, but which does not comprise the a chain constant domain, the a chain variable domain of the TCR forming a heterodimer with the β chain.

The TCRs of the invention may also be provided in the form of multivalent complexes. Multivalent TCR complexes of the invention comprise polymers formed by association of two, three, four or more TCRs of the invention, such as might be formed by tetramer formation with the tetrameric domain of p53, or complexes 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, where 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. biotoxics (Chaudhary et al, 1989, nature 339, 394, epel et al, 2002, cancer Immunology and Immunotherapy) 51, 565); 3. cytokines such as IL-2 etc (Gillies et al, 1992, national institute of sciences (PNAS) 89, 1428, card et al, 2004, cancer Immunology and Immunotherapy) 53, 345, haiin et al, 2003, cancer Research (Cancer Research) 63, 3202; 4. antibody Fc fragment (Mosquera et al, 2005, journal Of Immunology 174, 4381); 5. antibody scFv fragments (Zhu et al, 1995, international Journal of Cancer 62,319); 6. gold nanoparticles/nanorods (Lapotko et al, 2005, cancer letters (Cancer letters) 239, 36, huang et al, 2006, journal of the American Chemical Society 128, 2115); 7. viral particles (Peng et al, 2004, gene therapy) 11, 1234); 8. liposomes (Mamot et al, 2005, cancer research 65, 11631); 9. nano magnetic particles; 10. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, and the like.

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

It should be understood that the amino acid names herein are expressed in terms of international single-letter or three-letter english letters, and the single-letter english letter and three-letter english letters of the amino acid names correspond to the following relationships: ala (A), arg (R), asn (N), asp (D), cys (C), gln (Q), glu (E), gly (G), his (H), ile (I), leu (L), lys (K), met (M), phe (F), pro (P), ser (S), thr (T), trp (W), tyr (Y), val (V).

Nucleic acid molecules

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

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

αCDR1-gtaggaataagtgcc(SEQ ID NO:16)

αCDR2-ctgagctcagggaag(SEQ ID NO:17)

αCDR3-gctgtcgaaacctcctacgacaaggtgata(SEQ ID NO:18)

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

βCDR1-tcgggtcatgtatcc(SEQ ID NO:19)

βCDR2-ttcaattatgaagcccaa(SEQ ID NO:20)

βCDR3-gccagcagctacggagcgggagggcctttagatacgcagtat(SEQ ID NO:21)

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

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

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

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

The full-length sequence of the nucleic acid molecule of the present invention or a fragment thereof can be obtained by, but not limited to, PCR amplification, recombination, or artificial synthesis. At present, DNA sequences encoding the TCRs of the invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or e.g., vectors) 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 that have been engineered with 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 (PBMCs) and may be CD4 ⁺ Helper T cell or CD8 ⁺ Cytotoxic T cells. The cells can be in CD4 ⁺ Helper T cell/CD 8 ⁺ A mixed population of cytotoxic T cells. Generally, the cells can be activated with antibodies (e.g., anti-CD 3 or anti-CD 28 antibodies) to make them more receptive to transfection, e.g.Transfection is performed 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 TCRs of the invention may be used for adoptive immunotherapy. One skilled in the art will be aware of many suitable methods for adoptive therapy (e.g., rosenberg et al, (2008) Nat Rev Cancer8 (4): 299-308).

AFP antigen associated diseases

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

The AFP-specific T cells of the invention can be used to treat any AFP-related disease that presents 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 having a disease associated with AFP antigen and introducing the TCRs of the invention into such T cells, followed by transfusion of these genetically engineered cells back 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 is capable of binding to the AFP antigen short peptide complex FMNKFIYEI-HLA A0201, while cells transduced with the inventive TCR are capable of being 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 indicated in the following examples, are generally carried out according to conventional conditions, for example as described in Sambrook and Russell et al, molecular Cloning: A Laboratory Manual (third edition) (2001) CSHL Press, or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

Example 1 cloned AFP antigen short peptide specific T cells

Peripheral Blood Lymphocytes (PBLs) from healthy volunteers of genotype HLA-A0201 were stimulated with synthetic short peptide FMNKFIYEI (SEQ ID No.:9; baisheng Gene technologies, beijing Sai Ltd.). The FMNKFIYEI short peptide is renatured with HLA-A0201 with biotin label to prepare pHLA haploid. These haploids were 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 in this example are T cell clones obtained in the present invention, the target cells are T2 cells loaded with the short peptide of the present invention, and the control group are T2 cells loaded with other short peptides and T2 cells not loaded with any short peptide.

