CN116135878A - TCR for recognizing AFP antigen - Google Patents

Abstract

The present invention provides a T Cell Receptor (TCR) capable of specifically binding to a short peptide TSSELMAITR derived from AFP antigen, which short peptide TSSELMAITR can form a complex with HLA a1101 and be presented on the cell surface together. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules. In addition, the invention provides cells transduced with the TCRs of the invention.

Description

TCR for recognizing AFP antigen

Technical Field

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

Background

AFP (alpha Fetoprotein), also known as alpha Fetoprotein, is a protein expressed during embryo development and is the main component of embryo serum. During development, AFP has relatively high expression levels in the yolk sac and liver, which are subsequently inhibited. In hepatocellular carcinoma, AFP expression is activated (Butterfield et al J immunol.,2001, apr 15;166 (8): 5300-8). AFP is degraded into small molecule polypeptides after intracellular production and is presented on the cell surface in association with MHC (major histocompatibility complex) molecules to form complexes. TSSELMAITR (SEQ ID NO: 9) is a short peptide derived from an AFP antigen, and is a target for the treatment of AFP-related diseases.

T cell adoptive immunotherapy involves transferring reactive T cells specific for a target cell antigen into a patient to act against the target cell. The T Cell Receptor (TCR) is a membrane protein on the surface of T cells that is capable of recognizing the corresponding antigenic short peptide on the surface of target cells. In the immune system, the direct physical contact of T cells and Antigen Presenting Cells (APCs) is initiated by the binding of antigen-short peptide specific TCRs to the short peptide-major histocompatibility complex (pMHC complex), and then the interaction of T cells and other cell membrane surface molecules of both APCs occurs, causing 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. Accordingly, those skilled in the art have focused on isolating TCRs specific for AFP antigen peptides and transducing T cells with the TCRs to obtain T cells specific for AFP antigen peptides, thereby allowing them to play a role in cellular immunotherapy.

Disclosure of Invention

The invention aims at providing a T cell receptor for recognizing AFP antigen short peptide.

In a first aspect of the invention there is provided a T Cell Receptor (TCR) capable of binding to the TSSELMAITR-HLA a1101 complex.

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

αCDR1-DRGSQS(SEQ ID NO:10)

αCDR2-IYSNGD(SEQ ID NO:11)

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

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

βCDR1-SEHNR(SEQ ID NO:13)

βCDR2-FQNEAQ(SEQ ID NO:14)

βCDR3-ASSPVGEQY(SEQ ID NO:15)。

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

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

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

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

In another preferred embodiment, the TCR is soluble.

In another preferred embodiment, the TCR is isolated or purified.

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 a linkage of an alpha chain variable domain and a beta chain variable domain via a peptide linker sequence.

In another preferred embodiment, the constant regions of the α and β chains of the TCR are those of murine origin, respectively.

In another preferred embodiment, the TCR has one or more mutations in the alpha chain variable region amino acids 11, 13, 19, 21, 53, 76, 89, 91, or 94, and/or the alpha chain J gene short peptide amino acid position 3, 5, or 7; and/or the TCR has one or more mutations in amino acid 11, 13, 19, 21, 53, 76, 89, 91, or 94 of the β chain variable region, and/or the β chain J gene short peptide amino acid position 2, 4, or 6, wherein the amino acid position numbers are numbered as listed in IMGT (international immunogenetic information system).

In another preferred embodiment, the alpha chain variable domain amino acid sequence of the TCR comprises SEQ ID NO:32 and or the beta chain variable domain amino acid sequence of the TCR comprises SEQ ID NO:34.

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

In another preferred embodiment, the TCR comprises (a) all or part of a TCR a chain other than a transmembrane domain; and (b) all or part of the TCR β chain except 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 α and β 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 of the sets of sites selected from:

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

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

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

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

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

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

TRAC.01 exon 1 Pro89 and TRBC 1.01 or TRBC 2.01 exon 1 Ala19; and Tyr10 of TRAC x 01 exon 1 and Glu20 of TRBC1 x 01 or TRBC2 x 01 exon 1.

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

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

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

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

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

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

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

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, except the transmembrane domain, but 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 C-or N-terminus of the alpha and/or beta chain of the TCR is conjugated to a conjugate.

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 TCR molecule 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 of the first aspect of the invention or a complement thereof.

In another preferred embodiment, the nucleic acid molecule is isolated or purified.

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

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

in another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 4 and/or comprises the nucleotide sequence of 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 nucleic acid molecule according to the third aspect of the invention integrated into the genome.

In a sixth aspect of the invention, there is provided a cell transduced with 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, NK cell, NKT cell or stem cell.

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

In an eighth aspect of the invention there is provided 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, or a cell according to the sixth aspect of the invention, for the manufacture of a medicament for the treatment of a tumour or autoimmune disease, preferably the tumour is an AFP positive tumour.

In a ninth aspect of the invention there is provided a T cell receptor according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, or a cell according to the sixth aspect of the invention, for use as a medicament in the treatment of a tumour or an autoimmune disease; preferably, the tumor is an AFP-positive tumor.

In a tenth 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, or a cell according to the sixth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention; preferably, the disease is a tumor, preferably the tumor is an AFP positive tumor.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

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

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

FIG. 3 is a CD8 of a monoclonal cell ⁺ -APC and tetramer-PE biscationic staining results.

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

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

FIGS. 6a and 6b are gel diagrams of soluble TCR obtained after purification. The right lanes in fig. 6a and 6b are respectively a reducing gel and a non-reducing gel, and the left lanes are molecular weight markers.

FIGS. 7a and 7b are the amino acid and nucleotide sequences of a single chain TCR, respectively, the amino acid and nucleotide sequences of the linker sequence (linker) are underlined.

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

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

FIGS. 10a and 10b are gel diagrams of soluble single chain TCR obtained after purification. The right lanes in fig. 10a and 10b are respectively a reducing gel and a non-reducing gel, and the left lanes are molecular weight markers.

FIG. 11 is a chart showing BIAcore kinetics of binding of soluble TCR of the invention to TSSELMAITR-HLA A A1101 complex.

FIG. 12 is a BIAcore kinetic profile of binding of soluble single chain TCR of the invention to TSSELMAITR-HLA A A1101 complex.

FIG. 13 shows the results of ELISPOT activation function verification of the resulting T cell clones.

FIG. 14 shows the results of ELISPOT activation function verification of effector cells transfected with TCR of the invention against T2 cells loaded with a short peptide.

FIG. 15 shows the results of ELISPOT activation function verification of effector cells transfected with TCRs of the invention against tumor cell lines.

