CN108659114B - TCR for recognizing PASD1 antigen short peptide - Google Patents

Abstract

The invention relates to a TCR for recognizing PASD1 antigen short peptide. The present invention provides a T Cell Receptor (TCR) capable of specifically binding a short peptide qlerterwll derived from the PASD1 antigen, which can form a complex with HLA a0201 and be presented together on the cell surface. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules. In addition, the invention provides cells that transduce a TCR of the invention.

Description

TCR for recognizing PASD1 antigen short peptide

Technical Field

The invention relates to a TCR capable of recognizing a short peptide derived from a PASD1 antigen, and PASD 1-specific T cells obtained by transducing the TCR, and use of the T cells in prevention and treatment of PASD 1-related diseases.

Background

As an endogenous tumor antigen, PASD1 is degraded into small polypeptides after intracellular production and is presented on the cell surface as a complex with MHC (major histocompatibility complex) molecules. Studies have shown that qlertpll is a short peptide derived from PASD 1. PASD1 antigen is common in diffuse large B-cell lymphomas and multiple myeloma, but is not expressed in most normal tissues except testis (Joseph-Pietraras D1, gao Y, zojer N, ait-Tahar K, banham AH, pulford K, rice J, savelyeva N, sahota SS. Leukemia.2010 Nov;24 (11): 1951-9 Cooper CD, liggins AP, ait-Tahar K, roncador G, banham AH, pulford K. PASD1, leukemia 20 2172-2174.. For the treatment of the above diseases, chemotherapy, radiotherapy and the like can be used, but both of them cause damages to normal cells themselves.

T cell adoptive immunotherapy is the transfer of reactive T cells specific for a target cell antigen into a patient to act on the target cell. The T Cell Receptor (TCR) is a membrane protein on the surface of T cells that recognizes a corresponding short peptide antigen on the surface of a target cell. In the immune system, the direct physical contact between T cells and Antigen Presenting Cells (APC) is initiated by the binding of antigen short peptide specific TCR and short peptide-major histocompatibility complex (pMHC complex), and then other cell membrane surface molecules of the T cells and APC interact to cause a series of subsequent cell signaling and other physiological reactions, so that T cells with different antigen specificities exert immune effects on their target cells. Therefore, those skilled in the art have focused on the isolation and action of TCR specific for the PASD1 antigen short peptide, or the transduction of T cells with the TCR to obtain T cells specific for the PASD1 antigen short peptide, thereby allowing them to play a role in cellular immunotherapy.

Disclosure of Invention

The invention aims to provide a T cell receptor for recognizing the short peptide of the PASD1 antigen.

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

In another preferred embodiment, the TCR comprises a TCR α chain variable domain and a TCR β chain variable domain, the amino acid sequence of CDR3 of the TCR α chain variable domain being CAMRAIQGAQKLVF (SEQ ID No. 12); and/or the amino acid sequence of CDR3 of the variable domain of the TCR β chain is CASSRGQGEAFF (SEQ ID No. 15).

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

αCDR1-NSAFQY(SEQ ID NO.10)

αCDR2-TYSSGN(SEQ ID NO.11)

α CDR3-CAMRAIQGAQKLVF (SEQ ID NO. 12); and/or

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

βCDR1-PRHDT(SEQ ID NO.13)

βCDR2-FYEKMQ(SEQ ID NO.14)

βCDR3-CASSRGQGEAFF(SEQ ID NO.15)。

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

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 β 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 amino acid sequence of the beta chain of the TCR is SEQ ID No.7.

In another preferred embodiment, the TCR is soluble.

In another preferred embodiment, the TCR is single chain.

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

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

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

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

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

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

thr48 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 of TRAC × 01 exon 1 and TRBC1 × 01 or TRBC2 × 01 exon 1 Ser17;

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.

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 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 comprises a conjugate attached to the C-or N-terminus of the α chain and/or β chain.

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

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

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

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

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

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

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

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

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

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

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

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

preferably, the disease is a tumor, preferably the tumor comprises diffuse large B-cell lymphoma, multiple myeloma, melanoma, and other solid tumors such as gastric cancer, lung cancer, esophageal cancer, bladder cancer, head and neck squamous cell carcinoma, prostate cancer, breast cancer, colon cancer, ovarian cancer, and the like.

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

Drawings

FIG. 1a, FIG. 1b, FIG. 1c, FIG. 1d, FIG. 1e and FIG. 1f are the 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 shows CD8 of monoclonal cells ⁺ And tetramer-PE double positive staining results.

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

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

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

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

FIG. 8 shows the results of transfection assays of T cell receptor lentivirus packaging and primary T cells.

FIG. 9 shows the results of the ELISPOT assay.

FIG. 10 is a graph of the results of a nonradioactive cytotoxicity assay of effector cells transduced with a TCR of the invention.

Detailed Description

The present inventors have extensively and intensively studied to find a TCR capable of specifically binding to the PASD1 antigen short peptide qlertpll (SEQ ID No. 9) which can form a complex with HLA a0201 and be presented together on the cell surface. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules. In addition, the invention provides cells that transduce a TCR of the invention.

Term(s) for

MHC molecules are proteins of the immunoglobulin superfamily, which may be MHC class I or class II molecules. Therefore, it is specific for antigen presentation, different individuals have different MHC, and different short peptides in one protein antigen can be presented on the cell surface of respective APC. Human MHC is often referred to as HLA gene or HLA complex.

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

TCRs are cell membrane surface glycoproteins that exist as heterodimers from either the α chain/β chain or the γ chain/δ chain. In 95% of T cells the TCR heterodimer consists of α and β chains, while 5% of T cells have 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, which is located differently from 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 within cells and then carried to the cell surface by MHC molecules. T cell receptors are capable of recognizing peptide-MHC complexes on the surface of antigen presenting cells. Accordingly, in a first aspect the invention provides a TCR molecule capable of binding to the qlerterwll-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-NSAFQY(SEQ ID NO.10)

αCDR2-TYSSGN(SEQ ID NO.11)

α CDR3-CAMRAIQGAQKLVF (SEQ ID NO. 12); and/or

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

βCDR1-PRHDT(SEQ ID NO.13)

βCDR2-FYEKMQ(SEQ ID NO.14)

βCDR3-CASSRGQGEAFF(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 the alpha chain variable domain amino acid sequence SEQ ID No.1. More preferably, the amino acid sequence of the α chain variable domain of the TCR molecule is SEQ ID No.1. In another aspect, the β chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the β chain variable domain amino acid sequence comprises CDR1 (SEQ ID NO. 13), CDR2 (SEQ ID NO: 14), and CDR3 (SEQ ID NO. 15) of the above-described β chain. Preferably, the TCR molecule comprises the β 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. One skilled in the art can readily construct single chain TCR molecules comprising the CDRs regions of the present invention, based on the teachings in the literature. In particular, the single chain TCR molecule comprises V α, V β and C β, preferably linked in order from N-terminus to C-terminus.