Firstly, preparing an ELISPOT plate, wherein the ELISPOT experiment steps are as follows: the components of the assay were added to the ELISPOT plate in the following order: 40 u l T cells 5X 10 ⁵ After each cell/ml (i.e., 20,000T 2 cells/well), 40. Mu.l of effector cells (2000T cell clones/well), 20. Mu.l of specific short peptide was added to the experimental group, 20. Mu.l of nonspecific short peptide was added to the control group, 20. Mu.l of medium (test medium) was added to the blank group, and 2 replicate wells were set. Then incubated overnight (37 ℃,5% 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.). The results of the experiment are shown in FIG. 14, and the obtained T cell clone specific to the specific antigen has a specific response to the T2 cell loaded with the short peptide of the present invention, but has no substantial response to the T2 cell loaded with other irrelevant peptides and unloaded with the short peptide.

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

Using Quick-RNA ^TM MiniPrep (ZYMO research) extracted the total RNA of the antigen short peptide FMNKFIYEI specific, HLA-A0201 restricted T cell clone selected in example 1. The cDNA was synthesized using SMART RACE cDNA amplification kit from clontech, using primers designed in the C-terminal conserved 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 TCR beta 0 chain variable domain, the nucleotide sequence of the TCR beta chain variable domain, the amino acid sequence of the TCR beta chain, TC, respectivelyAn R 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 with the following amino acid sequences:

αCDR1-VGISA (SEQ ID NO:10)

αCDR2-LSSGK (SEQ ID NO:11)

αCDR3-AVETSYDKVI (SEQ ID NO:12)

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

βCDR1-SGHVS (SEQ ID NO:13)

βCDR2-FNYEAQ (SEQ ID NO:14)

βCDR3-ASSYGAGGPLDTQY (SEQ ID NO:15)。

the full-length genes of the TCR α and β chains were cloned into the lentiviral expression vector pllenti (addendum) by overlap (overlap) PCR, respectively. The method specifically comprises the following steps: 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 domain and part of the constant domain 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 desired gene sequences for the TCR alpha and beta chains were synthesized and inserted into the expression vector pET28a + (Novagene) by standard methods described in Molecular Cloning A Laboratory Manual (third edition, sambrook and Russell), and the upstream and downstream Cloning sites were NcoI and NotI, respectively. The insert was confirmed by sequencing without error.

The expression vectors of TCR alpha and beta chains are respectively transformed into expression bacteria BL21 (DE 3) by a chemical transformation method, and the bacteria grow by LB culture solution and OD ₆₀₀ At 0.6 induction with final concentration of 0.5mM IPTG, 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, 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 beta-merimepoethylamine (4 ℃ C.) to a final concentration of 60mg/mL. After mixing, the solution was dialyzed against 10 times the volume 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 α and β dimers that were successfully renatured 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

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 flexible short peptide (linker) using site-directed mutagenesis as described in WO 2014/206304. 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 the nucleotide sequence of the alpha chain variable domain are shown in FIG. 8a and FIG. 8b respectively; the amino acid sequence and the nucleotide sequence of the beta chain variable domain are shown in FIG. 9a and FIG. 9b respectively; the amino acid sequence and the nucleotide sequence of the 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 to 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 correct sequence was determined, recombinant plasmids were extracted and transformed into e.coli BL21 (DE 3) for expression.

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

The BL21 (DE 3) colonies containing the recombinant plasmid pET28 a-template strand prepared in example 4 were all inoculated in 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 a Bugbuster Master Mix (Merck), the inclusion bodies were recovered by centrifugation at 6000rpm for 15min, and 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 (20 mM Tris-HCl pH 8.0,8M urea), the insoluble material was removed by high speed centrifugation, the supernatant was quantified by BCA method and then split charged, and stored at-80 ℃ for further use.