Detailed Description

The present inventors have conducted extensive and intensive studies to find a TCR capable of specifically binding to AFP antigen short peptide TSSELMAITR (SEQ ID NO: 9), which antigen short peptide TSSELMAITR can form a complex with HLA A A1101 and be presented on the cell surface together. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules. In addition, the invention provides cells transduced with the TCRs of the invention.

Terminology

The MHC molecules are proteins of the immunoglobulin superfamily, which may be class I or class II MHC molecules. Thus, it is specific for antigen presentation, and different individuals have different MHCs, which are capable of presenting different short peptides of a single protein antigen to the respective APC cell surfaces. Human MHC is commonly referred to as an HLA gene or HLA complex.

T Cell Receptor (TCR), the only receptor for specific antigenic peptides presented on the Major Histocompatibility Complex (MHC). In the immune system, direct physical contact of T cells with Antigen Presenting Cells (APCs) is initiated by binding of antigen-specific TCRs to pMHC complexes, and then interaction of T cells with other cell membrane surface molecules of both APCs occurs, 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 glycoproteins on the surface of cell membranes that exist as heterodimers from either the alpha/beta or gamma/delta chain. TCR heterodimers consist of alpha and beta chains in 95% of T cells, while 5% of T cells have TCRs consisting of gamma and delta chains. The native αβ heterodimeric TCR has an α chain and a β chain, which constitute subunits of the αβ heterodimeric TCR. In a broad sense, each of the α and β chains comprises a variable region, a linking region, and a constant region, and the β chain also typically comprises a short variable region between the variable region and the linking region, but the variable region is often considered part of the linking region. Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, which are chimeric in a framework structure (framework regions). The CDR regions determine the binding of the TCR to the pMHC complex, wherein CDR3 is recombined from the variable region and the linking region, known as the hypervariable region. The α and β chains of TCRs are generally regarded as having two "domains" each, i.e., a variable domain and a constant domain, the variable domain being composed of linked variable and linking regions. The sequence of the TCR constant domain can be found in published databases of the international immunogenetic information system (IMGT), for example the constant domain sequence of the α chain of a TCR molecule is "TRAC x 01" and the constant domain sequence of the β chain of a TCR molecule is "TRBC1 x 01" or "TRBC2 x 01". In addition, the α and β chains of TCRs also contain transmembrane and cytoplasmic regions, which are 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 inter-chain disulfide bonds and artificial inter-chain disulfide bonds

A set of disulfide bonds exist between the near membrane regions cα and cβ of a native TCR, referred to herein as "native interchain disulfide bonds". In the present invention, an inter-chain covalent disulfide bond, which is artificially introduced at a position different from that of a natural inter-chain disulfide bond, is referred to as an "artificial inter-chain disulfide bond".

For convenience of description of disulfide bond positions, TRAC.sub.01 and TRBC.sub.1.sub.01 or TRBC.sub.2.sub.01 amino acid sequences are sequentially numbered from N-terminal to C-terminal, for example, TRBC.sub.1.sub.01 or TRBC.sub.2.sub.01 is P (proline) as the 60 th amino acid in the sequence from N-terminal to C-terminal, and can be described as Pro60 of TRBC.sub.1.sub.01 or TRBC.sub.2.sub.01 exon 1, it may also be expressed as amino acid 60 of exon 1 TRBC1 x 01 or TRBC2 x 01, and as in TRBC1 x 01 or TRBC2 x 01, amino acid 61 in the order from N-terminal to C-terminal is Q (glutamine), and it may be expressed as Gln61 of exon 1 TRBC1 x 01 or TRBC2 x 01, or as amino acid 61 of exon 1 TRBC1 x 01 or TRBC2 x 01, and so on. In the present invention, the position numbers of the amino acid sequences of the variable regions TRAV and TRBV are according to the position numbers listed in IMGT. If an amino acid in TRAV is numbered 46 in IMGT, it is described in the present invention as TRAV amino acid 46, and so on. In the present invention, the sequence position numbers of other amino acids are specifically described, and are specifically described.

Detailed Description

TCR molecules

During antigen processing, the antigen is degraded inside the cell 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 TSSELMAITR-HLA a1101 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 following amino acid sequences:

αCDR1-DRGSQS(SEQ ID NO:10)

αCDR2-IYSNGD(SEQ ID NO:11)

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

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

βCDR1-SEHNR(SEQ ID NO:13)

βCDR2-FQNEAQ(SEQ ID NO:14)

βCDR3-ASSPVGEQY(SEQ ID NO:15)。

chimeric TCRs may be prepared by embedding the CDR region amino acid sequences of the invention described above into any suitable framework structure. As long as the framework structure is compatible with the CDR regions of the TCRs of the present invention, one skilled in the art will be able to design or synthesize TCR molecules having corresponding functions based on the CDR regions disclosed herein. Accordingly, a TCR molecule of the invention refers to a TCR molecule 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 identical to SEQ ID NO: 5 has an amino acid sequence 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 alpha and beta chains. Specifically, in one aspect the alpha chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the alpha chain variable domain amino acid sequence comprises CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the above alpha chain. Preferably, the TCR molecule comprises the alpha chain variable domain amino acid sequence SEQ ID NO. 1. More preferably, the alpha chain variable domain amino acid sequence 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 β chain described above. Preferably, the 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 TCR molecule is SEQ ID No. 5.

In a preferred embodiment of the invention, the TCR molecule of the invention is a single chain TCR molecule consisting of part or all of the alpha chain and/or part or all of the beta chain. For descriptions of single chain TCR molecules, reference may be made to Chung et al (1994) Proc.Natl. Acad.Sci.USA 91,12654-12658. From the literature, one skilled in the art can readily construct single chain TCR molecules comprising the CDRs regions of the invention. In particular, the single chain TCR molecule comprises vα, vβ and cβ, preferably linked in order from the N-terminus to the 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 above alpha chain. Preferably, the single chain TCR molecule comprises the alpha chain variable domain amino acid sequence SEQ ID NO. 1. More preferably, the alpha chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO. 1. The β chain variable domain amino acid sequence 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 β 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 the public database of related books or IMGT (international immunogenetic information system). For example, the constant domain sequence of the α chain of the TCR molecule of the invention may be "TRAC x 01", and the constant domain sequence of the β chain of the TCR molecule may be "TRBC1 x 01" or "TRBC2 x 01". Arg at position 53 of the amino acid sequence given in TRAC 01 of IMGT, denoted herein as: TRAC.01 Arg53 of exon 1, and so on. Preferably, the amino acid sequence of the alpha chain of the TCR molecule of the invention is SEQ ID NO. 3 and/or the amino acid sequence of the beta chain is SEQ ID NO. 7.

