CN108929378B - T cell receptor for recognizing PRAME antigen and nucleic acid for encoding receptor - Google Patents

Abstract

The present invention provides a T Cell Receptor (TCR) capable of specifically binding short peptide QLLALLPSL derived from PRAME antigen, said antigen short peptide QLLALLPSL being capable of forming a complex with HLA a0201 and being presented together on the cell surface. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules. In addition, the invention provides cells that transduce a TCR of the invention.

Description

T cell receptor for recognizing PRAME antigen and nucleic acid for encoding receptor

Technical Field

The present invention relates to a TCR capable of recognising a short peptide derived from the PRAME antigen, and to PRAME specific T cells obtained by transduction of such TCRs, and their use in the prevention and treatment of PRAME related diseases.

Background

PRAME is a melanoma-specific antigen (PRAME) that is expressed in 88% of primary and 95% of metastatic melanomas (Ikeda H, et al. immunity,1997,6(2):199- "208), while normal skin tissue and benign melanocytes are not expressed. PRAME is degraded into small polypeptides after intracellular production and is presented on the cell surface as a complex by binding to MHC (major histocompatibility complex) molecules. QLLALLPSL is a short peptide derived from the PRAME antigen, which is a target for the treatment of PRAME related diseases. In addition to melanoma, PRAME is expressed in a variety of tumors including lung squamous cell carcinoma, breast cancer, renal cell carcinoma, head and neck tumors, Hodgkin's lymphoma, sarcoma, medulloblastoma, etc. (van't Veer LJ, et al Nature,2002,415(6871): 530-. For the treatment of the above diseases, chemotherapy, radiotherapy and the like can be used, but both of them cause damages to normal cells themselves.

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

Disclosure of Invention

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

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

In another preferred embodiment, the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, the amino acid sequence of CDR3 of the TCR alpha chain variable domain is AMSNDKII (SEQ ID NO: 12); and/or the amino acid sequence of CDR3 of the variable domain of the TCR beta chain is ASSWGGNEQF (SEQ ID NO: 15).

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

α CDR1-NSAFQY (SEQ ID NO:10)

α CDR2-TYSSGN(SEQ ID NO:11)

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

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

β CDR1-SGHAT (SEQ ID NO:13)

β CDR2-FQNNGV(SEQ ID NO:14)

β CDR3-ASSWGGNEQF (SEQ ID NO:15)。

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

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

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

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

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

In another preferred embodiment, the TCR is soluble.

In another preferred embodiment, the TCR is single chain.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

preferably, the disease is a tumor, preferably the tumor includes melanoma, as well as other tumors such as squamous cell carcinoma of the lung, breast cancer, renal cell carcinoma, head and neck tumors, hodgkin's lymphoma, sarcoma and medulloblastoma, acute lymphocytic leukemia, acute myelocytic leukemia, 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 amino acid sequence of the TCR α chain variable domain, the nucleotide sequence of the TCR α chain variable domain, the amino acid sequence of the TCR α chain, the nucleotide sequence of the TCR α chain, the amino acid sequence of the TCR α chain with leader sequence and the nucleotide sequence of the TCR α chain with leader sequence, respectively.

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

3a and 3b are the amino acid and nucleotide sequences, respectively, of the soluble TCR alpha chain.

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

Figure 5 is a gel diagram of the soluble TCR obtained after purification. The leftmost lane is reducing gel, the middle lane is molecular weight marker (marker), and the rightmost lane is non-reducing gel.

FIG. 6 is a BIAcore kinetic profile of binding of soluble TCRs of the invention to the QLLALLPSL-HLA A0201 complex.

FIG. 7 shows the results of IFN- γ release from T cells transduced with the TCR of the invention in response to target cells loaded with their specific short peptides.

FIG. 8 shows the results of IFN-. gamma.release in response by T cells expressing PRAME transduced with the TCRs of the invention.

Detailed Description

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

Term(s) for

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

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

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

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

Natural interchain disulfide bond and artificial interchain disulfide bond

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

For convenience of description of the positions of disulfide bonds, the positions of the amino acid sequences of TRAC 01 and TRBC1 × 01 or TRBC2 × 01 are numbered in the order from the N-terminus to the C-terminus, such as in TRBC1 × 01 or TRBC2 × 01, and the 60 th amino acid in the order from the N-terminus to the C-terminus is P (proline), and thus in the present invention it can be described as Pro60 of TRBC1 × 01 or TRBC2 × 01 exon 1, and also as the 60 th amino acid of TRBC1 × 01 or TRBC2 × 01 exon 1, and as in 737bc 3 × 01 or TRBC2 × 01, and the 61 th amino acid in the order from the N-terminus to the C-terminus is Q (glutamine), and thus in the present invention it can be described as TRBC1 × 01 or TRBC 6301 × 01, or TRBC 8501, and similarly as TRBC 8261 or glbc 891. In the present invention, the position numbering of the amino acid sequences of the variable regions TRAV and TRBV follows the position numbering listed in IMGT. If an amino acid in TRAV, the position listed in IMGT is numbered 46, it is described herein as the 46 th amino acid of TRAV, and so on. In the present invention, the sequence position numbers of other amino acids are specifically described.