To 5mg of solubilized single-chain TCR inclusion body protein, 2.5mL of buffer (6M Gua-HCl,50mM Tris-HCl pH 8.1, 100mM NaCl,10mM EDTA) was added, DTT was added to a final concentration of 10mM, and treatment was carried out at 37 ℃ for 30min. The treated single-chain TCR was added dropwise to 125mL of renaturation buffer (100 mM 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 dialysis 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 (20 mM Tris-HCl pH 8.0), dialysis was continued for 8h at 4 ℃ and then the dialysate was changed to the same fresh buffer and dialysis was continued overnight. After 17 hours, the sample was filtered through a 0.45 μ M filter, vacuum degassed, passed through an anion exchange column (HiTrap Q HP, GE Healthcare), protein purified by a linear gradient of 0-1M NaCl in 20mM Tris-HCl pH 8.0, the fractions collected were analyzed by SDS-PAGE, fractions containing single-chain TCR concentrated and then further purified by a gel filtration column (Superdex 75/300, GE Healthcare), the target fraction was also analyzed by SDS-PAGE.

The eluted fractions for BIAcore analysis were further tested for purity using gel filtration. The conditions are as follows: the chromatographic column Agilent Bio SEC-3 (300A, phi 7.8X 300 mM) 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 214nm.

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 FMNKFIYEI-HLA a0201 complex.

Binding activity of the TCR molecules obtained in examples 3 and 5 to FMNKFIYEI-HLA a0201 complex was tested using a BIAcore T200 real-time assay system. Anti-streptavidin antibody (GenScript) was added to coupling buffer (10 mM 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.

And (2) enabling low-concentration streptavidin to flow through the surface of the chip coated with the antibody, then enabling FMNKFIYEI-HLA A0201 complex to flow through a detection channel, enabling the other channel to serve as a reference channel, and enabling 0.05mM biotin to flow through the chip at the flow rate of 10 mu L/min for 2min to block the remaining binding sites of the streptavidin.

The FMNKFIYEI-HLA A0201 complex is prepared as follows:

a. purification of

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

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

b. Renaturation

The synthesized short peptide FMNKFIYEI (Beijing Saibance Gene technology Co., ltd.) was dissolved in DMSO to a concentration of 20 mg/ml. Inclusion of light and heavy chains was solubilized using 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 oxidized glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4 ℃) at 25mg/L (final concentration), followed by the addition of 20mg/L light chain and 90mg/L heavy chain in sequence (final concentration, heavy chain was added in three portions, 8 h/time), renaturation was carried out at 4 ℃ for at least 3 days until completion, and SDS-PAGE was examined to see whether the renaturation was successful or not.

c. Purification after renaturation

The renaturation buffer was replaced by dialysis against 10 volumes of 20mM Tris pH 8.0, at least twice to reduce the ionic strength of the solution sufficiently. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and then loaded onto a HiTrap Q HP (GE general electric) anion exchange column (5 ml bed volume). Using an Akta purifier (GE general electric Co., ltd.), a linear gradient of 0-400mM NaCl prepared at 20mM Tris pH 8.0 was used to elute proteins, and pMHC was eluted at about 250mM NaCl, and 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 replacing the buffer with 20mM Tris pH 8.0, followed by addition of biotinylation reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50. Mu. M D-Biotin, 100. Mu.g/ml BirA enzyme (GST-BirA), incubation of the mixture overnight at room temperature, and SDS-PAGE to determine whether biotinylation was complete.

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 an Akta purifier (GE general electric Co., ltd.) ^TM A16/60S 200HR column (GE general electric) was loaded with 1ml of concentrated biotinylated pMHC molecules and then eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules appeared as a unimodal elution at approximately 55 ml. The fractions containing the protein were pooled, concentrated using 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 are calculated by using BIAcore Evaluation software, and the obtained soluble TCR molecule and the kinetic maps of the combination of the soluble single-chain TCR molecule and the FMNKFIYEI-HLA A0201 compound are respectively shown in FIG. 12 and FIG. 13. The map shows that the soluble TCR molecule and the soluble single-chain TCR molecule obtained by the invention can be combined with 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 assays were performed to demonstrate the specific activation response of T cells transduced by the inventive TCR on target cells. IFN-. Gamma.production as measured by the ELISPOT assay was used as a readout for T cell activation.

Reagent

Test medium: 10% of FBS (Gibco, catalog number 16000-044), RPMI1640 (Gibco, catalog number C11875500 bt)

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

PBS (Gibbo Co., catalog number C10010500 BT)

PVDF ELISPOT 96-well plate (Merck Millipore, cat. No. MSIPS 4510)

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 specific short peptides. Target cells were prepared in experimental media: the concentration of the target cells is adjusted to 2.0X 10 ⁵ One/ml, 100. Mu.l/well to obtain 2.0X 10 ⁴ Individual cells/well.