A naturally occurring TCR is a membrane protein, which is stabilised by its transmembrane region. Like immunoglobulins (antibodies) as antigen recognition molecules, TCRs may also be developed for diagnostic and therapeutic applications, where soluble TCR molecules are desired. Soluble TCR molecules do not include their transmembrane region. Soluble TCRs have a wide range of uses, not only for studying the interaction of TCRs with pMHC, 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 specific antigen. The invention also provides soluble TCRs specific for AFP antigen peptides.

To obtain a soluble TCR, in one aspect, the TCR of the invention may be a TCR in which an artificial disulfide bond is introduced between residues of its alpha and beta chain constant domains. Cysteine residues form artificial interchain disulfide bonds between the α and β chain constant domains of the TCR. Cysteine residues may be substituted for other amino acid residues at suitable sites in the native TCR to form artificial interchain disulfide bonds. For example, a disulfide bond is formed by substituting Thr48 of TRAC x 01 exon 1 and substituting cysteine residue of Ser57 of TRBC1 x 01 or TRBC2 x 01 exon 1. Other sites for introducing cysteine residues to form disulfide bonds may also be: thr45 of tranc x 01 exon 1 and Ser77 of TRBC1 x 01 or TRBC2 x 01 exon 1; tyr10 of TRAC x 01 exon 1 and Ser17 of TRBC1 x 01 or TRBC2 x 01 exon 1; thr45 of TRAC x 01 exon 1 and Asp59 of TRBC1 x 01 or TRBC2 x 01 exon 1; ser15 of TRAC x 01 exon 1 and Glu15 of TRBC1 x 01 or TRBC2 x 01 exon 1; arg53 of TRAC x 01 exon 1 and Ser54 of TRBC1 x 01 or TRBC2 x 01 exon 1; TRAC.01 exon 1 Pro89 and TRBC 1.01 or TRBC 2.01 exon 1 Ala19; or Tyr10 of TRAC x 01 exon 1 and Glu20 of TRBC1 x 01 or TRBC2 x 01 exon 1. I.e., a cysteine residue replaces any of the set of sites in the constant domains of the alpha and beta chains described above. The deletion of the native disulfide bond may be achieved by truncating up to 50, or up to 30, or up to 15, or up to 10, or up to 8 or less amino acids at one or more of the C-termini of the TCR constant domains of the present invention, such that they do not include a cysteine residue, or by mutating the cysteine residue forming the 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 the alpha and beta chains thereof. It should be noted that the TCRs of the invention may each contain a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, with or without the introduced artificial disulfide bond as described above. The TRAC constant domain sequence and TRBC1 or TRBC2 constant domain sequence of the TCR can be linked by a native disulfide bond present in the TCR.

To obtain a soluble TCR, on the other hand, the inventive TCRs also include TCRs having mutations in their hydrophobic core region, preferably mutations that result in an improved stability of the inventive soluble TCRs, as described in the patent publication No. WO 2014/206304. Such TCRs may be mutated at the following variable domain hydrophobic core positions: (α and/or β chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or the α chain J gene (TRAJ) short peptide amino acid position reciprocal 3,5,7, and/or the β chain J gene (TRBJ) short peptide amino acid position reciprocal 2,4,6, wherein the position numbers of the amino acid sequences are as listed in the international immunogenetic information system (IMGT). The person skilled in the art is aware of the above-mentioned international immunogenetic information system and can derive the position numbers of amino acid residues of different TCRs in IMGT from this database.

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

In addition, patent document 201680003540.2 discloses that the introduction of an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of a TCR can significantly improve the stability of the TCR. Thus, the TCRs of the present invention may also contain artificial interchain disulfide bonds between the α chain variable and β chain constant regions. 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: 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 TRBC1 x 01 or TRBC2 x 01 exon 1. Preferably, such TCRs may comprise (i) all or part of the TCR a chain except for its transmembrane domain, and (ii) all or part of the TCR β chain except for its transmembrane domain, wherein (ii) and (ii) each comprise a variable domain and at least part of a constant domain of the TCR chain, the a chain forming a heterodimer with the β chain. More preferably, such TCRs may comprise an alpha chain variable domain and a beta chain variable domain and all or part of a beta chain constant domain other than the transmembrane domain, but they do not comprise an alpha chain constant domain, the alpha chain variable domain of the TCR forming a heterodimer with the beta chain.

The TCRs of the present invention may also be provided in the form of multivalent complexes. The multivalent TCR complexes of the invention comprise a multimer of two, three, four or more TCRs of the invention bound, e.g., a tetramer may be generated using the tetramer domain of p53, or a complex of a plurality of TCRs of the invention bound to 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, as well as to generate intermediates for other multivalent TCR complexes having such applications.

The TCRs of the present invention may be used alone or may be covalently or otherwise bound to the conjugate, preferably covalently. The conjugates include a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the TSSELMAITR-HLA a1101 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination or coupling of any of the above.

Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or enzymes capable of producing a detectable product.

Therapeutic agents that may be conjugated or coupled to a TCR of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, cancer metastasis reviews (Cancer metastasis reviews) 24, 539); 2. biotoxicity (Chaudhary et al, 1989, nature 339, 394; epel et al, 2002, cancer immunology and immunotherapy (Cancer Immunology and Immunotherapy) 51, 565); 3. cytokines such as IL-2 et al (Gillies et al, 1992, proc. Natl. Acad. Sci. USA (PNAS) 89, 1428; card et al, 2004, cancer immunology and immunotherapy (Cancer Immunology and Immunotherapy) 53, 345; halin et al, 2003, cancer Research (Cancer Research) 63, 3202); 4. antibody Fc fragments (Mosquera et al, 2005, journal of immunology (The Journal Of Immunology) 174, 4381); 5. antibody scFv fragments (Zhu et al, 1995, J.cancer International (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 American society of chemistry (Journal of the American Chemical Society) 128, 2115); 7. viral particles (Peng et al, 2004, gene therapy (Gene therapy) 11, 1234); 8. liposomes (Mamot et al 2005, cancer research 65, 11631); seventhly, 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 any form of nanoparticle, and the like.

In addition, the TCRs of the present 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, TCRs of the invention may comprise a human variable domain and a murine constant domain. The disadvantage of this approach is the possibility of eliciting an immune response. Thus, there should be a regulatory regime for immunosuppression when it is used in adoptive T cell therapy to allow implantation of T cells expressing murine species.