Detailed Description

TCR molecules

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

α CDR1-NSAFQY (SEQ ID NO:10)

α CDR2-TYSSGN(SEQ ID NO:11)

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

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

β CDR1-SGHAT(SEQ ID NO:13)

β CDR2-FQNNGV(SEQ ID NO:14)

β CDR3-ASSWGGNEQF(SEQ ID NO:15)。

chimeric TCRs can be prepared by embedding the above-described amino acid sequences of the CDR regions of the invention into any suitable framework. One skilled in the art can design or synthesize a TCR molecule with the corresponding function based on the CDR regions disclosed herein, so long as the framework structure is compatible with the CDR regions of the TCR of the invention. Thus, the TCR molecules of the invention are those which comprise the above-described α and/or β chain CDR region sequences and any suitable framework structure. The TCR α chain variable domain of the invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity to SEQ ID No. 1; and/or the TCR β chain variable domain of the invention is a variant of SEQ ID NO:5, having at least 90%, preferably 95%, more preferably 98% sequence identity.

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

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

The alpha chain variable domain amino acid sequence of the single chain TCR molecule comprises the CDR1(SEQ ID NO: 10), CDR2(SEQ ID NO: 11) and CDR3(SEQ ID NO:12) of the alpha chain described above. Preferably, the single chain TCR molecule comprises the alpha chain variable domain amino acid sequence SEQ ID NO 1. More preferably, the α chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO 1. The amino acid sequence of the beta chain variable domain of the single chain TCR molecule comprises the CDR1(SEQ ID NO:13), CDR2(SEQ ID NO: 14) and CDR3(SEQ ID NO:15) of the above-described beta chain. Preferably, the single chain TCR molecule comprises the beta chain variable domain amino acid sequence SEQ ID NO 5. More preferably, the amino acid sequence of the β chain variable domain of the single chain TCR molecule is SEQ ID NO 5.

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

Naturally occurring TCRs are membrane proteins that are stabilized by their transmembrane regions. Like immunoglobulins (antibodies) as antigen recognition molecules, TCRs can also be developed for diagnostic and therapeutic applications, where soluble TCR molecules are required. Soluble TCR molecules do not include their transmembrane regions. Soluble TCRs have a wide range of uses, not only for studying the interaction of TCRs with pmhcs, but also as diagnostic tools for detecting infections or as markers for autoimmune diseases. Similarly, soluble TCRs can be used to deliver therapeutic agents (e.g., cytotoxic or immunostimulatory compounds) to cells presenting a specific antigen, and in addition, soluble TCRs can be conjugated to other molecules (e.g., anti-CD 3 antibodies) to redirect T cells to target them to cells presenting a particular antigen. The invention also provides soluble TCRs specific for PRAME antigen short peptides.

To obtain a soluble TCR, in one aspect, the inventive TCR may be one in which an artificial disulfide bond is introduced between residues of the constant domains of its alpha and beta chains. Cysteine residues form an artificial interchain disulfide bond between the alpha and beta chain constant domains of the TCR. Cysteine residues may be substituted for other amino acid residues at appropriate positions in native TCRs to form artificial interchain disulfide bonds. For example, a disulfide bond is formed by substituting Thr48 of exon 1 of TRAC × 01 and a cysteine residue of Ser57 of exon 1 of TRBC1 × 01 or TRBC2 × 01. Other sites for introducing cysteine residues to form disulfide bonds may also be: thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser77 of TRBC2 × 01 exon 1; tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC2 x 01; thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1; ser15 and TRBC1 × 01 of TRAC × 01 exon 1 or Glu15 of TRBC2 × 01 exon 1; arg53 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser54 of TRBC2 × 01 exon 1; pro89 and TRBC1 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01; or Tyr10 and TRBC1 and 01 of TRAC 01 exon 1 or Glu20 of TRBC2 and 01 exon 1. I.e., a cysteine residue, in place of any of the above-described alpha and beta chain constant domains. The TCR constant domains of the invention may be truncated at one or more of their C-termini by up to 50, or up to 30, or up to 15, or up to 10, or up to 8 or fewer amino acids, so as not to include a cysteine residue for the purpose of deleting the native disulphide bond, or by mutating the cysteine residue forming the native disulphide bond to another amino acid.

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

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

The TCR with the mutated hydrophobic core region of the invention can be a stable soluble single chain TCR formed by connecting the variable domains of the alpha and beta chains of the TCR by a flexible peptide chain. It should be noted that the flexible peptide chain of the present invention can be any peptide chain suitable for linking the TCR α and β chain variable domains.

In addition, for stability, patent document PCT/CN2016/077680 also discloses that the introduction of an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR can significantly improve the stability of the TCR. Thus, the high affinity TCRs of the invention may also contain an artificial interchain disulfide bond between the α chain variable region and the β chain constant region. Specifically, the cysteine residues that form the artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR are substituted for: amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01; amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01; amino acid 46 of TRAV and amino acid 61 of TRBC1 x 01 or TRBC2 x 01 exon 1; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01. Preferably, such a TCR may comprise (i) all or part of a TCR α chain, excluding its transmembrane domain, and (ii) all or part of a TCR β chain, excluding its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least part of the constant domain of the TCR chain, the α chain forming a heterodimer with the β chain. More preferably, such a TCR may comprise the a chain variable domain and the β chain variable domain and all or part of the β chain constant domain, excluding the transmembrane domain, but which does not comprise the a chain constant domain, the a chain variable domain of the TCR forming a heterodimer with the β chain.