The alpha chain variable domain amino acid sequence of the single chain TCR molecule comprises CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the alpha chain described above. Preferably, the single chain TCR molecule comprises the alpha chain variable domain amino acid sequence SEQ ID No.1. More preferably, the α chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID No.1. The amino acid sequence of the 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 described above. Preferably, the single chain TCR molecule comprises the β chain variable domain amino acid sequence SEQ ID No.5. More preferably, the β chain variable domain amino acid sequence 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 person skilled in the art knows or can obtain the human constant domain amino acid sequence by consulting public 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 molecules of the invention is SEQ ID No.3, and/or the amino acid sequence of the β chain is SEQ ID No.7.

Naturally occurring TCRs are membrane proteins that are stabilized by their transmembrane regions. Like immunoglobulins (antibodies) as antigen recognition molecules, TCRs can also be developed for diagnostic and therapeutic applications, where 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 infection or as markers for autoimmune diseases. Similarly, soluble TCRs can be used to deliver therapeutic agents (e.g., cytotoxic or immunostimulatory compounds) to cells presenting a specific antigen, and in addition, soluble TCRs can be conjugated to other molecules (e.g., anti-CD 3 antibodies) to redirect T cells to target them to cells presenting a particular antigen. The invention also obtains soluble TCR with specificity to the PASD1 antigen short peptide.

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 TRAC × 01 exon 1 and TRBC1 × 01 or Ala19 of TRBC2 × 01 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 has substituted any of the above-mentioned groups of positions in the constant domains of the alpha and beta chains. Deletion of the native disulfide bond can be achieved by truncating at most 50, or at most 30, or at most 15, or at most 10, or at most 8 or fewer amino acids at one or more of the C-termini of the TCR constant domains of the invention such that they do not include a cysteine residue, or by mutating a cysteine residue that forms a native disulfide bond to another amino acid.

As described above, the TCRs of the invention may comprise artificial disulfide bonds introduced between residues of the constant domains of their alpha and beta chains. It should be noted that the TCRs of the invention may each contain both TRAC constant domain sequences and TRBC1 or TRBC2 constant domain sequences, with or without the artificial disulfide bonds introduced as described above between the constant domains. The TRAC constant domain sequence and 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 TCR may, on the other hand, also comprise a TCR having a mutation in its hydrophobic core region, preferably a mutation that results in an improved stability of the inventive soluble TCR, as described in the patent publication WO 2014/206304. Such TCRs may be mutated at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or positions 3,5,7 from the last amino acid position of the short peptide of the alpha chain J gene (TRAJ) and/or positions 2,4,6 from the last amino acid position of the short peptide of the beta chain J gene (TRBJ), wherein the position numbers of the amino acid sequences are according to the position numbers 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 invention can be a stable soluble single chain TCR formed by connecting the variable domains of the alpha and beta chains of the TCR by a flexible peptide chain. It should be noted that the flexible peptide chain of the present invention can be any peptide chain suitable for linking the variable domains of TCR α and β chains.

In addition, for stability, PCT/CN2016/077680 also discloses that the introduction of an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of TCR can significantly improve TCR stability. 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 may be covalently or otherwise associated, preferably covalently, with a conjugate. The conjugates include a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the qlerthtl-HLA a0201 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.

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

Therapeutic agents that may be associated with or coupled to the TCRs of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, cancer metastasis reviews (Cancer metastasis) 24, 539); 2. 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, journal of the national academy of sciences (PNAS) 89, 1428, card et al, 2004, cancer Immunology and Immunotherapy) 53, 345, hain 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 communications (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 TCR of the invention may also be a hybrid TCR comprising sequences derived from more than one species. For example, studies have shown that murine TCRs are more efficiently expressed in human T cells than human TCRs. Thus, the inventive TCR may comprise a human variable domain and a murine constant domain. The drawback of this approach is the possibility of eliciting an immune response. Thus, there should be a regulatory regimen to immunosuppresse when it is used for adoptive T cell therapy to allow for the engraftment of murine expressing T cells.

It is understood that the amino acid names herein are expressed in the single english alphabet or the three english alphabets of the international common usage, and the single english alphabet and the three english alphabets of the amino acid names correspond to each other as follows: 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 CDR regions of the α chain of the TCR molecule of the first aspect of the invention is as follows:

αCDR1-aacagtgcttttcaatac(SEQ ID NO.16)

αCDR2-acatactccagtggtaac(SEQ ID NO.17)

αCDR3-tgtgcaatgagggcaattcagggagcccagaagctggtattt(SEQ ID NO.18)

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

βCDR1-cctagacacgacact(SEQ ID NO.19)

βCDR2-ttttatgaaaagatgcag(SEQ ID NO.20)

βCDR3-tgtgccagcagccgcggacagggtgaagctttcttt(SEQ ID NO.21)

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

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

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

The nucleotide sequence may be codon optimized. Different cells differ in the utilization of specific codons, and the expression level can be increased by changing the codons in the sequence according to the type of the cell. Codon usage tables for mammalian cells as well as for a variety of 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 PASD1 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 the expression of the inventionIsolated cells, particularly T cells, of the subject TCRs. The T cell may be derived from a T cell isolated from a subject, or may be part of a mixed population of cells isolated from a subject, such as a population of Peripheral Blood Lymphocytes (PBLs). For example, the cells may be isolated from Peripheral Blood Mononuclear Cells (PBMC), which may be CD4 ⁺ Helper T cell or CD8 ⁺ A cytotoxic T cell. 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 an antibody (e.g., an anti-CD 3 or anti-CD 28 antibody) to render them more amenable to transfection, e.g., transfection with a vector comprising a nucleotide sequence encoding a TCR molecule of the invention.

Alternatively, the cells of the invention may also be or be derived from stem cells, such as Hematopoietic Stem Cells (HSCs). Gene transfer to HSCs does not result in TCR expression on the cell surface, since the CD3 molecule is not expressed on the stem cell surface. However, when stem cells differentiate into lymphoid precursors (lymphoid precursors) that migrate to the thymus, expression of the CD3 molecule will initiate expression of the introduced TCR molecule on the surface of the thymocytes.

There are many methods suitable for T cell transfection using DNA or RNA encoding the TCR of the invention (e.g., robbins et al, (2008) J.Immunol.180: 6116-6131). T cells expressing the inventive TCR can be used in adoptive immunotherapy. Those skilled in the art will be able to recognize many suitable methods for adoptive therapy (e.g., rosenberg et al, (2008) Nat Rev Cancer8 (4): 299-308).