It should be understood that, in this document, the amino acid names are represented by international single english letters or three english letters, and the correspondence between the single english letters and the three english letters of the amino acid names is 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

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

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:

CDR1α-gaccgaggttcccagtcc(SEQ ID NO:16)

CDR2α-atatactccaatggtgac(SEQ ID NO:17)

CDR3α-gccgtgaaggttggaggatacagcaccctcacc(SEQ ID NO:18)

The nucleotide sequence encoding the CDR region of the β chain of the TCR molecule of the first aspect of the invention is as follows:

CDR1β-tctgaacacaaccgc(SEQ ID NO:19)

CDR2β-ttccagaatgaagctcaa(SEQ ID NO:20)

CDR3β-gccagcagcccggtgggggagcagtac(SEQ ID NO:21)

thus, the nucleotide sequences of the nucleic acid molecules of the invention encoding the TCR alpha chain of the invention include SEQ ID NO. 16, SEQ ID NO. 17 and SEQ ID NO. 18, and/or the nucleotide sequences of the nucleic acid molecules of the invention encoding the TCR beta chain of the invention include 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 introns. Preferably, the nucleotide sequence of the nucleic acid molecule of the invention does not comprise an intron but is capable of encoding the polypeptide of the invention, e.g. the nucleotide sequence of the nucleic acid molecule of the invention encoding the variable domain of the TCR alpha chain of the invention comprises SEQ ID NO. 2 and/or the nucleotide sequence of the nucleic acid molecule of the invention encoding the variable domain of the TCR beta chain of the invention comprises SEQ ID NO. 6. Alternatively, the nucleotide sequence of the nucleic acid molecule of the invention encoding the variable domain of the TCR α chain of the invention comprises the sequence set forth in seq ID NO:33 and/or the nucleic acid molecule of the invention encoding a variable domain of a TCR β chain of the invention comprises a sequence of SEG ID NO:35. preferably, the nucleotide sequence of the nucleic acid molecule according to the invention comprises SEQ ID NO. 4 and/or SEQ ID NO. 8. Alternatively, the nucleotide sequence of the nucleic acid molecule of the invention is SEQ ID NO:31.

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

The nucleotide sequence may be codon optimized. Different cells differ in the use of specific codons, and the amount of expression can be increased by changing codons in the sequence depending on the cell type. Codon usage tables for mammalian cells and 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 generally obtained by, but not limited to, PCR amplification, recombinant methods or artificial synthesis. At present, it is already possible to obtain the DNA sequence encoding the TCR of the invention (or a fragment or derivative thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. The DNA may be a coding strand or a non-coding strand.

Carrier body

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, phages and animal and plant viruses.

Viral delivery systems include, but are not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, retrovirus vectors, lentivirus vectors, baculovirus vectors.

Preferably, the vector may transfer the nucleotide of the invention into a cell, such as a T cell, such that the cell expresses an AFP antigen-specific TCR. Ideally, the vector should be capable of sustained high level expression in T cells.

Cells

The invention also relates to host cells genetically engineered with the vectors or coding sequences of the invention. The host cell contains the vector or chromosome of the present invention integrated with the nucleic acid molecule of the present 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 expressing the TCRs of the invention, which may be but are not limited to T cells, NK cells, NKT cells, stem cells, and in particular T cells. The T cells may be derived from T cells isolated from a subject, or may be part of a mixed cell population 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), and may be CD4 ⁺ Helper T cells or CD8 ⁺ Cytotoxic T cells. The cell can be in CD4 ⁺ Helper T cell/CD 8 ⁺ In a mixed population of cytotoxic T cells. Generally, the cells will be activated with an antibody (e.g., an anti-CD 3 or anti-CD 28 antibody) to render them more susceptible to transfection, for example, 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, as the stem cell surface does not express CD3 molecules. However, when stem cells differentiate into lymphoid precursors that migrate to the thymus (lymphoid precursor), expression of the CD3 molecule will initiate expression of the introduced TCR molecule on the surface of the thymocytes.

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

AFP antigen-related diseases

The invention also relates to a method of treating and/or preventing an AFP-associated disease in a subject comprising the step of adoptively transferring AFP-specific T cells to the subject. The AFP-specific T cells recognize the TSSELMAITR-HLA A A1101 complex.

The AFP-specific T cells of the invention can be used to treat any AFP-related disease presenting the AFP antigen oligopeptide TSSELMAITR-HLA A A1101 complex, including but not limited to tumors, such as liver cancer, etc.

Therapeutic method

Treatment may be performed by isolating T cells from a patient or volunteer suffering from a disease associated with AFP antigen and introducing the TCR of the invention into the T cells and then reinjecting the genetically modified cells back into the patient. Accordingly, the present invention provides a method of treating an AFP-related disorder comprising administering to a patient isolated T cells expressing a TCR of the invention, preferably derived from the patient itself. Generally, this involves (1) isolating T cells from a patient, (2) transducing T cells outside the patient with a nucleic acid molecule of the invention or a nucleic acid molecule capable of encoding a TCR molecule of the invention, and (3) introducing genetically modified T cells into the patient. The number of isolated, transfected and reinfused cells can be determined by the physician.

The invention has the main advantages that:

(1) The TCR of the invention can specifically bind to AFP antigen short peptide complex TSSELMAITR-HLA A1101, and effector cells transduced with the TCR of the invention can be specifically activated.

The following specific examples further illustrate the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address specific conditions in the examples below, is generally followed by conventional conditions, for example those described in the laboratory Manual (Molecular Cloning-A Laboratory Manual) (third edition) (2001) CSHL Press, or by the manufacturer's recommendations (Sambrook and Russell et al, molecular cloning). Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.

EXAMPLE 1 cloning of AFP antigen-short peptide-specific T cells

Peripheral Blood Lymphocytes (PBLs) from healthy volunteers of genotype HLA-A1101 were stimulated with synthetic short peptide TSSELMAITR (SEQ ID NO:9; jiangsu St. Biotech Co., ltd.). The TSSELMAITR short peptide was renatured with HLA-A1101 carrying a biotin label to prepare a pMHC haploid. These haploids are combined with PE-labeled streptavidin (BD company) to form PE-labeled tetramers, which are sorted together with anti-CD 8-APC biscationic cells. The sorted cells were expanded and subjected to secondary sorting as described above, followed by monoclonal by limiting dilution. Monoclonal cells were stained with tetramers and the selected biscationic clones are shown in FIG. 3. The double-positive clones obtained by layer-by-layer screening are also required to meet further functional tests.

IFN-gamma is a potent immunomodulator produced by activated T lymphocytes, and therefore this example demonstrates the activation function and antigen specificity of cells transfected with TCRs of the invention by detecting IFN-gamma numbers by ELISPOT assays well known to those skilled in the art. The function and specificity of the T cell clone was further examined by ELISPOT experiments. The effector cells used in the IFN-. Gamma.ELISPOT experiments were the T cell clones obtained in the present invention, the target cells were T2-A11 loaded with TSSELMAITR short peptide (refer to T2 cells transfected with HLA-A 1101), SK-MEL-28-AFP (refer to SK-MEL-28 cells transfected with AFP), and the control groups were T2-A11 and SK-MEL-28 loaded with other antigen short peptides. Wherein the T2 cells were purchased from ATCC and SK-MEL-28 cells were purchased from Sakuku Biotechnology Co., guangzhou.