Effector cell preparation

The effector cells (T cells) of this experiment were CD8 expressing TCR specific for the AFP antigen oligopeptide of the invention ⁺ T cells and CD8 cells of the same volunteer untransfected with a 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 AFP antigen short peptide specific TCR genes, expanded in 10% FBS containing 1640 medium containing 50IU/ml IL-2 and 10ng/ml IL-7 until 9-12 days post transduction, then placed in assay medium and washed by centrifugation at 300g for 10 minutes 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) experimental group to give a final concentration of 1. Mu.g/ml of short peptide in the ELISPOT well plate.

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. Add 100. Mu.l/well RPMI1640 medium containing 10% FBS and incubate the well plates at room temperature for 2 hours to close the well plates. The media was then washed from the well plate, and any residual wash buffer was removed by flicking and tapping the ELISPOT well plate on paper.

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

100 microliter of target cells 2 x 10 ⁵ Cells/ml (total of about 2 x 10 was obtained) ⁴ 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 plates were then incubated overnight (37 ℃/5% CO) ₂ ) The next day, the medium was discarded, the well plate was washed 2 times with double distilled water and 3 times with wash buffer, and tapped on a paper towel to remove residual wash buffer. Then, the mixture was diluted with 10-vol FBS-containing PBS in a proportion 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.

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

Results

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

As shown in FIG. 15, T cells (effector cells) transduced with the TCR of the invention were very responsive to activation of target cells loaded with their specific short peptides, whereas T cells (effector cells) transduced with other TCRs were substantially 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> T cell receptor for identifying AFP antigen short peptide and its coding sequence

<130> P2018-0967

<160> 37

<170> PatentIn version 3.5

<210> 1

<211> 109

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 1

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

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

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His Trp Leu Gln Gln His Pro Gly Gly Gly Ile Val Ser Leu Phe Met

35 40 45

Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Ile Ala Thr Ile Asn

50 55 60

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

65 70 75 80

Asp Ser Ala Val Tyr Ile Cys Ala Val Glu Thr Ser Tyr Asp Lys Val

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Ile Phe Gly Pro Gly Thr Ser Leu Ser Val Ile Pro Asn

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<210> 2

<211> 327

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

aaaaatgaag tggagcagag tcctcagaac ctgactgccc aggaaggaga atttatcaca 60

atcaactgca gttactcggt aggaataagt gccttacact ggctgcaaca gcatccagga 120

ggaggcattg tttccttgtt tatgctgagc tcagggaaga agaagcatgg aagattaatt 180

gccacaataa acatacagga aaagcacagc tccctgcaca tcacagcctc ccatcccaga 240

gactctgccg tctacatctg tgctgtcgaa acctcctacg acaaggtgat atttgggcca 300

gggacaagct tatcagtcat tccaaat 327

<210> 3

<211> 249

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 3

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

1 5 10 15

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

20 25 30

His Trp Leu Gln Gln His Pro Gly Gly Gly Ile Val Ser Leu Phe Met

35 40 45

Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Ile Ala Thr Ile Asn

50 55 60

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

65 70 75 80

Asp Ser Ala Val Tyr Ile Cys Ala Val Glu Thr Ser Tyr Asp Lys Val

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Leu Met Thr Leu Arg Leu Trp Ser Ser