PASD1 antigen associated diseases

The invention also relates to a method of treating and/or preventing a disease associated with PASD1 in a subject comprising the step of adoptively transferring PASD 1-specific T cells to the subject. The PASD1 specific T cells can recognize QLEERTFLL-HLA A0201 complex.

The PASD1 specific T cells of the invention can be used for treating any PASD1 related diseases presenting the PASD1 antigen short peptide QLEERTFLL-HLA A0201 complex. Including but not limited to tumors such as melanoma, and other solid tumors such as gastric cancer, lung cancer, esophageal cancer, bladder cancer, head and neck squamous cell carcinoma, prostate cancer, breast cancer, colon cancer, ovarian cancer, and the like.

Method of treatment

Treatment may be effected by isolating T cells from patients or volunteers suffering from a disease associated with the PASD1 antigen and introducing the TCR of the invention into such T cells, followed by reinfusion of these genetically engineered cells into the patient. Accordingly, the present invention provides a method of treating a PASD1 related disease comprising infusing into a patient isolated T cells expressing a TCR of the invention, preferably derived from the patient himself. Generally, this involves (1) isolating T cells from the patient, (2) transducing T cells in vitro with a nucleic acid molecule of the invention or a nucleic acid molecule capable of encoding a TCR molecule of the invention, and (3) infusing the genetically modified T cells into the patient. The number of cells isolated, transfected and transfused can be determined by a physician.

The main advantages of the invention are:

(1) The TCR can be combined with a PASD1 antigen short peptide complex QLEERTWLL-HLA A0201, and cells transduced with the TCR can be specifically activated and have strong killing effect on target cells.

The following specific examples further illustrate the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example as described in Sambrook and 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 cloning of specific T cells

Peripheral Blood Lymphocytes (PBLs) from healthy volunteers of genotype HLA-A02 were stimulated using the synthetic short peptide QLEERTFLL (Nanjing Kingskan Biotech Co., ltd.). The qlerterwll short peptide was renatured with HLA-base:Sub>A 0201 with biotin label to preparebase:Sub>A pHLA homopolymer. These monomers were combined with streptavidin labeled with PE (BD Co.) to form PE-labeled tetramers, which were then sorted from anti-CD 8-APC double positive cells. The sorted cells were expanded and subjected to secondary sorting as described above, followed by monoclonal culture by limiting dilution. Monoclonal cells were stained with tetramer and double positive clones were selected as shown in FIG. 3.

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

Using Quick-RNA ^TM Total RNA from QLEERTLL-specific, HLA-A02-restricted T cell clones selected in example 1 was extracted by MiniPrep (ZYMO research). cDNA was synthesized using the SMART RACE cDNA amplification kit from clontech, using primers designed to be conserved at the C-terminus of the human TCR gene. The sequences were cloned into the T vector (TAKARA) and sequenced. The alpha chain and beta chain sequence structures of the TCR expressed by the double positive clone are respectively shown in figure 1 and figure 2 after sequencing, and figure 1a, figure 1b, figure 1c and figure 1d are respectively a TCR alpha chain variable domain amino acid sequence, a TCR alpha chain variable domain nucleotide sequence, a TCR alpha chain amino acid sequence and a TCR alpha chain nucleotide sequence; fig. 2a, fig. 2b, fig. 2c and fig. 2d are a TCR β chain variable domain amino acid sequence, a TCR β chain variable domain nucleotide sequence, a TCR β chain amino acid sequence and a TCR β chain nucleotide sequence, respectively.

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

αCDR1-NSAFQY(SEQ ID NO.10)

αCDR2-TYSSGN(SEQ ID NO.11)

αCDR3-CAMRAIQGAQKLVF(SEQ ID NO.12)

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

βCDR1-PRHDT(SEQ ID NO.13)

βCDR2-FYEKMQ(SEQ ID NO.14)

βCDR3-CASSRGQGEAFF(SEQ ID NO.15)。

variable domains of TCR α chain and β chain were spliced to conserved domains of mouse TCR α chain and β chain, respectively, to full-length genes by overlap (overlap) PCR and ligated to lentiviral expression vector pLenti (addendum). The method comprises the following specific steps: the TCR alpha chain and the TCR beta chain are connected by overlap PCR to obtain the TCR alpha-2A-TCR beta segment. The lentivirus expression vector and the TCR alpha-2A-TCR beta are connected by enzyme digestion to obtain pLenti-PASD1TRA-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 PASD1 antigen short peptide specific soluble TCR

To obtain soluble TCR molecules, the α and β chains of the TCR molecules of the invention may comprise only the variable and part of the constant domains thereof, respectively, and a cysteine residue is introduced into the constant domains of the α and β chains, respectively, to form artificial interchain disulfide bonds, at the positions Thr48 of TRAC × 01 exon 1 and Ser57 of TRBC2 × 01 exon 1, respectively; the amino acid sequence and nucleotide sequence of the α chain are shown in FIGS. 4a and 4b, respectively, and the amino acid sequence and nucleotide sequence of the β chain are shown in FIGS. 5a and 5b, respectively, with the introduced cysteine residues indicated in bold and underlined letters. 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 in LB culture solution and grow on OD ₆₀₀ Induction with final concentration 0.5mM IPTG at 0.6 inclusion bodies formed after α and β chain expression of TCR were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution for several times, the inclusion bodies finally being 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 are separated by a 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 method. The SDS-PAGE gel of the soluble TCR of the invention is shown in FIG. 6.

Example 4 binding characterization

ProteOn analysis

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

The binding activity of the TCR molecules obtained in example 3 to the QLEERTLL-HLA A0201 complex was examined using the ProteOn XPR36 real-time assay system. The corresponding channel of the CM5 chip was activated using EDC/NHS, and 50. Mu.g/ml streptavidin (dissolved in 10mM acetate buffer, pH 4.5) was immobilized on the corresponding channel of the CM5 chip. The unreacted activated surface was blocked by injection of 1M ethanolamine solution (pH 8.0) to complete the coupling process. The amount of streptavidin coupled is generally greater than 1000RU. The TCR was flowed through the pMHC-coated chip at several different concentrations with an association time of 30-60s and an dissociation time of 30-60s.