First, prepare an ELISPOT plate, the ELISPOT experiment procedure is as follows: the individual components tested were added to the ELISPOT plates in the following order: after 20000 target cells/well and 2000 effector cells/well, 20. Mu.l of the corresponding short peptide was added to the experimental group and the control group, and the final concentration of the T2-A11-loaded short peptide was 10 ^-5 M, blank was added with 20. Mu.l of medium (test medium) and 2 wells were set. Then incubated overnight (37 ℃,5% co) ₂ ). The plates were then washed and subjected to secondary detection and development, and the plates were dried for 1 hour, and spots formed on the films were counted using an immunoblotter plate reader (ELISPOT READER system; AID company). As shown in FIG. 13, the obtained T cell clone has high IFN-gamma release to T2-A11 and SK-MEL-28-AFP loaded with TSSELMAITR short peptide, but has no response to T2-A11 and SK-MEL-28 loaded with other antigen short peptide.

EXAMPLE 2 acquisition of AFP antigen short peptide-specific T cell clone TCR Gene

With Quick-RNA ^TM MiniPrep (ZYMO research) Total RNA from antigen-short peptide TSSELMAITR-specific, HLA-A 1101-restricted T cell clones selected in example 1 was extracted. The cDNA was synthesized using a clontech SMART RACE cDNA amplification kit using primers designed on the C-terminal conserved region of the human TCR gene. The sequences were cloned into a T vector (TAKARA) for sequencing. It should be noted that the sequence is a complementary sequence and does not contain an intron. The alpha chain and beta chain sequence structures of the double-positive clone expressed TCR are respectively shown in the figure 1 and the figure 2, and the figure 1a, the figure 1b, the figure 1c, the figure 1d, the figure 1e and the figure 1f are respectively TCR alpha chain variable domain amino acid sequences, TCR alpha chain variable domain nucleotide sequences, TCR alpha chain amino acid sequences, TCR alpha chain nucleotide sequences, TCR alpha chain amino acid sequences with leader sequences and TCR alpha chain nucleotide sequences with leader sequences; FIGS. 2a, 2b, 2c, 2d, 2e and 2f are, respectively, TCR.beta.0 chain variable domain amino acid sequence, TCR.beta.1 chain variable domain nucleotide sequence, TCR.beta.2 chain amino acid sequence, TCR.beta.3 chain nucleotide sequence, TCR.beta.chain amino acid sequence with leader sequence, and TCR.beta.chain nucleotide sequence with leader sequence.

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

αCDR1-DRGSQS(SEQ ID NO:10)

αCDR2-IYSNGD(SEQ ID NO:11)

αCDR3-AVKVGGYSTLT(SEQ ID NO:12)

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

βCDR1-SEHNR(SEQ ID NO:13)

βCDR2-FQNEAQ(SEQ ID NO:14)

βCDR3-ASSPVGEQY(SEQ ID NO:15)。

EXAMPLE 3 expression, refolding and purification of AFP antigen short peptide-specific soluble TCR

To obtain soluble TCR molecules, the α and β chains of the TCR molecules of the invention may comprise only their variable domains and part of their constant domains, respectively, and a cysteine residue is introduced in the constant domains of the α and β chains to form an artificial inter-chain disulphide bond, the amino acid sequence and nucleotide sequence of the α chain of which are shown in figures 4a and 4b, respectively, and the amino acid sequence and nucleotide sequence of the β chain of which are shown in figures 5a and 5b, respectively. The target gene sequences of the above TCR alpha and beta chains were synthesized and inserted into the expression vector pET28a+ (Novagene) by standard methods described in molecular cloning laboratory Manual (Molecular Cloning a Laboratory Manual) (third edition, sambrook and Russell), and cloning sites upstream and downstream were NcoI and NotI, respectively. The insert was confirmed by sequencing to be error-free.

The expression vectors of TCR alpha and beta chains are respectively transformed into expression bacteria BL21 (DE 3) by a chemical transformation method, the bacteria are grown by LB culture solution, and the bacteria are grown on OD ₆₀₀ At 0.6, inclusion bodies formed after expression of the α and β chains of TCR were extracted by bugbaster Mix (Novagene) and washed repeatedly with bugbaster solution, and finally the inclusion bodies were dissolved in 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT), 10mM ethylenediamine tetraacetic acid (EDTA), 20mM Tris (pH 8.1), induced with a final concentration of 0.5mM IPTG.

The TCR alpha and beta chains after dissolution were found to be 1:1 in mass ratio in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine,6.6mM beta-mercapoethylamine (4 ℃ C.) at a final concentration of 60mg/mL. After mixing the solution was dialyzed (4 ℃) in 10 volumes of deionized water, after 12 hours the deionized water was changed to buffer (20 mM Tris, pH 8.0) and dialysis was continued at 4℃for 12 hours. The dialyzed solution was filtered through a 0.45 μm filter and purified by an anion exchange column (HiTrap Q HP,5ml,GE Healthcare). The elution peak contains the successfully renatured alpha and beta dimer TCR as confirmed by SDS-PAGE gel. The TCR was then 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. SDS-PAGE gel of soluble TCR obtained according to the invention is shown in FIGS. 6a and 6 b.

EXAMPLE 4 production of soluble single chain TCR specific for AFP antigen short peptide

The variable domains of the tcra and β chains of example 2 were constructed as a stable soluble single chain TCR molecule linked by flexible short peptides (linker) using site-directed mutagenesis, as described in WO 2014/206304. The amino acid sequence and nucleotide sequence of the single chain TCR molecule are shown in figures 7a and 7b, respectively, with the amino acid sequence and nucleotide sequence of the linker underlined. The amino acid sequence and the nucleotide sequence of the alpha chain variable domain are shown in FIG. 8a and FIG. 8b respectively; the amino acid sequence and nucleotide sequence of the β chain variable domain are shown in fig. 9a and 9b, respectively.

The target gene is subjected to double digestion by Nco I and Not I, and is connected with a pET28a vector subjected to double digestion by Nco I and Not I. The ligation product was transformed into E.coli DH 5. Alpha. And the ligation product was spread on LB plates containing kanamycin, incubated at 37℃overnight in an inverted position, positive clones were picked up for PCR screening, positive recombinants were sequenced, and after the correct sequence was confirmed, the recombinant plasmid was extracted and transformed into E.coli BL21 (DE 3) for expression.