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

The TCRs of the invention may be used alone or in covalent or other association, preferably covalently, with a conjugate. The conjugates include a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the QLLALLPSL-HLA a0201 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.

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

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

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

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

Nucleic acid molecules

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

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

α CDR1-aacagtgcttttcaatac(SEQ ID NO:16)

α CDR2-acatactccagtggtaac(SEQ ID NO:17)

α CDR3-gcaatgagtaatgacaagatcatc(SEQ ID NO:18)

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

β CDR1-tctggccatgctacc(SEQ ID NO:19)

β CDR2-tttcagaataacggtgta(SEQ ID NO:20)

β CDR3-gccagcagctggggggggaatgagcagttc(SEQ ID NO:21)

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

The nucleotide sequence of the nucleic acid molecule of the invention may be single-stranded or double-stranded, the nucleic acid molecule may be RNA or DNA, and may or may not comprise an intron. Preferably, the nucleotide sequence of the nucleic acid molecule of the invention does not comprise an intron but is capable of encoding a polypeptide of the invention, e.g. the nucleotide sequence of the nucleic acid molecule of the invention encoding a TCR alpha chain variable domain of the invention comprises SEQ ID NO 2 and/or the nucleotide sequence of the nucleic acid molecule of the invention encoding a TCR beta chain variable domain of the invention comprises SEQ ID NO 6. More preferably, the nucleotide sequence of the nucleic acid molecule of the invention comprises SEQ ID NO. 4 and/or SEQ ID NO. 8. It will be appreciated that, due to the degeneracy of the genetic code, different nucleotide sequences may encode the same polypeptide. Thus, the nucleic acid sequence encoding the TCR of the present invention may be identical to or a degenerate variant of the nucleic acid sequences shown in the figures of the present invention. As illustrated by one of the examples herein, a "degenerate variant" refers to a nucleic acid sequence that encodes a protein sequence having SEQ ID NO. 1, but differs from the sequence of SEQ ID NO. 2.

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

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

Carrier

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

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

Preferably, the vector can transfer the nucleotide of the invention into a cell, e.g., a T cell, such that the cell expresses a TCR specific for the PRAME antigen. Ideally, the vector should be capable of sustained high level expression in T cells.

Cells

The invention also relates to genetically engineered host cells that have been engineered with the vectors or coding sequences of the invention. The host cell comprises a vector of the invention or has integrated into its chromosome a nucleic acid molecule of the invention. The host cell is selected from: prokaryotic and eukaryotic cells, such as E.coli, yeast cells, CHO cells, and the like.

In addition, the invention also includes isolated cells, particularly T cells, that express the TCRs of the invention. The T cell may be derived from a T cell isolated from a subject, or may be part of a mixed population of cells isolated from a subject, such as a population of Peripheral Blood Lymphocytes (PBLs). For example, the cells may be isolated from Peripheral Blood Mononuclear Cells (PBMC), which may be CD4⁺Helper T cell or CD8⁺Cytotoxic T cells. The cell may be in CD4⁺Helper T cell/CD 8⁺A mixed population of cytotoxic T cells. Generally, the cells can be activated with an antibody (e.g., an anti-CD 3 or anti-CD 28 antibody) to render them more amenable to transfection, e.g., transfection with a vector comprising a nucleotide sequence encoding a TCR molecule of the invention.

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

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

PRAME antigen associated diseases

The invention also relates to a method of treating and/or preventing a PRAME-associated disease in a subject comprising the step of adoptively transferring PRAME-specific T cells to the subject. The PRAME-specific T cells recognized QLLALLPSL-HLA A0201 complex.

The PRAME specific T cells of the invention may be used to treat any PRAME-associated disease that presents the PRAME antigen short peptide QLLALLPSL-HLA a0201 complex. Including but not limited to tumors such as melanoma, as well as squamous cell carcinoma of the lung, breast carcinoma, renal cell carcinoma, head and neck tumors, hodgkin's lymphoma, sarcoma, medulloblastoma, 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 PRAME 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 PRAME-related disease comprising infusing into a patient an isolated T cell expressing a TCR of the invention, preferably the T cell is derived from the patient per se. Generally, this involves (1) isolating T cells from the patient, (2) transducing T cells in vitro with a nucleic acid molecule of the invention or a nucleic acid molecule capable of encoding a TCR molecule of the invention, and (3) infusing the genetically modified T cells into the patient. The number of cells isolated, transfected and transfused can be determined by a physician.

The main advantages of the invention are:

(1) the TCR disclosed by the invention can be combined with a PRAME antigen short peptide complex QLLALLPSL-HLA A0201, and cells transduced with the TCR disclosed by the invention can be specifically activated and have a strong killing effect on target cells.