245

<210> 4

<211> 747

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

aaaaatgaag tggagcagag tcctcagaac ctgactgccc aggaaggaga atttatcaca 60

atcaactgca gttactcggt aggaataagt gccttacact ggctgcaaca gcatccagga 120

ggaggcattg tttccttgtt tatgctgagc tcagggaaga agaagcatgg aagattaatt 180

gccacaataa acatacagga aaagcacagc tccctgcaca tcacagcctc ccatcccaga 240

gactctgccg tctacatctg tgctgtcgaa acctcctacg acaaggtgat atttgggcca 300

gggacaagct tatcagtcat tccaaatatc cagaaccctg accctgccgt gtaccagctg 360

agagactcta aatccagtga caagtctgtc tgcctattca ccgattttga ttctcaaaca 420

aatgtgtcac aaagtaagga ttctgatgtg tatatcacag acaaaactgt gctagacatg 480

aggtctatgg acttcaagag caacagtgct gtggcctgga gcaacaaatc tgactttgca 540

tgtgcaaacg ccttcaacaa cagcattatt ccagaagaca ccttcttccc cagcccagaa 600

agttcctgtg atgtcaagct ggtcgagaaa agctttgaaa cagatacgaa cctaaacttt 660

caaaacctgt cagtgattgg gttccgaatc ctcctcctga aagtggccgg gtttaatctg 720

ctcatgacgc tgcggctgtg gtccagc 747

<210> 5

<211> 116

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 5

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

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Leu Thr Val Leu

115

<210> 6

<211> 348

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

ggtgctggag tctcccagtc tcccaggtac aaagtcacaa agaggggaca ggatgtagct 60

ctcaggtgtg atccaatttc gggtcatgta tccctttatt ggtaccgaca ggccctgggg 120

cagggcccag agtttctgac ttacttcaat tatgaagccc aacaagacaa atcagggctg 180

cccaatgatc ggttctctgc agagaggcct gagggatcca tctccactct gacgatccag 240

cgcacagagc agcgggactc ggccatgtat cgctgtgcca gcagctacgg agcgggaggg 300

cctttagata cgcagtattt tggcccaggc acccggctga cagtgctc 348

<210> 7

<211> 295

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 7

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

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Lys Arg Lys Asp Ser Arg Gly

290 295

<210> 8

<211> 885

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

ggtgctggag tctcccagtc tcccaggtac aaagtcacaa agaggggaca ggatgtagct 60

ctcaggtgtg atccaatttc gggtcatgta tccctttatt ggtaccgaca ggccctgggg 120

cagggcccag agtttctgac ttacttcaat tatgaagccc aacaagacaa atcagggctg 180

cccaatgatc ggttctctgc agagaggcct gagggatcca tctccactct gacgatccag 240

cgcacagagc agcgggactc ggccatgtat cgctgtgcca gcagctacgg agcgggaggg 300

cctttagata cgcagtattt tggcccaggc acccggctga cagtgctcga ggacctgaaa 360

aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 420

caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 480

tggtgggtga atgggaagga ggtgcacagt ggggtcagca cagacccgca gcccctcaag 540

gagcagcccg ccctcaatga ctccagatac tgcctgagca gccgcctgag ggtctcggcc 600

accttctggc agaacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660

gagaatgacg agtggaccca ggatagggcc aaacctgtca cccagatcgt cagcgccgag 720

gcctggggta gagcagactg tggcttcacc tccgagtctt accagcaagg ggtcctgtct 780

gccaccatcc tctatgagat cttgctaggg aaggccacct tgtatgccgt gctggtcagt 840

gccctcgtgc tgatggccat ggtcaagaga aaggattcca gaggc 885

<210> 9

<211> 9

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 9

Phe Met Asn Lys Phe Ile Tyr Glu Ile

1 5

<210> 10

<211> 5

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 10

Val Gly Ile Ser Ala

1 5

<210> 11

<211> 5

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 11

Leu Ser Ser Gly Lys

1 5

<210> 12

<211> 10

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 12

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

1 5

<210> 15

<211> 14

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 15

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

1 5 10

<210> 16

<211> 15

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 16

gtaggaataa gtgcc 15

<210> 17

<211> 15

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 17

ctgagctcag ggaag 15

<210> 18

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 18

gctgtcgaaa cctcctacga caaggtgata 30

<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

ttcaattatg aagcccaa 18

<210> 21

<211> 42

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 21

gccagcagct acggagcggg agggccttta gatacgcagt at 42

<210> 22

<211> 271

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 22

Met Val Lys Ile Arg Gln Phe Leu Leu Ala Ile Leu Trp Leu Gln Leu

1 5 10 15

Ser Cys Val Ser Ala Ala Lys Asn Glu Val Glu Gln Ser Pro Gln Asn

20 25 30

Leu Thr Ala Gln Glu Gly Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser

35 40 45

Val Gly Ile Ser Ala Leu His Trp Leu Gln Gln His Pro Gly Gly Gly

50 55 60

Ile Val Ser Leu Phe Met Leu Ser Ser Gly Lys Lys Lys His Gly Arg

65 70 75 80

Leu Ile Ala Thr Ile Asn Ile Gln Glu Lys His Ser Ser Leu His Ile

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

<210> 23

<211> 813

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 23

atggtgaaga tccggcaatt tttgttggct attttgtggc ttcagctaag ctgtgtaagt 60

gccgccaaaa atgaagtgga gcagagtcct cagaacctga ctgcccagga aggagaattt 120

atcacaatca actgcagtta ctcggtagga ataagtgcct tacactggct gcaacagcat 180

ccaggaggag gcattgtttc cttgtttatg ctgagctcag ggaagaagaa gcatggaaga 240

ttaattgcca caataaacat acaggaaaag cacagctccc tgcacatcac agcctcccat 300

cccagagact ctgccgtcta catctgtgct gtcgaaacct cctacgacaa ggtgatattt 360

gggccaggga caagcttatc agtcattcca aatatccaga accctgaccc tgccgtgtac 420

cagctgagag actctaaatc cagtgacaag tctgtctgcc tattcaccga ttttgattct 480

caaacaaatg tgtcacaaag taaggattct gatgtgtata tcacagacaa aactgtgcta 540

gacatgaggt ctatggactt caagagcaac agtgctgtgg cctggagcaa caaatctgac 600

tttgcatgtg caaacgcctt caacaacagc attattccag aagacacctt cttccccagc 660

ccagaaagtt cctgtgatgt caagctggtc gagaaaagct ttgaaacaga tacgaaccta 720

aactttcaaa acctgtcagt gattgggttc cgaatcctcc tcctgaaagt ggccgggttt 780

aatctgctca tgacgctgcg gctgtggtcc agc 813

<210> 24

<211> 314

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 24

Met Gly Thr Ser 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 Thr

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

50 55 60

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

65 70 75 80

Asn Asp Arg Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Ala Met Val Lys Arg Lys Asp Ser Arg Gly