EXAMPLE 5 expression, renaturation and purification of AFP antigen short peptide specific soluble single chain TCR

BL21 (DE 3) colonies prepared in example 4 and containing the recombinant plasmid pET28 a-template strand were all inoculated into LB medium containing kanamycin, cultured at 37℃until OD600 was 0.6-0.8, added with IPTG to a final concentration of 0.5mM, and cultured at 37℃for 4 hours. Cell pellet was harvested by centrifugation at 5000rpm for 15min, cell pellet was lysed by Bugbuster Master Mix (Merck), inclusion bodies were recovered by centrifugation at 6000rpm for 15min, washed with Bugbuster (Merck) to remove cell debris and membrane components, and collected by centrifugation at 6000rpm for 15 min. The inclusion bodies were dissolved in buffer (20 mM Tris-HCl pH 8.0,8M urea), high-speed centrifuged to remove insoluble material, and the supernatant was quantified by BCA method and then sub-packaged and stored at-80℃for further use.

To 5mg of solubilized single chain TCR inclusion body protein, 2.5mL of buffer (6M ua-HCl,50 mM Tris-HCl pH 8.1, 100mM NaCl,10mM EDTA) was added, followed by addition of DTT to a final concentration of 10mM and treatment at 37℃for 30min. The single-chain TCR after the treatment was added dropwise to 125mL of renaturation buffer (100 mM Tris-HCl pH 8.1,0.4M L-arginine, 5M urea, 2mM EDTA,6.5mM EDTA,6.5mM. Beta. -mercapthoethylamine, 1.87mM Cystamine) with a syringe, stirred at 4℃for 10min, and then the renaturation solution was put into a cellulose membrane dialysis bag with a retention of 4kDa, and the dialysis bag was placed in 1L of pre-chilled water, and stirred slowly at 4℃overnight. After 17 hours, the dialysate was changed to 1L of pre-chilled buffer (20 mM Tris-HCl pH 8.0), dialysis was continued for 8 hours at 4℃and then the dialysate was changed to the same fresh buffer and dialysis continued overnight. After 17 hours, the sample was filtered through a 0.45 μm filter, vacuum degassed and passed through an anion exchange column (HiTrap Q HP, GE Healthcare) and the protein was purified using a linear gradient of 0-1M NaCl from 20mM Tris-HCl pH8.0, the collected eluted fractions were subjected to SDS-PAGE analysis, the fractions containing single chain TCR were concentrated and further purified using a gel filtration column (Superdex 7510/300,GE Healthcare), and the target fractions were also subjected to SDS-PAGE analysis.

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

The gel diagram of SDS-PAGE of soluble single chain TCR obtained according to the invention is shown in FIGS. 10a and 10b

Example 6 characterization in combination

This example demonstrates by BIAcore analysis that soluble TCR molecules of the invention are able to bind specifically to the TSSELMAITR-HLA a1101 complex.

The binding activity of the TCR molecule obtained in example 3 to the TSSELMAITR-HLA A A1101 complex was examined using a BIAcore T200 real-time assay system. The coupling process was completed by adding anti-streptavidin antibody (GenScript) to coupling buffer (10 mM sodium acetate buffer, pH 4.77), then flowing the antibody through CM5 chips previously activated with EDC and NHS to immobilize the antibody on the chip surface, and finally blocking the unreacted activated surface with ethanolamine in hydrochloric acid solution at a coupling level of about 15,000 RU.

The low concentration of streptavidin was flowed over the surface of the antibody-coated chip, then TSSELMAITR-HLA A A1101 complex was flowed over the detection channel, the other channel was used as a reference channel, and 0.05 mM biotin was flowed over the chip at a flow rate of 10. Mu.L/min for 2min, blocking the remaining binding sites for streptavidin.

The preparation process of the TSSELMAITR A1101 compound is as follows:

a. purification

Collecting 100ml E.coli bacterial liquid for inducing expression of heavy chain or light chain, centrifuging at 8000g at 4 ℃ for 10min, washing the bacterial body once with 10ml PBS, then severely shaking and re-suspending the bacterial body with 5ml BugBuster Master Mix Extraction Reagents (Merck), rotating at room temperature for 20min, centrifuging at 6000g at 4 ℃ for 15min, discarding the supernatant, and collecting inclusion bodies.

The inclusion body is resuspended in 5ml BugBuster Master Mix and incubated for 5min at room temperature; adding 30ml BugBuster diluted 10 times, mixing, and centrifuging at 4deg.C for 15min at 6000 g; removing the supernatant, adding 30ml of BugBuster diluted 10 times, mixing, centrifuging at 4 ℃ for 15min, repeating twice, adding 30ml of 20mM Tris-HCl pH 8.0, mixing, centrifuging at 4 ℃ for 15min, dissolving the inclusion body with 20mM Tris-HCl 8M urea, detecting purity of the inclusion body by SDS-PAGE, and detecting concentration by BCA kit.

b. Renaturation

The synthesized short peptide TSSELMAITR was dissolved in DMSO to a concentration of 20 mg/ml. The inclusion bodies of the light and heavy chains were solubilized with 8M urea, 20mM Tris pH 8.0, 10mM DTT, and further denatured by adding 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA prior to renaturation. TSSELMAITR peptide was added to a renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4 ℃) at 25mg/L (final concentration), followed by sequential addition of 20mg/L light chain and 90mg/L heavy chain (final concentration, three heavy chain additions, 8 h/time), renaturation was performed at 4℃for at least 3 days to completion, and SDS-PAGE was examined for success of renaturation.

c. Purification after renaturation

The renaturation buffer was exchanged with 10 volumes of 20mM Tris pH 8.0 for dialysis, at least twice to sufficiently reduce the ionic strength of the solution. 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 company) anion exchange column (5 ml bed volume). Using an Akta purifier (GE general electric), proteins were eluted with a linear gradient of 0-400mM NaCl in 20mM Tris pH 8.0, pMHC eluted at about 250mM NaCl, and fractions were collected for SDS-PAGE to check purity.

d. Biotinylation

Purified pMHC molecules were concentrated using Millipore ultrafiltration tubes while buffer was replaced with 20mM Tris pH 8.0, and then biotinylated reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50. Mu. M D-Biotin, 100. Mu.g/ml birA enzyme (GST-birA), the mixture incubated overnight at room temperature and SDS-PAGE was performed to determine whether biotinylation was complete.

e. Purification of biotinylated complexes

Biotinylated pMHC molecules were concentrated to 1ml using a Millipore ultrafiltration tube, biotinylated pMHC was purified using gel filtration chromatography, hiPrep was pre-equilibrated with filtered PBS using an Akta purifier (GE general electric company) ^TM 16/60S200 HR column (GE general electric company), loaded with 1ml of concentrated biotinylated pMHC molecule, and eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules appeared as a single peak elution at about 55 ml. The protein-containing fractions were pooled, concentrated by Millipore ultrafiltration tube, protein concentration was determined by BCA method (Thermo), and biotinylated pMHC molecules were stored in aliquots at-80℃with the addition of protease inhibitor cocktail (Roche).