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

b. Renaturation

The synthetic short peptide qlertpll (nanjing kingkon biotechnology limited) 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. QLEERTLLL peptide was added at 25mg/L (final concentration) to a renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidative glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4 ℃ C.), followed by the addition of 20mg/L of light chain and 90mg/L of heavy chain in sequence (final concentration, heavy chain was added in three portions, 8 h/time), renaturation was carried out at 4 ℃ for at least 3 days until completion, and SDS-PAGE was checked for success of the renaturation.

c. Purification after renaturation

The renaturation buffer was replaced by dialysis against 10 volumes of 20mM Tris pH 8.0, at least twice to reduce the ionic strength of the solution sufficiently. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and then loaded onto a HiTrap Q HP (GE general electric) anion exchange column (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

Biotinylated pMHC molecules were applied to Millipore ultrafiltration tubesConcentrated to 1ml, 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 were calculated to obtain kinetic profiles of binding of soluble TCR molecules of the invention to QLEERTLL-HLA A0201 complex as shown in FIG. 7. The map shows that the soluble TCR molecule obtained by the invention can be combined with the QLEERTLL-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 5T cell receptor Lentiviral packaging and Primary T cell transfection

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

A third generation lentiviral packaging system was used to package lentiviruses containing the gene encoding the desired TCR. 293T/17 cells were transfected with 4 plasmids (a lentiviral vector containing pLenti-PASD1TRA-2A-TRB-IRES-NGFR described In example 2, and 3 plasmids containing other components necessary for the construction of infectious but non-replicating lentiviral particles) using rapid-mediated transient transfection (Express-In-mediated transfection) (Open Biosystems).

For transfection, cells were seeded at day 0 on a 15 cm petri dish at 1.7X 10 ⁷ 293T/17 cells, which were distributed evenly on the culture dish with a degree of confluence slightly higher than 50%. The plasmid was transfected on day 1, pLenti-PASD1TRA-2A-TRB-IRES-NGFR and pLenti-eGFP pseudoviruses were packaged, and the above expression plasmids were combined with packaging plasmids pMDLg/pRRE, pRSV-REV andpMD.2G was mixed well and the amount used for one 15 cm diameter plate was as follows: 22.5 microgram: 15 microgram: 7.5 micrograms. The ratio of the transfection reagent PEI-MAX to the plasmid was 2. The specific operation is as follows: the expression plasmid and the packaging plasmid were added to 1800. Mu.l of OPTI-MEM (Gibco, cat. No. 31985-070) medium, mixed uniformly, and left to stand at room temperature for 5 minutes to obtain a DNA mixture, a corresponding amount of PEI was added to 1800. Mu.l of OPTI-MEM medium, and left to stand at room temperature for 5 minutes to obtain a PEI mixture, the DNA mixture and PEI mixture were mixed and left to stand at room temperature for 30 minutes, 3150. Mu.l of OPTI-MEM medium was added, and after mixing uniformly, the mixture was added to 293T/17 cells which had been converted into 11.25 ml of OPTI-MEM, and the culture dish was gently shaken to mix the media uniformly, 37 ℃/5 CO was added ₂ And (5) culturing. 5-7 hours of transfection, removal of transfection medium, replacement with DMEM (Gibbco, cat. No. C11995500 bt)) complete medium containing 10% fetal bovine serum, 37 ℃/5% CO ₂ And (5) culturing. Culture supernatants containing packaged lentiviruses were collected on days 3 and 4. To harvest the packaged lentivirus, the collected culture supernatant was centrifuged at 3000g for 15min to remove cell debris, filtered through a 0.22 micron filter (Merck Millipore, catalog number SLGP033 RB), and finally concentrated using a 50KD cut-off concentration tube (Merck Millipore, catalog number UFC 905096), to remove most of the supernatant, and finally concentrated to 1ml, and aliquots were frozen at-80 ℃. Pseudovirus samples were taken for virus titer determination, procedures were referenced to p24ELISA (Clontech, catalog number 632200) kit instructions. As a control, a pseudovirus transformed with pLenti-eGFP was also included.

(b) Transduction of Primary T cells with lentiviruses containing the T cell receptor genes of the invention

Isolation of CD8 from blood of healthy volunteers ⁺ T cells, then transduced with the packaged lentivirus. These cells were counted in 48-well plates in 1640 (Gibbco (Gibco), cat. No. C11875500 BT) medium containing 10% FBS (Gibbo, cat. No. C10010500 BT) with 50IU/ml IL-2 and 10ng/ml IL-7 ⁶ Single cell/ml (0.5 ml/well) and pre-mixWashed anti-CD 3/CD28 antibody-coated beads (T cell amplicons, life technologies, cat. No.)11452D) Co-incubation overnight stimulation, cells: bead =3:1.

after overnight stimulation, lentiviruses of the concentrated PASD 1-specific T cell receptor gene were added at MOI =10 and centrifuged at 32 ℃ and 900g for 1 hour, depending on the viral titer measured with the p24ELISA kit. After completion of infection, the lentivirus infection solution was removed, the cells were suspended in a 10% FBS-containing 1640 medium supplemented with 50IU/ml IL-2 and 10ng/ml IL-7, 37 ℃/5% CO ₂ The cells were cultured for 3 days. Cells were counted 3 days after transduction and diluted to 0.5X 10 ⁶ Individual cells/ml. Cells were counted every two days, and fresh medium containing 50IU/ml IL-2 and 10ng/ml IL-7 was replaced or added to maintain the cells at 0.5X 10 ⁶ -1×10 ⁶ Individual cells/ml. Cells were analyzed by flow cytometry starting on day 3 and starting on day 5 for functional assays (e.g., ELISPOT for IFN- γ release and non-radioactive cytotoxicity assays). Cryopreserving aliquots of cells, at least 4X 10, from day 10 or as the cells slow division and become smaller in size ⁶ Individual cell/tube (1X 10) ⁷ Individual cells/ml, 90% FBS/10% DMSO).

(c) Tetramer staining of TCR transduced primary T cells

The PASD1PX 248-391 QLEERTFLL short peptide is renatured with HLA-A0201 with biotin labels to preparebase:Sub>A pHLA unimer. These monomers were combined with PE-labeled streptavidin (BD) into a PE-labeled tetramer, designated PX248-tetramer-PE. The tetramer can label T cells expressing the T cell receptor gene of the present invention as positive cells. Mixing the transduced T cell sample of (b) with PX248-tetramer-PE and incubating on ice for 30 minutes, then adding anti-mouse beta chain-APC antibody and continuing the incubation on ice for 15 minutes. Samples were washed 2 times with PBS containing 2% fbs and then PX248-tetramer-PE expressing the PASD 1-specific T cell receptor gene and anti-mouse β -chain-APC double positive T cells were detected OR sorted using Millipore guava OR BD Arial and data analysis was analyzed using guavaSoft 3.1 software ((Merck Millipore) OR FlowJo software (Tree Star Inc, ashland, OR).