305 310

<210> 25

<211> 942

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 25

atgggcacca gtctcctatg ctgggtggtc ctgggtttcc tagggacaga tcacacaggt 60

gctggagtct cccagtctcc caggtacaaa gtcacaaaga ggggacagga tgtagctctc 120

aggtgtgatc caatttcggg tcatgtatcc ctttattggt accgacaggc cctggggcag 180

ggcccagagt ttctgactta cttcaattat gaagcccaac aagacaaatc agggctgccc 240

aatgatcggt tctctgcaga gaggcctgag ggatccatct ccactctgac gatccagcgc 300

acagagcagc gggactcggc catgtatcgc tgtgccagca gctacggagc gggagggcct 360

ttagatacgc agtattttgg cccaggcacc cggctgacag tgctcgagga cctgaaaaac 420

gtgttcccac ccgaggtcgc tgtgtttgag ccatcagaag cagagatctc ccacacccaa 480

aaggccacac tggtgtgcct ggccacaggc ttctaccccg accacgtgga gctgagctgg 540

tgggtgaatg ggaaggaggt gcacagtggg gtcagcacag acccgcagcc cctcaaggag 600

cagcccgccc tcaatgactc cagatactgc ctgagcagcc gcctgagggt ctcggccacc 660

ttctggcaga acccccgcaa ccacttccgc tgtcaagtcc agttctacgg gctctcggag 720

aatgacgagt ggacccagga tagggccaaa cctgtcaccc agatcgtcag cgccgaggcc 780

tggggtagag cagactgtgg cttcacctcc gagtcttacc agcaaggggt cctgtctgcc 840

accatcctct atgagatctt gctagggaag gccaccttgt atgccgtgct ggtcagtgcc 900

ctcgtgctga tggccatggt caagagaaag gattccagag gc 942

<210> 26

<211> 203

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 26

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

1 5 10 15

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

20 25 30

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

35 40 45

Met Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Ile Ala Thr Ile

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

195 200

<210> 27

<211> 609

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 27

atgaaaaacg aagtggaaca gagtcctcag aacctgactg cccaggaagg agaatttatc 60

acaatcaact gcagttactc ggtaggaata agtgccttac actggctgca acagcatcca 120

ggaggaggca ttgtttcctt gtttatgctg agctcaggga agaagaagca tggaagatta 180

attgccacaa taaacataca ggaaaagcac agctccctgc acatcacagc ctcccatccc 240

agagactctg ccgtctacat ctgtgctgtc gaaacctcct acgacaaggt gatatttggg 300

ccagggacaa gcttatcagt cattccaaat atccagaacc ctgaccctgc cgtgtaccag 360

ctgagagact ctaagtcgag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 420

acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaatg tgtgctagac 480

atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacaa atctgacttt 540

gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca 600

gaaagttcc 609

<210> 28

<211> 247

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 28

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Glu Ala Trp Gly Arg Ala Asp

245

<210> 29

<211> 741

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 29

atgggtgcag gtgttagcca gtctcccagg tacaaagtca caaagagggg acaggatgta 60

gctctcaggt gtgatccaat ttcgggtcat gtatcccttt attggtaccg acaggccctg 120

gggcagggcc cagagtttct gacttacttc aattatgaag cccaacaaga caaatcaggg 180

ctgcccaatg atcggttctc tgcagagagg cctgagggat ccatctccac tctgacgatc 240

cagcgcacag agcagcggga ctcggccatg tatcgctgtg ccagcagcta cggagcggga 300

gggcctttag atacgcagta ttttggccca ggcacccggc tgacagtgct cgaggacctg 360

aaaaacgtgt tcccacccga ggtcgctgtg tttgagccat cagaagcaga gatctcccac 420

acccaaaagg ccacactggt gtgcctggcc accggtttct accccgacca cgtggagctg 480

agctggtggg tgaatgggaa ggaggtgcac agtggggtct gcacagaccc gcagcccctc 540