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

Peripheral Blood Lymphocytes (PBLs) from healthy volunteers of genotype HLA-A0201 were stimulated with synthetic short peptide QLLALLPSL (SEQ ID NO. 9; Peking Saibance Gene technology, Inc.). QLLALLPSL short peptide and HLA-A0201 with biotin label are renatured to prepare pHLA haploid. These haploids were combined with streptavidin labeled with PE (BD Co.) to form PE-labeled tetramers, which were sorted for double positive anti-CD 8-APC cells. The sorted cells were expanded and subjected to secondary sorting as described above, followed by single cloning by limiting dilution. Monoclonal cells were stained with tetramer and double positive clones were selected.

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

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

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

α CDR1-NSAFQY(SEQ ID NO:10)

α CDR2-TYSSGN(SEQ ID NO:11)

α CDR3-AMSNDKII(SEQ ID NO:12)

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

β CDR1-SGHAT(SEQ ID NO:13)

β CDR2-FQNNGV(SEQ ID NO:14)

β CDR3-ASSWGGNEQF(SEQ ID NO:15)

the full length genes for the TCR α and β chains were cloned into the lentiviral expression vector pllenti (addendum) by overlap (overlap) PCR, respectively. The method specifically comprises the following steps: the TCR alpha chain and the TCR beta chain are connected by overlap PCR to obtain the TCR alpha-2A-TCR beta segment. And (3) carrying out enzyme digestion and connection on the lentivirus expression vector and the TCR alpha-2A-TCR beta to obtain pLenti-TRA-2A-TRB-IRES-NGFR plasmid. As a control, a lentiviral vector pLenti-eGFP expressing eGFP was also constructed. The pseudovirus was then packaged again at 293T/17.

Example 3 expression, refolding and purification of PRAME antigen short peptide specific soluble TCR

To obtain soluble TCR molecules, the α and β chains of the TCR molecules of the invention may comprise only the variable and part of the constant domains thereof, respectively, and a cysteine residue has been introduced into the constant domains of the α and β chains, respectively, to form artificial interchain disulfide bonds, at the positions Thr48 of exon 1 TRAC × 01 and Ser57 of exon 1 TRBC2 × 01, respectively; the amino acid sequence and nucleotide sequence of the alpha chain are shown in FIGS. 3a and 3b, respectively, and the amino acid sequence and nucleotide sequence of the beta chain are shown in FIGS. 4a and 4b, respectively, with the introduced cysteine residues indicated in bold and underlined letters. The above-mentioned gene sequences of interest for the TCR alpha and beta chains were synthesized and inserted into the expression vector pET28a + (Novagene) by standard methods described in Molecular Cloning A Laboratory Manual (third edition, Sambrook and Russell), and the upstream and downstream Cloning sites were NcoI and NotI, respectively. The insert was confirmed by sequencing without error.

The expression vectors of TCR alpha and beta chains are transformed into expression bacteria BL21(DE3) by chemical transformation method, and the bacteria are grown in LB culture solution and OD₆₀₀Final concentration when not equal to 0.6Inclusion bodies formed after expression of the α and β chains of the TCR were induced with 0.5mM IPTG, extracted with a BugBuster Mix (Novagene) and washed repeatedly with a BugBuster solution several times, and finally dissolved in 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT),10mM ethylenediaminetetraacetic acid (EDTA),20mM Tris (pH 8.1).

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

Example 4 binding characterisation

BIAcore analysis

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

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

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

The QLLALLPSL-HLA A0201 complex is prepared as follows:

a. purification of

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

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

b. Renaturation

The synthesized short peptide QLLALLPSL (Beijing Baisheng Gene technology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg/ml. Inclusion of light and heavy chains was solubilized with 8M Urea, 20mM Tris pH 8.0, 10mM DTT and further denatured by addition of 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA prior to renaturation. QLLALLPSL peptide was added to a renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidative glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4 ℃) at 25mg/L (final concentration), followed by the addition of 20mg/L of light chain and 90mg/L of heavy chain in sequence (final concentration, heavy chain was added in three portions, 8 h/time), and renaturation was carried out at 4 ℃ for at least 3 days until completion, and SDS-PAGE checked for success or failure of the renaturation.

c. Purification after renaturation

The renaturation buffer was replaced by dialysis against 10 volumes of 20mM Tris pH 8.0, at least twice to reduce the ionic strength of the solution sufficiently. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and then loaded onto a HiTrap Q HP (GE general electric) anion exchange column (5ml bed volume). The protein was eluted using an Akta purifier (GE general electric) with a 0-400mM NaCl linear gradient prepared in 20mM Tris pH 8.0, pMHC was eluted at about 250mM NaCl, the peak fractions were collected, and the purity was checked by SDS-PAGE.

d. Biotinylation of the compound

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

e. Purification of biotinylated complexes

The biotinylated pMHC molecules were concentrated to 1ml using Millipore ultrafiltration tubes, the biotinylated pMHC was purified by gel filtration chromatography, and HiPrep was pre-equilibrated with filtered PBS using Akta purifier (GE general electric Co., Ltd.)^TM16/60S200HR column (GE general electric) was loaded with 1ml of concentrated biotinylated pMHC molecules and then eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules appeared as a unimodal elution at approximately 55 ml. The fractions containing the protein were pooled, concentrated using Millipore ultrafiltration tubes, protein concentration was determined by BCA (Thermo), and biotinylated pMHC molecules were stored in aliquots at-80 ℃ by addition of the protease inhibitor cocktail (Roche).