The results of the detection and analysis are shown in fig. 8, after the cells are stained with PX248-tetramer-PE and anti-mouse beta chain-APC antibody, the T cells of the blank control group without TCR lentivirus infection have no PX248-tetramer-PE and anti-mouse beta chain-APC double-positive cells, while the T cells infected with TCR lentivirus have PX248-tetramer-PE and anti-mouse beta chain-APC double-positive cells expressing TCR, and when the T cells are stained with other tetramer-PE than PX248-tetramer-PE, only a small amount of non-specific double-positive cells exist.

Example 6 ELISPOT functional validation of TCRs of the invention

ELISPOT scheme

The following assay was performed to demonstrate activation of TCR-transduced T cells in response specifically to target cells. IFN-. Gamma.production as measured by the ELISPOT assay was used as a readout for T cell activation.

Reagent

Test medium: 10% of FBS (Gibco, catalog number 16000-044), RPMI 1640 (Gibco, catalog number C11875500 bt)

Washing buffer solution: 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 of this example were Epstein-Barr virus (EBV) transformed immortalized Lymphoblast Cell Lines (LCLs). B95-8 cells are induced by phorbol myristate acetate (TPA) to produce culture medium supernatant containing EBV, centrifuged at 4 deg.C/600 g for 10min to remove impurities, filtered by 0.22 μm filter, and stored at-70 deg.C. From Peripheral Blood Lymphocytes (PBLs) of healthy volunteers of the genotype HLA-A11/A02/A24 (including homozygote and heterozygote), 10ml of 2X 10-ml of the cells were collected ⁷ One ml PBL suspension in 25 cm square culture flask, adding cyclosporine at 37 deg.C/CO ₂ Incubating for 1 hour in incubator, rapidly thawing an EBV aliquot, diluting with 1/10 of EBV, adding to the cells, shaking gently, and standing the flask at 37 deg.C/CO ₂ Culturing in an incubator. After 12 days of culture, 10ml of medium was added to continue the culture, and after about 30 days, the culture was further expanded and subjected to flow assay, in which CD19 was present ⁺ CD23 ^hi CD58 ⁺ Immortalized Lymphoblastoid Cell Lines (LCLs). The ELISPOT test uses HLA-A02 as a specific target cell.

Effector cell preparation

The effector cells (T cells) of this assay were CD8 expressing a PASD1 specific TCR as analyzed by flow cytometry in example 3 ⁺ T cells and CD8 cells of the same volunteer ⁺ T served as negative control effector cells. T cells were stimulated with anti-CD 3/CD28 coated beads (T cell amplicons, life technologies), transduced with lentiviruses carrying the TCR gene of the invention (according to example 5), 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 10min at RT. The cells were then resuspended in the test medium at 2 × the desired final concentration. Negative control effector cells were treated as well.

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 RPMI 1640 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 plates, and any residual wash buffer was removed by flicking and tapping the ELISPOT well plates on paper.

QL6CD8 ⁺ T cells (T cells) (T cells transduced by the TCR of the invention, effector cells), CD8+ T cells (negative control effector cells) and LCL A02/LCL A24 (target cells) were prepared as described in example 3 and the corresponding short peptides were added to the corresponding experimental groups, wherein PX248 is PASD1PX 383-391 QLEERTL short peptide, PA02-1 and PA24-1 are TCR specific binding short peptides not provided by the invention.

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

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

50 microliter of effector cells (1000 PASD1TCR double positive T cells).

20 microliter 10 ^-4 Molar/liter PASD1PX248 383-391 QLEERTLLL/nonspecific short peptide solution (final concentration is 10) ^-5 Moles/liter).

All wells were made in triplicate for addition.

The plates were then incubated overnight (37 ℃/5% 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. Primary antibodies were then detected by dilution with 10% FBS-containing PBS 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 at room temperature for 1 hour. The plate was then washed 3 times with PBS 2 times, 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 release of IFN-. Gamma.from T cells transduced with the PASD1TCR in response to target cells loaded with the short peptide of PASD1PX 248-391 QLEERTLLL and non-specific short peptide was examined by ELISPOT assay (described above). The number of ELSPOT spots observed in each well was plotted using a graphpad prism 6.

The results of the experiment are shown in FIG. 9, PASD1 CD8 ⁺ T cells (QL 6CD 8) ⁺ T Cell, effector Cell) alone releases little IFN- γ with LCL cells (target cells).

PASD1 CD8 ⁺ T cells (QL 6CD 8) ⁺ T Cell, effector Cell) can react with LCLA02 cells supplemented with PX248 to release more IFN- γ.

PASD1 CD8 ⁺ T cells (QL 6CD 8) ⁺ T Cell, effector cells) with very little IFN- γ release when LCL cells with nonspecific short peptides were added.

CD8 ⁺ T cells (CD 8) ⁺ T Cell, negative control effector cells) had very little IFN- γ release when LCLA02 cells of PX248 were added.

Example 7 non-radioactive cytotoxicity assay of TCRs of the invention

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

Material

CytoTox

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

Test medium: 10% FBS (heat-inactivated, gibbo, cat. No. 16000-044), 95% RPMI 1640 (Gibbo, cat. No. 11835-030) without phenol red.

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

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

Method

Target cell preparation

The test adopts three tumor cell lines of U266B1, NCI-H1299 and A375 as target cells. Target cells were prepared in assay medium: the concentration of the target cells was adjusted to 334/ml, and 45. Mu.l/well was used to obtain 1.5X 10 ⁴ Individual cells/well.

Effector cell preparation

The effector cells (T cells) of this assay were CD8 expressing a PASD1 specific TCR as analyzed by flow cytometry in example 3 ⁺ T cells. Effector cell to target cell ratio 10:1. 1, 2.5. Setting source CD8 ⁺ T cells plus target cell control group (10.

PASD1 specific TCR transduced CD8 ⁺ T cell specific killing tumor cell assay

Preparation of the test

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

45ul of target cells (prepared as described above) were added to each well

45ul of effector cells (prepared as described above) were added to each well

A control group was prepared as follows:

the effector cells release spontaneously: there were only 45ul of effector cells.

Target cells release: there were only 45ul of target cells.

Maximum release of target cells: there were only 45ul of target cells.

-media control: there were only 90ul of medium.

All wells were made in triplicate and the final volume was 90ul (insufficient media make up).

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

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

Calculation results

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

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

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

Results

The PASD1TCR transduced T cells were tested for LDH release in response to specific target cells by a non-radioactive cytotoxicity assay (as described above). The 490nm visible absorbance in each well was plotted using graphpad prism 6.