aaggagcagc ccgccctcaa tgactccaga tacgctctga gcagccgcct gagggtctcg 600

gccaccttct ggcaggaccc ccgcaaccac ttccgctgtc aagtccagtt ctacgggctc 660

tcggagaatg acgagtggac ccaggatagg gccaaacccg tcacccagat cgtcagcgcc 720

gaggcctggg gtagagcaga c 741

<210> 30

<211> 249

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 30

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

1 5 10 15

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

20 25 30

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

35 40 45

Met Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Asn Ala Thr Ile

50 55 60

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

65 70 75 80

Arg Asp Ser Ala Val Tyr Phe Cys Ala Val Glu Thr Ser Tyr Asp Lys

85 90 95

Val Ile Phe Gly Pro Gly Thr Ser Leu Ser Val Thr Pro Gly Gly Gly

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Leu Pro Asn Asp Arg Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser

195 200 205

Thr Leu Thr Ile Gln Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe

210 215 220

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

225 230 235 240

Gly Pro Gly Thr Arg Leu Thr Val Asp

245

<210> 31

<211> 747

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 31

gctaaaaatg aagttgaaca gagcccgcaa aacctgaccg tgcaggaagg tgaaaacgtt 60

accatcaact gcagctacag cgtgggtatt agcgcgctgc attggctgcg tcaagatccg 120

ggtggcggta tcgttagcct gttcatgctg agcagcggca agaaaaagca cggtcgtctg 180

aacgcgacca tcaacattca agagaaacac agcagcctgc acattaccga cgttcacccg 240

cgtgatagcg cggtgtactt ttgcgcggtt gaaaccagct atgacaaagt gatttttggt 300

ccgggtacca gcctgagcgt taccccgggc ggtggcagcg agggtggcgg tagcgaaggc 360

ggtggcagcg agggtggcgg tagcgaaggc ggtaccggcg gtgcgggtgt gagccagagc 420

ccgcgttacc tgagcgtgaa acgtggccaa gacgttaccc tgcgttgcga tccgatcagc 480

ggtcacgtta gcctgtactg gtaccgtcaa gatccgggtc aaggtccgga gttcctgacc 540

tactttaact atgaagcgca gcaagacaag agcggcctgc cgaacgatcg tttcagcgcg 600

gagcgtccgg aaggtagcat cagcaccctg accattcagc gtgtggagcc gcgtgatagc 660

gcgatgtatt tttgcgcgag cagctacggt gcgggcggtc cgctggacac ccaatatttt 720

ggcccgggta cccgtctgac cgttgat 747

<210> 32

<211> 109

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 32

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

1 5 10 15

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

20 25 30

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

35 40 45

Met Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Asn Ala Thr Ile

50 55 60

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

65 70 75 80

Arg Asp Ser Ala Val Tyr Phe Cys Ala Val Glu Thr Ser Tyr Asp Lys

85 90 95

Val Ile Phe Gly Pro Gly Thr Ser Leu Ser Val Thr Pro

100 105

<210> 33

<211> 327

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 33

gctaaaaatg aagttgaaca gagcccgcaa aacctgaccg tgcaggaagg tgaaaacgtt 60

accatcaact gcagctacag cgtgggtatt agcgcgctgc attggctgcg tcaagatccg 120

ggtggcggta tcgttagcct gttcatgctg agcagcggca agaaaaagca cggtcgtctg 180

aacgcgacca tcaacattca agagaaacac agcagcctgc acattaccga cgttcacccg 240

cgtgatagcg cggtgtactt ttgcgcggtt gaaaccagct atgacaaagt gatttttggt 300

ccgggtacca gcctgagcgt taccccg 327

<210> 34

<211> 116

<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 Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Leu Thr Val Asp