Kinetic parameters were calculated using BIAcore Evaluation software to obtain kinetic profiles of the binding of the soluble TCR molecules of the invention to the TSSELMAITR-HLA a1101 complex, as shown in figures 11 and 12, respectively. The pattern shows that the soluble TCR molecules obtained by the invention can bind to TSSELMAITR-HLA A A1101 complex. Meanwhile, the binding activity of the soluble TCR molecules of the invention and other irrelevant antigens of short peptides and HLA complexes is also detected by using the method, and the result shows that the TCR molecules of the invention are not bound with other irrelevant antigens.

Example 7 effector cell activation experiments on short peptide loaded T2 cells transfected with TCRs of the invention

The effector cells used in this experiment were CD3 transfected with TCR of the invention ⁺ T cells and transfection of CD3 of other TCRs (A6) with the same volunteer ⁺ T cells served as control. The target cells used were T2-A11 loaded with AFP antigen short peptide TSSELMAITR, and T2-A11 loaded with other antigen short peptides and empty were used as controls.

First, ELISPOT plates were prepared, ethanol-activated coated, and at 4℃overnight. On day 1 of the experiment, the coating was removed, the block was washed, incubated at room temperature for two hours, the block was removed, and the individual components of the experiment were added to the ELISPOT plate: target cells 1X 10 ⁴ Per well, effector cells were 2×10 ³ Each well (calculated as transfection positive rate) and two duplicate wells were set. Then adding TSSELMAITR short peptide into corresponding well to make final concentration of short peptide in ELISPOT pore plate 10 ^-6 M. Incubation was carried out overnight (37 ℃,5% co 2). On day 2 of the experiment, the plates were washed and subjected to secondary detection and development, the plates were dried, and spots formed on the membrane were counted using an immunoblotter plate reader (ELISPOT READER system; AID 20).

As shown in FIG. 14, the T cells transfected with the TCR of the invention had a significant activating effect on T2-A11 loaded with TSSELMAITR short peptide, while T2 cells loaded with other short peptides or empty had no activating effect on T cells transfected with the TCR of the invention.

Example 8 experiment of activation function of effector cells transfected with TCR of the invention against tumor cell lines

This example also examined the function and specificity of the TCRs of the invention in cells by ELISPOT experiments. The effector cells used were CD3 expressing the inventive AFP antigen-short peptide-specific TCR ⁺ T cells and cd3+ T cells transfected with other TCRs (A6) from the same volunteer were used as control. The positive tumor cell line used was SK-MEL-28-AFP (SK-MEL-28 cells transfected with AFP); the negative tumor cell lines used were SK-MEL-28, SNU423, JHH7 and HUCCT1 as control groups. Wherein SK-MEL-28, SNU423 and HUCCT1 were purchased from Guangzhou Sakuku Biotechnology Co., ltd, and JHH7 was purchased from Nanjing Corp.

First, an ELISPOT plate was prepared. ELISPOT plate ethanol activation coating, 4 ℃ overnight. On day 1 of the experiment, the coating was removed, the block was washed, incubated at room temperature for two hours, the block was removed, and the individual components of the experiment were added to the ELISPOT plate: target cells were 2X 10 ⁴ Per well, effector cells were 2×10 ³ Each well (calculated as transfection positive rate) and two duplicate wells were set. Incubation was carried out overnight (37 ℃,5% co 2). On day 2 of the experiment, the plates were washed and subjected to secondary detection and development, the plates were dried, and spots formed on the membrane were counted using an immunoblotter plate reader (ELISPOT READER system; AID 20).

The experimental results are shown in fig. 15, where effector cells transfected with other TCRs were inactive against all cell lines; effector cells transfected with the TCRs of the present invention are specifically activated by positive tumor cell lines, while being inactive against negative tumor cell lines.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

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<213> Artificial sequence (Artificial Sequence)

<400> 4

cagaaggagg tggagcagaa ttctggaccc ctcagtgttc cagagggagc cattgcctct 60

ctcaactgca cttacagtga ccgaggttcc cagtccttct tctggtacag acaatattct 120

gggaaaagcc ctgagttgat aatgttcata tactccaatg gtgacaaaga agatggaagg 180

tttacagcac agctcaataa agccagccag tatgtttctc tgctcatcag agactcccag 240

cccagtgatt cagccaccta cctctgtgcc gtgaaggttg gaggatacag caccctcacc 300

tttgggaagg ggactatgct tctagtctct ccagatatcc agaaccctga ccctgccgtg 360

taccagctga gagactctaa atccagtgac aagtctgtct gcctattcac cgattttgat 420

tctcaaacaa atgtgtcaca aagtaaggat tctgatgtgt atatcacaga caaaactgtg 480

ctagacatga ggtctatgga cttcaagagc aacagtgctg tggcctggag caacaaatct 540

gactttgcat gtgcaaacgc cttcaacaac agcattattc cagaagacac cttcttcccc 600

agcccagaaa gttcctgtga tgtcaagctg gtcgagaaaa gctttgaaac agatacgaac 660

ctaaactttc aaaacctgtc agtgattggg ttccgaatcc tcctcctgaa agtggccggg 720

tttaatctgc tcatgacgct gcggctgtgg tccagc 756

<210> 5

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 6

<211> 333

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

gatactggag tctcccagga ccccagacac aagatcacaa agaggggaca gaatgtaact 60

ttcaggtgtg atccaatttc tgaacacaac cgcctttatt ggtaccgaca gaccctgggg 120

cagggcccag agtttctgac ttacttccag aatgaagctc aactagaaaa atcaaggctg 180

ctcagtgatc ggttctctgc agagaggcct aagggatctt tctccacctt ggagatccag 240

cgcacagagc agggggactc ggccatgtat ctctgtgcca gcagcccggt gggggagcag 300

tacttcgggc cgggcaccag gctcacggtc aca 333

<210> 7

<211> 290

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Asp Leu Lys Asn 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 Tyr 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 Glu 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 Ser