Experimental data statistics the results are shown in FIG. 10, and as the ratio of effective targets increases, the PASD1TCR transduced T cells (QL 6CD 8) ⁺ T Cell) enhanced killing of specific target Cell U266B 1; the killing effect on nonspecific target cells NCI-H1299 and A375 is weak. Homologous CD8 ⁺ T(CD8 ⁺ T Cell) cells had significantly lower kill rates for the target Cell U266B1 than the group of T cells transduced by PASD1 TCR.

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

Sequence listing

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

<120> TCR recognizing PASD1 antigen short peptide

<130> P2017-0445

<160> 29

<170> PatentIn version 3.5

<210> 1

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> TCR alpha chain variable Domain

<400> 1

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

1 5 10 15

Ala Ile Val Ser Leu Asn Cys Thr Tyr Ser Asn Ser Ala Phe Gln Tyr

20 25 30

Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys Gly Pro Glu Leu Leu Met

35 40 45

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

50 55 60

Val Asp Lys Ser Ser Lys Tyr Ile Ser Leu Phe Ile Arg Asp Ser Gln

65 70 75 80

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

85 90 95

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

100 105 110

<210> 2

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> TCR alpha chain variable domain

<400> 2

cagaaggagg tggagcagga tcctggacca ctcagtgttc cagagggagc cattgtttct 60

ctcaactgca cttacagcaa cagtgctttt caatacttca tgtggtacag acagtattcc 120

agaaaaggcc ctgagttgct gatgtacaca tactccagtg gtaacaaaga agatggaagg 180

tttacagcac aggtcgataa atccagcaag tatatctcct tgttcatcag agactcacag 240

cccagtgatt cagccaccta cctctgtgca atgagggcaa ttcagggagc ccagaagctg 300

gtatttggcc aaggaaccag gctgactatc aaccca 336

<210> 3

<211> 253

<212> PRT

<213> Artificial sequence

<220>

<223> TCR alpha chain

<400> 3

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

1 5 10 15

Ala Ile Val Ser Leu Asn Cys Thr Tyr Ser Asn Ser Ala Phe Gln Tyr

20 25 30

Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys Gly Pro Glu Leu Leu Met

35 40 45

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

50 55 60

Val Asp Lys Ser Ser Lys Tyr Ile Ser Leu Phe Ile Arg Asp Ser Gln

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250

<210> 4

<211> 759

<212> DNA

<213> Artificial sequence

<220>

<223> TCR alpha chain

<400> 4

cagaaggagg tggagcagga tcctggacca ctcagtgttc cagagggagc cattgtttct 60

ctcaactgca cttacagcaa cagtgctttt caatacttca tgtggtacag acagtattcc 120

agaaaaggcc ctgagttgct gatgtacaca tactccagtg gtaacaaaga agatggaagg 180

tttacagcac aggtcgataa atccagcaag tatatctcct tgttcatcag agactcacag 240

cccagtgatt cagccaccta cctctgtgca atgagggcaa ttcagggagc ccagaagctg 300

gtatttggcc aaggaaccag gctgactatc aacccaaata tccagaaccc tgaccctgcc 360

gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 420

gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 480

gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540

tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600

cccagcccag aaagttcctg tgatgtcaag ctggtcgaga aaagctttga aacagatacg 660

aacctaaact ttcaaaacct gtcagtgatt gggttccgaa tcctcctcct gaaagtggcc 720

gggtttaatc tgctcatgac gctgcggctg tggtccagc 759

<210> 5

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> TCR beta chain variable domain

<400> 5

Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg

1 5 10 15

Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val

20 25 30

Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser

35 40 45

Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe

50 55 60

Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Arg Gly

85 90 95

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

100 105 110

<210> 6

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> TCR beta chain variable domain