Kinetic parameters were calculated by BIAcore Evaluation software, and kinetic profiles of the binding of soluble TCR molecules of the invention to QLLALLPSL-HLA A0201 complex were obtained as shown in FIG. 6. The map shows that the soluble TCR molecule obtained by the invention can be combined with QLLALLPSL-HLA A0201 complex. Meanwhile, the method is used for detecting the binding activity of the soluble TCR molecule and the short peptides of other unrelated antigens and the HLA complex, and the result shows that the TCR molecule is not bound with other unrelated antigens.

Example 5 validation of the function of effector cells transducing TCRs of the invention Using T2 cells

ELISPOT scheme

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

Reagent

Test medium: 10% FBS (Gibbo, catalog number 16000-

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

PBS (Gibbo Co., catalog number C10010500BT)

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

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

Method

Target cell preparation

The target cells used in this experiment were T2 cells. Target cells were prepared in experimental media: the concentration of the target cells is adjusted to 2.0X 10⁵One/ml, 100. mu.l/well to obtain 2.0X 10⁴Individual cells/well.

Effector cell preparation

The effector cells (T cells) of this experiment were CD8+ T cells transduced with the TCR of the invention, and CD8+ T cells of the same volunteer, which were not transfected with the TCR of the invention, were used as a control. 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, expanded in 1640 medium containing 10% FBS with 50IU/ml IL-2 and 10ng/ml IL-7 until 9-12 days post transduction, then placed in 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.

Preparation of short peptide solution

The corresponding short peptide was added to the corresponding target cell (T2) test group so that the final concentrations of the short peptide in the ELISPOT well plate were 1. mu.g/ml, 0.1. mu.g/ml, 0.01. mu.g/ml, 0.001. mu.g/ml and 0. mu.g/ml, respectively, and further, the non-specific short peptide was also added to the test group, which was labeled (NC) as a control group and the final concentration of the non-specific short peptide in the ELISPOT well plate was 1. mu.g/ml.

ELISPOT

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

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

100 microliter of target cells 2 x 10⁵Cells/ml (total of about 2 x 10 was obtained)⁴Individual target cells/well).

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

All wells were prepared in duplicate for addition.

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

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

Results

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

The experimental results are shown in fig. 7, and the T cells transduced with the TCR of the present invention showed a good activation response to the target cells loaded with their specific short peptides, and showed no activation response to the target cells not loaded with the corresponding short peptides and the target cells loaded with non-specific short peptides.

Example 6 functional validation of effector cells transducing TCRs of the invention Using cell lines

ELISPOT experiments were performed according to the ELISPOT protocol described in example 5, using cell lines to verify the function of effector cells transducing the TCR of the invention.

Method

Target cell preparation

The target cells used in this experiment were SW620, SW620-PRAME (PRAME over-expressed) and K526-A24. The expression level of the PRAME antigen in the cell line is measured according to nanostring technology, wherein SW620 basically does not express the PRAME antigen, SW620-PRAME is a PRAME over-expression cell line, and the genotype of K526-A24 is A2402 and is not A0201 although the expression level is more. Therefore, SW620 and K526-A24 served as controls. Target cells will be prepared in experimental media: the concentration of the target cells is adjusted to 2.0X 10⁵One/ml, 100. mu.l/well to obtain 2.0X 10⁴Individual cells/well.

Effector cell preparation

The effector cells (T cells) of this experiment were CD8+ T cells transduced with the TCR of the invention, and the same volunteer was used as a pair with CD8+ T cells transduced with GFPPhotograph group (NC). Effector cells were prepared in experimental media: the effector cell concentration is adjusted to 2.0 × 10⁴Each positive cell/ml, 100. mu.l/well to obtain 2.0X 10³Individual positive cells/well.

ELISPOT

The well plate was prepared as follows according to the manufacturer's instructions: 5ml of sterile PBS per plate 1: anti-human IFN-. gamma.capture antibody was diluted at 200, and 50. 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. 200 PBS containing 5% FBS was added and the well plate was incubated at room temperature for 2 hours to close the well plate. The blocking solution was decanted, and the ELISPOT plate was flicked and tapped to remove any residual blocking solution.

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

100 microliter of target cells 2 x 10⁵Cells/ml (total of about 2 x 10 was obtained)⁴Individual target cells/well).

100 microliters of effector cells (yielding a total of about 2.0X 10³Individual positive cells/well and control effector cells/well).

All wells were prepared in duplicate for addition.

The well plates were then incubated overnight (37 ℃/5% CO)₂) The next day, the medium was discarded, the well plate was washed 2 times with double distilled water and 3 times with wash buffer, and tapped on a paper towel to remove residual wash buffer. Then, the mixture was mixed with PBS containing 5% FBS at a ratio of 1: the detection antibody was diluted at 200 and added to each well at 50. mu.l/well. The well plate was incubated at room temperature for 2 hours, washed 3 times with wash buffer and the well plate was tapped on a paper towel to remove excess wash buffer.