115

<210> 35

<211> 348

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 35

ggtgcgggtg tgagccagag cccgcgttac ctgagcgtga aacgtggcca agacgttacc 60

ctgcgttgcg atccgatcag cggtcacgtt agcctgtact ggtaccgtca agatccgggt 120

caaggtccgg agttcctgac ctactttaac tatgaagcgc agcaagacaa gagcggcctg 180

ccgaacgatc gtttcagcgc ggagcgtccg gaaggtagca tcagcaccct gaccattcag 240

cgtgtggagc cgcgtgatag cgcgatgtat ttttgcgcga gcagctacgg tgcgggcggt 300

ccgctggaca cccaatattt tggcccgggt acccgtctga ccgttgat 348

<210> 36

<211> 24

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 36

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

1 5 10 15

Gly Gly Ser Glu Gly Gly Thr Gly

20

<210> 37

<211> 72

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 37

ggcggtggca gcgagggtgg cggtagcgaa ggcggtggca gcgagggtgg cggtagcgaa 60

ggcggtaccg gc 72

Claims (36)

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

αCDR1-VGISA (SEQ ID NO:10)

αCDR2-LSSGK (SEQ ID NO:11)

α CDR3-AVETSYDKVI (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-FNYEAQ (SEQ ID NO:14)

βCDR3-ASSYGAGGPLDTQY (SEQ ID NO:15)。

2. a TCR as claimed in claim 1 wherein the TCR α chain variable domain is an amino acid sequence which has 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 x 01 or TRBC2 x 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/or the beta chain amino acid sequence of the TCR is SEQ ID NO 7.

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

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

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

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

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

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

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

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

15. A TCR as claimed in claim 14 in which the cysteine residues form an artificial disulphide bond between the α and β chain constant domains of the TCR.

16. A TCR as claimed in claim 15 wherein the cysteine residues which form the artificial disulphide bond in the TCR are substituted at one or more groups selected from:

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

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

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

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

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

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

17. A TCR as claimed in claim 16 wherein 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.

18. A TCR as claimed in claim 14 which comprises an artificial interchain disulphide bond between the α chain variable domain and the β chain constant domain of the TCR.

19. A TCR as claimed in claim 18 wherein the cysteine residues which form the artificial interchain disulphide bond in the TCR are substituted at one or more groups 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 exon 1 of TRBC1 x 01 or TRBC2 x 01; or

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

20. A TCR as claimed in claim 18 or 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.

21. 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.

22. A TCR as claimed in claim 21 wherein the conjugate to which the TCR is bound is a detectable label, a therapeutic agent, a PK modifying moiety or a combination thereof.

23. A TCR as claimed in claim 22 wherein the therapeutic agent is an anti-CD 3 antibody.

24. 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 23.

25. A nucleic acid molecule comprising a nucleic acid sequence encoding a TCR according to any one of claims 1 to 3, or the complement thereof.

26. The nucleic acid molecule of claim 25, wherein said nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:33.

27. The nucleic acid molecule of claim 25 or 26, wherein said nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:6 or SEQ ID NO 35.

28. The nucleic acid molecule of claim 25, comprising the nucleotide sequence encoding a TCR α chain of SEQ ID NO:4 and/or a nucleic acid sequence comprising the nucleotide sequence encoding the TCR β chain SEQ ID NO:8.

29. a vector comprising the nucleic acid molecule of any one of claims 25-28.

30. The vector of claim 29, wherein said vector is a viral vector.

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

32. An isolated host cell comprising the vector of claim 29 or a nucleic acid molecule of any one of claims 25-28 integrated into the chromosome.

33. A cell transduced with the nucleic acid molecule of any one of claims 25 to 28 or the vector of claim 29.

34. The cell of claim 33, wherein the cell is a T cell or a stem cell.

35. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to any one of claims 1 to 23, a TCR complex according to claim 24 or a cell according to claim 33.

36. Use of a TCR as claimed in any of claims 1 to 23, or a TCR complex as claimed in claim 24 or a cell as claimed in claim 33, for 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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