<400> 6

gctgctggag tcatccagtc cccaagacat ctgatcaaag aaaagaggga aacagccact 60

ctgaaatgct atcctatccc tagacacgac actgtctact ggtaccagca gggtccaggt 120

caggaccccc agttcctcat ttcgttttat gaaaagatgc agagcgataa aggaagcatc 180

cctgatcgat tctcagctca acagttcagt gactatcatt ctgaactgaa catgagctcc 240

ttggagctgg gggactcagc cctgtacttc tgtgccagca gccgcggaca gggtgaagct 300

ttctttggac aaggcaccag actcacagtt gta 333

<210> 7

<211> 288

<212> PRT

<213> Artificial sequence

<220>

<223> TCR beta chain

<400> 7

Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg

1 5 10 15

Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val

20 25 30

Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser

35 40 45

Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe

50 55 60

Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Arg Gly

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

<210> 8

<211> 864

<212> DNA

<213> Artificial sequence

<220>

<223> TCR beta chain

<400> 8

gctgctggag tcatccagtc cccaagacat ctgatcaaag aaaagaggga aacagccact 60

ctgaaatgct atcctatccc tagacacgac actgtctact ggtaccagca gggtccaggt 120

caggaccccc agttcctcat ttcgttttat gaaaagatgc agagcgataa aggaagcatc 180

cctgatcgat tctcagctca acagttcagt gactatcatt ctgaactgaa catgagctcc 240

ttggagctgg gggactcagc cctgtacttc tgtgccagca gccgcggaca gggtgaagct 300

ttctttggac aaggcaccag actcacagtt gtagaggacc tgaacaaggt gttcccaccc 360

gaggtcgctg tgtttgagcc atcagaagca gagatctccc acacccaaaa ggccacactg 420

gtgtgcctgg ccacaggctt cttccccgac cacgtggagc tgagctggtg ggtgaatggg 480

aaggaggtgc acagtggggt cagcacggac ccgcagcccc tcaaggagca gcccgccctc 540

aatgactcca gatactgcct gagcagccgc ctgagggtct cggccacctt ctggcagaac 600

ccccgcaacc acttccgctg tcaagtccag ttctacgggc tctcggagaa tgacgagtgg 660

acccaggata gggccaaacc cgtcacccag atcgtcagcg ccgaggcctg gggtagagca 720

gactgtggct ttacctcggt gtcctaccag caaggggtcc tgtctgccac catcctctat 780

gagatcctgc tagggaaggc caccctgtat gctgtgctgg tcagcgccct tgtgttgatg 840

gccatggtca agagaaagga tttc 864

<210> 9

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> antigen short peptide

<400> 9

Gln Leu Glu Glu Arg Thr Trp Leu Leu

1 5

<210> 10

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> α CDR1

<400> 10

Asn Ser Ala Phe Gln Tyr

1 5

<210> 11

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> α CDR2

<400> 11

Thr Tyr Ser Ser Gly Asn

1 5

<210> 12

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> α CDR3

<400> 12

Cys Ala Met Arg Ala Ile Gln Gly Ala Gln Lys Leu Val Phe

1 5 10

<210> 13

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> β CDR1

<400> 13

Pro Arg His Asp Thr

1 5

<210> 14

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> β CDR2

<400> 14

Phe Tyr Glu Lys Met Gln

1 5

<210> 15

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> β CDR3

<400> 15

Cys Ala Ser Ser Arg Gly Gln Gly Glu Ala Phe Phe

1 5 10

<210> 16

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> α CDR1

<400> 16

aacagtgctt ttcaatac 18

<210> 17

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> α CDR2

<400> 17

acatactcca gtggtaac 18

<210> 18

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> α CDR3

<400> 18

tgtgcaatga gggcaattca gggagcccag aagctggtat tt 42

<210> 19

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> β CDR1

<400> 19

cctagacacg acact 15

<210> 20

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> β CDR2

<400> 20

ttttatgaaa agatgcag 18

<210> 21

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> β CDR3

<400> 21

tgtgccagca gccgcggaca gggtgaagct ttcttt 36

<210> 22

<211> 275

<212> PRT

<213> Artificial sequence

<220>

<223> TCR alpha chain with leader sequence

<400> 22

Met Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu

1 5 10 15

Ser Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asp Pro Gly Pro

20 25 30

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

35 40 45

Asn Ser Ala Phe Gln Tyr Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys

50 55 60

Gly Pro Glu Leu Leu Met Tyr Thr Tyr Ser Ser Gly Asn Lys Glu Asp

65 70 75 80

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

85 90 95

Phe Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala

100 105 110

Met Arg Ala Ile Gln Gly Ala Gln Lys Leu Val Phe Gly Gln Gly Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Trp Ser Ser