PBS containing 5% FBS was used at 1: streptavidin-alkaline phosphatase was diluted 100, 50 microliters of diluted streptavidin-alkaline phosphatase was added to each well and the wells were incubated for 1 hour at room temperature. The plate was then washed 3 times with wash buffer and 3 times with PBS, and the plate was tapped on a paper towel to remove excess wash buffer and PBS. After washing, 50 microliter/hole 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 function of the TCR-transduced T cells of the invention was examined by ELISPOT assay (as described above). The number of ELSPOT spots observed in each well was plotted using a graphipad prism 6.

The results are shown in FIG. 8, where T cells transduced with the TCR of the invention expressed PRAME were strongly activated and not activated in cell lines that did not express PRAME or in cell lines of different genotypes. Meanwhile, the NC group of GFP-transduced T cells had essentially no activation response.

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

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<120> T cell receptor recognizing PRAME antigen and nucleic acid encoding the receptor

<130> P2017-0843

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Leu Met Thr Leu Arg Leu Trp Ser Ser

245

<210> 4

<211> 747

<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 atgagtaatg acaagatcat ctttggaaaa 300

gggacacgac ttcatattct ccccaatatc cagaaccctg accctgccgt gtaccagctg 360

agagactcta aatccagtga caagtctgtc tgcctattca ccgattttga ttctcaaaca 420

aatgtgtcac aaagtaagga ttctgatgtg tatatcacag acaaaactgt gctagacatg 480

aggtctatgg acttcaagag caacagtgct gtggcctgga gcaacaaatc tgactttgca 540

tgtgcaaacg ccttcaacaa cagcattatt ccagaagaca ccttcttccc cagcccagaa 600

agttcctgtg atgtcaagct ggtcgagaaa agctttgaaa cagatacgaa cctaaacttt 660

caaaacctgt cagtgattgg gttccgaatc ctcctcctga aagtggccgg gtttaatctg 720

ctcatgacgc tgcggctgtg gtccagc 747

<210> 5

<211> 112

<212> PRT

<213> artificial sequence

<220>

<223> TCR beta chain variable domain

<400> 5

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

1 5 10 15

Gln Ser Val Ala Phe Trp Cys Asn Pro Ile Ser Gly His Ala Thr Leu

20 25 30

Tyr Trp Tyr Gln Gln Ile Leu Gly Gln Gly Pro Lys Leu Leu Ile Gln

35 40 45

Phe Gln Asn Asn Gly Val Val Asp Asp Ser Gln Leu Pro Lys Asp Arg

50 55 60

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

65 70 75 80

Pro Ala Lys Leu Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ser Trp

85 90 95

Gly Gly Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu

100 105 110

<210> 6

<211> 336

<212> DNA

<213> artificial sequence

<220>

<223> TCR beta chain variable domain

<400> 6

gaagctggag ttgcccagtc tcccagatat aagattatag agaaaaggca gagtgtggct 60

ttttggtgca atcctatatc tggccatgct accctttact ggtaccagca gatcctggga 120

cagggcccaa agcttctgat tcagtttcag aataacggtg tagtggatga ttcacagttg 180

cctaaggatc gattttctgc agagaggctc aaaggagtag actccactct caagatccag 240

cctgcaaagc ttgaggactc ggccgtgtat ctctgtgcca gcagctgggg ggggaatgag 300

cagttcttcg ggccagggac acggctcacc gtgcta 336

<210> 7

<211> 291

<212> PRT

<213> artificial sequence

<220>

<223> TCR beta chain

<400> 7

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

1 5 10 15

Gln Ser Val Ala Phe Trp Cys Asn Pro Ile Ser Gly His Ala Thr Leu

20 25 30

Tyr Trp Tyr Gln Gln Ile Leu Gly Gln Gly Pro Lys Leu Leu Ile Gln

35 40 45

Phe Gln Asn Asn Gly Val Val Asp Asp Ser Gln Leu Pro Lys Asp Arg

50 55 60

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

65 70 75 80

Pro Ala Lys Leu Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ser Trp

85 90 95

Gly Gly Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Ser Arg Gly

290

<210> 8

<211> 873

<212> DNA

<213> artificial sequence

<220>

<223> TCR beta chain

<400> 8

gaagctggag ttgcccagtc tcccagatat aagattatag agaaaaggca gagtgtggct 60

ttttggtgca atcctatatc tggccatgct accctttact ggtaccagca gatcctggga 120

cagggcccaa agcttctgat tcagtttcag aataacggtg tagtggatga ttcacagttg 180

cctaaggatc gattttctgc agagaggctc aaaggagtag actccactct caagatccag 240

cctgcaaagc ttgaggactc ggccgtgtat ctctgtgcca gcagctgggg ggggaatgag 300

cagttcttcg ggccagggac acggctcacc gtgctagagg acctgaaaaa cgtgttccca 360

cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 420

ctggtgtgcc tggccacagg cttctacccc gaccacgtgg agctgagctg gtgggtgaat 480

gggaaggagg tgcacagtgg ggtcagcaca gacccgcagc ccctcaagga gcagcccgcc 540

ctcaatgact ccagatactg cctgagcagc cgcctgaggg tctcggccac cttctggcag 600

aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 660

tggacccagg atagggccaa acctgtcacc cagatcgtca gcgccgaggc ctggggtaga 720

gcagactgtg gcttcacctc cgagtcttac cagcaagggg tcctgtctgc caccatcctc 780

tatgagatct tgctagggaa ggccaccttg tatgccgtgc tggtcagtgc cctcgtgctg 840

atggccatgg tcaagagaaa ggattccaga ggc 873

<210> 9

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> antigen short peptide

<400> 9

Gln Leu Leu Ala Leu Leu Pro Ser 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> 8