275

<210> 23

<211> 825

<212> DNA

<213> Artificial sequence

<220>

<223> TCR alpha chain having leader sequence

<400> 23

atgatgaaat ccttgagagt tttactggtg atcctgtggc ttcagttaag ctgggtttgg 60

agccaacaga aggaggtgga gcaggatcct ggaccactca gtgttccaga gggagccatt 120

gtttctctca actgcactta cagcaacagt gcttttcaat acttcatgtg gtacagacag 180

tattccagaa aaggccctga gttgctgatg tacacatact ccagtggtaa caaagaagat 240

ggaaggttta cagcacaggt cgataaatcc agcaagtata tctccttgtt catcagagac 300

tcacagccca gtgattcagc cacctacctc tgtgcaatga gggcaattca gggagcccag 360

aagctggtat ttggccaagg aaccaggctg actatcaacc caaatatcca gaaccctgac 420

cctgccgtgt accagctgag agactctaaa tccagtgaca agtctgtctg cctattcacc 480

gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 540

aaaactgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 600

aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 660

ttcttcccca gcccagaaag ttcctgtgat gtcaagctgg tcgagaaaag ctttgaaaca 720

gatacgaacc taaactttca aaacctgtca gtgattgggt tccgaatcct cctcctgaaa 780

gtggccgggt ttaatctgct catgacgctg cggctgtggt ccagc 825

<210> 24

<211> 317

<212> PRT

<213> Artificial sequence

<220>

<223> TCR beta chain with leader sequence

<400> 24

Met Leu Ser Pro Asp Leu Pro Asp Ser Ala Trp Asn Thr Arg Leu Leu

1 5 10 15

Cys Arg Val Met Leu Cys Leu Leu Gly Ala Gly Ser Val Ala Ala Gly

20 25 30

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

35 40 45

Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val Tyr Trp Tyr

50 55 60

Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser Phe Tyr Glu

65 70 75 80

Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe Ser Ala Gln

85 90 95

Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser Leu Glu Leu

100 105 110

Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Arg Gly Gln Gly Glu

115 120 125

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

130 135 140

Lys Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu

145 150 155 160

Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe

165 170 175

Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val

180 185 190

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

195 200 205

Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Phe

305 310 315

<210> 25

<211> 951

<212> DNA

<213> Artificial sequence

<220>

<223> TCR beta chain having leader sequence

<400> 25

atgcttagtc ctgacctgcc tgactctgcc tggaacacca ggctcctctg ccgtgtcatg 60

ctttgtctcc tgggagcagg ttcagtggct gctggagtca tccagtcccc aagacatctg 120

atcaaagaaa agagggaaac agccactctg aaatgctatc ctatccctag acacgacact 180

gtctactggt accagcaggg tccaggtcag gacccccagt tcctcatttc gttttatgaa 240

aagatgcaga gcgataaagg aagcatccct gatcgattct cagctcaaca gttcagtgac 300

tatcattctg aactgaacat gagctccttg gagctggggg actcagccct gtacttctgt 360

gccagcagcc gcggacaggg tgaagctttc tttggacaag gcaccagact cacagttgta 420

gaggacctga acaaggtgtt cccacccgag gtcgctgtgt ttgagccatc agaagcagag 480

atctcccaca cccaaaaggc cacactggtg tgcctggcca caggcttctt ccccgaccac 540

gtggagctga gctggtgggt gaatgggaag gaggtgcaca gtggggtcag cacggacccg 600

cagcccctca aggagcagcc cgccctcaat gactccagat actgcctgag cagccgcctg 660

agggtctcgg ccaccttctg gcagaacccc cgcaaccact tccgctgtca agtccagttc 720

tacgggctct cggagaatga cgagtggacc caggataggg ccaaacccgt cacccagatc 780

gtcagcgccg aggcctgggg tagagcagac tgtggcttta cctcggtgtc ctaccagcaa 840

ggggtcctgt ctgccaccat cctctatgag atcctgctag ggaaggccac cctgtatgct 900

gtgctggtca gcgcccttgt gttgatggcc atggtcaaga gaaaggattt c 951

<210> 26

<211> 198

<212> PRT

<213> Artificial sequence

<220>

<223> soluble TCR alpha chain

<400> 26

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

1 5 10 15

Ala Ile Val Ser Leu Asn Cys Thr Tyr Ser Asn Ser Ala Phe Gln Tyr

20 25 30

Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys Gly Pro Glu Leu Leu Met

35 40 45

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

50 55 60

Val Asp Lys Ser Ser Lys Tyr Ile Ser Leu Phe Ile Arg Asp Ser Gln

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Ile Ile Pro Glu Asp Thr

195

<210> 27

<211> 594

<212> DNA

<213> Artificial sequence

<220>

<223> soluble TCR alpha chain

<400> 27

cagaaagaag tggaacagga ccctggacca ctcagtgttc cagagggagc cattgtttct 60

ctcaactgca cttacagcaa cagtgctttt caatacttca tgtggtacag acagtattcc 120

agaaaaggcc ctgagttgct gatgtacaca tactccagtg gtaacaaaga agatggaagg 180

tttacagcac aggtcgataa atccagcaag tatatctcct tgttcatcag agactcacag 240

cccagtgatt cagccaccta cctctgtgca atgagggcaa ttcagggagc ccagaagctg 300

gtatttggcc aaggaaccag gctgactatc aacccatata tccagaatcc ggacccggcc 360

gtttatcagc tgcgtgatag caaaagcagc gataaaagcg tgtgcctgtt caccgatttt 420

gatagccaga ccaacgtgag ccagagcaaa gatagcgatg tgtacatcac cgataaatgc 480

gtgctggata tgcgcagcat ggatttcaaa agcaatagcg cggttgcgtg gagcaacaaa 540

agcgattttg cgtgcgcgaa cgcgtttaac aacagcatca tcccggaaga tacg 594

<210> 28

<211> 240

<212> PRT

<213> Artificial sequence

<220>

<223> soluble TCR beta chain

<400> 28

Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg Glu

1 5 10 15

Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val Tyr

20 25 30

Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser Phe

35 40 45

Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe Ser

50 55 60

Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser Leu

65 70 75 80

Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Arg Gly Gln

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

<210> 29

<211> 720

<212> DNA

<213> Artificial sequence

<220>

<223> soluble TCR beta chain

<400> 29

gcaggtgtta ttcagtcccc aagacatctg atcaaagaaa agagggaaac agccactctg 60

aaatgctatc ctatccctag acacgacact gtctactggt accagcaggg tccaggtcag 120

gacccccagt tcctcatttc gttttatgaa aagatgcaga gcgataaagg aagcatccct 180

gatcgattct cagctcaaca gttcagtgac tatcattctg aactgaacat gagctccttg 240

gagctggggg actcagccct gtacttctgt gccagcagcc gcggacaggg tgaagctttc 300

tttggacaag gcaccagact cacagttgta gaagatctga aaaatgtgtt tccgccggaa 360

gtcgcggtgt tcgaaccgtc ggaagccgaa attagccata cccagaaagc aacgctggtg 420

tgcctggcta ccggctttta tccggatcat gtggaactgt cctggtgggt taacggcaaa 480

gaagtgcact caggtgtttg tacggatccg cagccgctga aagaacaacc ggcactgaat 540

gactcgcgtt atgctctgag ttcccgtctg cgcgttagcg ccaccttctg gcaggatccg 600

cgtaaccact ttcgctgtca ggtccaattc tacggcctgt ccgaaaatga tgaatggacc 660

caggaccgtg caaaaccggt cacgcaaatc gtgtcagcag aagcttgggg tcgtgcagat 720

Claims (33)

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

αCDR1-NSAFQY (SEQ ID NO.10)

αCDR2-TYSSGN (SEQ ID NO.11)

α CDR3-CAMRAIQGAQKLVF (SEQ ID NO. 12); and

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

βCDR1-PRHDT (SEQ ID NO.13)

βCDR2-FYEKMQ (SEQ ID NO.14)

βCDR3-CASSRGQGEAFF (SEQ ID NO.15)。

2. a TCR as claimed in claim 1 which comprises a TCR α chain variable domain which is an amino acid sequence having at least 90% sequence identity to SEQ ID No.1, and a TCR β chain variable domain; and/or the TCR β chain variable domain is identical to SEQ ID NO:5 an amino acid sequence having at least 90% sequence identity.

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

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

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

6. A TCR as claimed in claim 5 wherein the α chain amino acid sequence of the TCR is as set out in SEQ ID NO:3 and/or the beta chain amino acid sequence of the TCR is SEQ ID No.7.

7. A TCR as claimed in claims 1 to 4 wherein the TCR is soluble.

8. A TCR as claimed in claim 7 which is single chain.

9. A TCR as claimed in claim 8 which is formed by the α chain variable domain linked to the β chain variable domain by a peptide linker sequence.

10. A TCR as claimed in claim 9 which has one or more mutations in the alpha chain variable region amino acid 11, 13, 19, 21, 53, 76, 89, 91 or 94 and/or the alpha chain J gene short peptide amino acid penultimate 3, penultimate 5 or penultimate 7 position; 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).

11. A TCR as claimed in claim 10 which comprises (a) all or part of a TCR α chain, excluding the transmembrane domain; and (b) all or part of a TCR β chain, excluding the transmembrane domain;

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

12. A TCR as claimed in claim 11 wherein (a) and (b) each further comprise at least part of the constant domain of the TCR chain.

13. A TCR as claimed in claim 11 in which the cysteine residues form an artificial disulphide bond between the α and β chain constant domains of the TCR.

14. A TCR as claimed in claim 13 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.

15. A TCR as claimed in claim 14 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.

16. A TCR as claimed in claim 11 which comprises an artificial interchain disulphide bond between the α chain variable region and the β chain constant region of the TCR.

17. A TCR as claimed in claim 16 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 of TRBC1 x 01 or TRBC2 x 01.

18. A TCR as claimed in claim 16 or claim 17 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.

19. 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.

20. A TCR as claimed in claim 19 wherein the conjugate to which the TCR is bound is a detectable label, a therapeutic agent, a PK modifying moiety or a combination thereof.

21. A TCR as claimed in claim 20 wherein the therapeutic agent is an anti-CD 3 antibody.

22. 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 21.

23. A nucleic acid molecule comprising a nucleic acid sequence encoding a TCR of any one of claims 1-21, or the complement thereof.

24. The nucleic acid molecule of claim 22, wherein said nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:2.

25. the nucleic acid molecule of claim 22, wherein said nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:6.

26. a vector comprising the nucleic acid molecule of any one of claims 23-25.

27. The vector of claim 26, wherein said vector is a viral vector.

28. The vector of claim 27, wherein said vector is a lentiviral vector.

29. An isolated host cell comprising the vector of any one of claims 26-28 or the chromosome of which is integrated an exogenous nucleic acid molecule of any one of claims 23-25.

30. A cell which transduces the nucleic acid molecule of any one of claims 23 to 25 or the vector of any one of claims 26 to 28.

31. The cell of claim 30, wherein the cell is a T cell or a stem cell.

32. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to any one of claims 1 to 21, a TCR complex according to claim 22, a nucleic acid molecule according to any one of claims 23 to 25, or a cell according to claim 30 or 31.

33. Use of a TCR as claimed in any one of claims 1 to 21 or a TCR complex as claimed in claim 22 or a cell as claimed in claim 30 or 31 in the manufacture of a medicament for the treatment of a tumour or autoimmune disease.

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