<212> PRT

<213> artificial sequence

<220>

<223> α CDR3

<400> 12

Ala Met Ser Asn Asp Lys Ile Ile

1 5

<210> 13

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> β CDR1

<400> 13

Ser Gly His Ala Thr

1 5

<210> 14

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> β CDR2

<400> 14

Phe Gln Asn Asn Gly Val

1 5

<210> 15

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> β CDR3

<400> 15

Ala Ser Ser Trp Gly Gly Asn Glu Gln 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> 24

<212> DNA

<213> artificial sequence

<220>

<223> α CDR3

<400> 18

gcaatgagta atgacaagat catc 24

<210> 19

<211> 15

<212> DNA

<213> artificial sequence

<220>

<223> β CDR1

<400> 19

tctggccatg ctacc 15

<210> 20

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> β CDR2

<400> 20

tttcagaata acggtgta 18

<210> 21

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> β CDR3

<400> 21

gccagcagct ggggggggaa tgagcagttc 30

<210> 22

<211> 271

<212> PRT

<213> artificial sequence

<220>

<223> TCR alpha chain having 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 Ser Asn Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

<210> 23

<211> 813

<212> DNA

<213> artificial sequence

<220>

<223> TCR alpha chain having leader sequence

<400> 23

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 gtaatgacaa gatcatcttt 360

ggaaaaggga cacgacttca tattctcccc aatatccaga accctgaccc tgccgtgtac 420

cagctgagag actctaaatc cagtgacaag tctgtctgcc tattcaccga ttttgattct 480

caaacaaatg tgtcacaaag taaggattct gatgtgtata tcacagacaa aactgtgcta 540

gacatgaggt ctatggactt caagagcaac agtgctgtgg cctggagcaa caaatctgac 600

tttgcatgtg caaacgcctt caacaacagc attattccag aagacacctt cttccccagc 660

ccagaaagtt cctgtgatgt caagctggtc gagaaaagct ttgaaacaga tacgaaccta 720

aactttcaaa acctgtcagt gattgggttc cgaatcctcc tcctgaaagt ggccgggttt 780

aatctgctca tgacgctgcg gctgtggtcc agc 813

<210> 24

<211> 310

<212> PRT

<213> artificial sequence

<220>

<223> TCR beta chain having leader sequence

<400> 24

Met Gly Thr Arg Leu Leu Cys Trp Ala Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

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

20 25 30

Glu Lys Arg Gln Ser Val Ala Phe Trp Cys Asn Pro Ile Ser Gly His

35 40 45

Ala Thr Leu Tyr Trp Tyr Gln Gln Ile Leu Gly Gln Gly Pro Lys Leu

50 55 60

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

65 70 75 80

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

85 90 95

Lys Ile Gln Pro Ala Lys Leu Glu Asp Ser Ala Val Tyr Leu Cys Ala

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Arg Lys Asp Ser Arg Gly

305 310

<210> 25

<211> 930

<212> DNA

<213> artificial sequence

<220>

<223> TCR beta chain having leader sequence

<400> 25

atgggcacca ggctcctctg ctgggcggcc ctctgtctcc tgggagcaga actcacagaa 60

gctggagttg cccagtctcc cagatataag attatagaga aaaggcagag tgtggctttt 120

tggtgcaatc ctatatctgg ccatgctacc ctttactggt accagcagat cctgggacag 180

ggcccaaagc ttctgattca gtttcagaat aacggtgtag tggatgattc acagttgcct 240

aaggatcgat tttctgcaga gaggctcaaa ggagtagact ccactctcaa gatccagcct 300

gcaaagcttg aggactcggc cgtgtatctc tgtgccagca gctggggggg gaatgagcag 360

ttcttcgggc cagggacacg gctcaccgtg ctagaggacc tgaaaaacgt gttcccaccc 420

gaggtcgctg tgtttgagcc atcagaagca gagatctccc acacccaaaa ggccacactg 480

gtgtgcctgg ccacaggctt ctaccccgac cacgtggagc tgagctggtg ggtgaatggg 540

aaggaggtgc acagtggggt cagcacagac ccgcagcccc tcaaggagca gcccgccctc 600

aatgactcca gatactgcct gagcagccgc ctgagggtct cggccacctt ctggcagaac 660

ccccgcaacc acttccgctg tcaagtccag ttctacgggc tctcggagaa tgacgagtgg 720

acccaggata gggccaaacc tgtcacccag atcgtcagcg ccgaggcctg gggtagagca 780

gactgtggct tcacctccga gtcttaccag caaggggtcc tgtctgccac catcctctat 840

gagatcttgc tagggaaggc caccttgtat gccgtgctgg tcagtgccct cgtgctgatg 900

gccatggtca agagaaagga ttccagaggc 930

<210> 26

<211> 203

<212> PRT

<213> artificial sequence

<220>

<223> soluble TCR alpha chain

<400> 26

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Gln Pro Ser Asp Ser Ala Thr Tyr Phe Cys Ala Met Ser Asn Asp Lys

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

195 200

<210> 27

<211> 609

<212> DNA

<213> artificial sequence

<220>

<223> soluble TCR alpha chain

<400> 27

ggtcaaaaag aagttgaaca agacccgggt ccgctgagcg tgccggaagg tgcgatcgtt 60

agcattaact gcacctacag caacagcgcg ttccagtatt ttatgtggta ccgtcaagat 120

ccgggcaagg gcccggaact gctgatgtac acctatagca gcggcaacaa agaggacggt 180

cgtttcaccg cgcaggtgga taagagcagc aaatacatca gcctgtttat tcgtgacgtt 240

caaccgagcg atagcgcgac ctatttctgc gcgatgagca acgataagat catttttggt 300

aaaggtaccc gtctgcacgt tctgccgaat atccagaacc ctgaccctgc cgtgtaccag 360

ctgagagact ctaagtcgag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 420

acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaatg tgtgctagac 480

atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacaa atctgacttt 540

gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca 600

gaaagttcc 609

<210> 28

<211> 242

<212> PRT

<213> artificial sequence

<220>

<223> soluble TCR beta chain

<400> 28

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

1 5 10 15

Gln Ser Val Ala Leu Trp Cys Asn Pro Ile Ser Gly His Ala Thr Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Lys Leu Leu Ile Gln

35 40 45

Phe Gln Asn Asn Gly Val Val Asp Asp Ser Gln Leu Pro Lys Asp Arg

50 55 60

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

65 70 75 80

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

85 90 95

Gly Gly Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Ala Asp

<210> 29

<211> 726

<212> DNA

<213> artificial sequence

<220>

<223> soluble TCR beta chain

<400> 29

gaagcaggtg ttgcacagag cccgcgttat ctgattgttg aaaaaggtca gagcgttgca 60

ctgtggtgta atccgattag cggtcatgca accctgtatt ggtatcgtca ggatccgggt 120

cagggtctga aactgctgat tcagtttcag aataatggtg ttgttgatga tagccagctg 180

ccgaaagatc gttttagcgc agaacgtctg aaaggtgttg atagcaccct gaaaattcag 240

ccggttaaac cggaagatag cgcagtttat ttttgtgcaa gcagctgggg tggtaatgaa 300

cagtttttcg gtccgggtac ccgtctgacc gttctggagg acctgaaaaa cgtgttccca 360

cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 420

ctggtgtgcc tggccaccgg tttctacccc gaccacgtgg agctgagctg gtgggtgaat 480

gggaaggagg tgcacagtgg ggtctgcaca gacccgcagc ccctcaagga gcagcccgcc 540

ctcaatgact ccagatacgc tctgagcagc cgcctgaggg tctcggccac cttctggcag 600

gacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 660

tggacccagg atagggccaa acccgtcacc cagatcgtca gcgccgaggc ctggggtaga 720

gcagac 726

Claims (35)

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

α CDR1- NSAFQY (SEQ ID NO: 10)

α CDR2- TYSSGN (SEQ ID NO: 11)

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

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

β CDR1- SGHAT (SEQ ID NO: 13)

β CDR2- FQNNGV (SEQ ID NO: 14)

β CDR3- ASSWGGNEQF (SEQ ID NO: 15)。

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

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

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

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

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

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

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

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

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

11. A TCR as claimed in claim 10 which has one or more mutations in amino acid 11, 13, 19, 21, 53, 76, 89, 91 or 94 of the 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).

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

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

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

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

15. A TCR as claimed in claim 14 wherein the cysteine residues which form the artificial disulphide bond in the TCR are substituted at one or more groups selected from:

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

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

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

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

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

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

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

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

16. A TCR as claimed in claim 15 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.

17. A TCR as claimed in claim 13 which comprises an artificial interchain disulphide bond between the α chain variable region and the β chain constant region of the TCR.

18. A TCR as claimed in claim 17 in which 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 TRBC1 x 01 or TRBC2 x 01 exon 1; or

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

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

20. 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.

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

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

23. 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 22.

24. A nucleic acid molecule comprising a nucleic acid sequence encoding a TCR according to any one of claims 1 to 22, or the complement thereof.

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

26. the nucleic acid molecule of claim 24 or 25, wherein said nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 6.

27. the nucleic acid molecule of claim 24, comprising the nucleotide sequence encoding a TCR α chain of SEQ ID NO:4 and/or comprises the nucleotide sequence encoding the TCR β chain SEQ ID NO: 8.

28. a vector comprising the nucleic acid molecule of any one of claims 24 to 27.

29. The vector of claim 28, wherein said vector is a viral vector.

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

31. An isolated host cell comprising the vector of claim 28 or a nucleic acid molecule of any one of claims 24-27 integrated into the chromosome.

32. A cell transduced with the nucleic acid molecule of any one of claims 24 to 27 or the vector of claim 28.

33. The cell of claim 32, wherein the cell is a T cell.

34. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to any one of claims 1 to 22, a TCR complex according to claim 23 or a cell according to claim 32.

35. Use of a TCR as claimed in any one of claims 1 to 22 or a TCR complex as claimed in claim 23 or a cell as claimed in claim 32 in the manufacture of a medicament for the treatment of a tumour.

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