275 280 285

Arg Gly

290

<210> 8

<211> 870

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

gatactggag tctcccagga ccccagacac aagatcacaa agaggggaca gaatgtaact 60

ttcaggtgtg atccaatttc tgaacacaac cgcctttatt ggtaccgaca gaccctgggg 120

cagggcccag agtttctgac ttacttccag aatgaagctc aactagaaaa atcaaggctg 180

ctcagtgatc ggttctctgc agagaggcct aagggatctt tctccacctt ggagatccag 240

cgcacagagc agggggactc ggccatgtat ctctgtgcca gcagcccggt gggggagcag 300

tacttcgggc cgggcaccag gctcacggtc acagaggacc tgaaaaacgt gttcccaccc 360

gaggtcgctg tgtttgagcc atcagaagca gagatctccc acacccaaaa ggccacactg 420

gtgtgcctgg ccacaggctt ctaccccgac cacgtggagc tgagctggtg ggtgaatggg 480

aaggaggtgc acagtggggt cagcacagac ccgcagcccc tcaaggagca gcccgccctc 540

aatgactcca gatactgcct gagcagccgc ctgagggtct cggccacctt ctggcagaac 600

ccccgcaacc acttccgctg tcaagtccag ttctacgggc tctcggagaa tgacgagtgg 660

acccaggata gggccaaacc tgtcacccag atcgtcagcg ccgaggcctg gggtagagca 720

gactgtggct tcacctccga gtcttaccag caaggggtcc tgtctgccac catcctctat 780

gagatcttgc tagggaaggc caccttgtat gccgtgctgg tcagtgccct cgtgctgatg 840

gccatggtca agagaaagga ttccagaggc 870

<210> 9

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

Thr Ser Ser Glu Leu Met Ala Ile Thr Arg

1 5 10

<210> 10

<211> 6

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 10

Asp Arg Gly Ser Gln Ser

1 5

<210> 11

<211> 6

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

Ile Tyr Ser Asn Gly Asp

1 5

<210> 12

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 12

Ala Val Lys Val Gly Gly Tyr Ser Thr Leu Thr

1 5 10

<210> 13

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 13

Ser Glu His Asn Arg

1 5

<210> 14

<211> 6

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 14

Phe Gln Asn Glu Ala Gln

1 5

<210> 15

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 15

Ala Ser Ser Pro Val Gly Glu Gln Tyr

1 5

<210> 16

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

gaccgaggtt cccagtcc 18

<210> 17

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

atatactcca atggtgac 18

<210> 18

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

gccgtgaagg ttggaggata cagcaccctc acc 33

<210> 19

<211> 15

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

tctgaacaca accgc 15

<210> 20

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

ttccagaatg aagctcaa 18

<210> 21

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

gccagcagcc cggtggggga gcagtac 27

<210> 22

<211> 272

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 22

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

1 5 10 15

Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser

20 25 30

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

35 40 45

Gly Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro

50 55 60

Glu Leu Ile Met Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

<210> 23

<211> 816

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

aaatccttga gagttttact agtgatcctg tggcttcagt tgagctgggt ttggagccaa 60

cagaaggagg tggagcagaa ttctggaccc ctcagtgttc cagagggagc cattgcctct 120

ctcaactgca cttacagtga ccgaggttcc cagtccttct tctggtacag acaatattct 180

gggaaaagcc ctgagttgat aatgttcata tactccaatg gtgacaaaga agatggaagg 240

tttacagcac agctcaataa agccagccag tatgtttctc tgctcatcag agactcccag 300

cccagtgatt cagccaccta cctctgtgcc gtgaaggttg gaggatacag caccctcacc 360

tttgggaagg ggactatgct tctagtctct ccagatatcc agaaccctga ccctgccgtg 420

taccagctga gagactctaa atccagtgac aagtctgtct gcctattcac cgattttgat 480

tctcaaacaa atgtgtcaca aagtaaggat tctgatgtgt atatcacaga caaaactgtg 540

ctagacatga ggtctatgga cttcaagagc aacagtgctg tggcctggag caacaaatct 600

gactttgcat gtgcaaacgc cttcaacaac agcattattc cagaagacac cttcttcccc 660

agcccagaaa gttcctgtga tgtcaagctg gtcgagaaaa gctttgaaac agatacgaac 720

ctaaactttc aaaacctgtc agtgattggg ttccgaatcc tcctcctgaa agtggccggg 780

tttaatctgc tcatgacgct gcggctgtgg tccagc 816

<210> 24

<211> 308

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 24

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Asp Ser Arg Gly

305

<210> 25

<211> 924

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

ggcaccagcc tcctctgctg gatggccctg tgtctcctgg gggcagatca cgcagatact 60

ggagtctccc aggaccccag acacaagatc acaaagaggg gacagaatgt aactttcagg 120

tgtgatccaa tttctgaaca caaccgcctt tattggtacc gacagaccct ggggcagggc 180

ccagagtttc tgacttactt ccagaatgaa gctcaactag aaaaatcaag gctgctcagt 240

gatcggttct ctgcagagag gcctaaggga tctttctcca ccttggagat ccagcgcaca 300

gagcaggggg actcggccat gtatctctgt gccagcagcc cggtggggga gcagtacttc 360

gggccgggca ccaggctcac ggtcacagag gacctgaaaa acgtgttccc acccgaggtc 420

gctgtgtttg agccatcaga agcagagatc tcccacaccc aaaaggccac actggtgtgc 480

ctggccacag gcttctaccc cgaccacgtg gagctgagct ggtgggtgaa tgggaaggag 540

gtgcacagtg gggtcagcac agacccgcag cccctcaagg agcagcccgc cctcaatgac 600

tccagatact gcctgagcag ccgcctgagg gtctcggcca ccttctggca gaacccccgc 660

aaccacttcc gctgtcaagt ccagttctac gggctctcgg agaatgacga gtggacccag 720

gatagggcca aacctgtcac ccagatcgtc agcgccgagg cctggggtag agcagactgt 780

ggcttcacct ccgagtctta ccagcaaggg gtcctgtctg ccaccatcct ctatgagatc 840

ttgctaggga aggccacctt gtatgccgtg ctggtcagtg ccctcgtgct gatggccatg 900

gtcaagagaa aggattccag aggc 924

<210> 26

<211> 205

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 26

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

1 5 10 15

Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Lys Val Gly Gly Tyr

85 90 95

Ser Thr Leu Thr Phe Gly Lys Gly Thr Met Leu Leu Val Ser Pro Asp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

<210> 27

<211> 615

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

cagaaagaag tggaacagaa ttctggaccc ctcagtgttc cagagggagc cattgcctct 60

ctcaactgca cttacagtga ccgaggttcc cagtccttct tctggtacag acaatattct 120

gggaaaagcc ctgagttgat aatgttcata tactccaatg gtgacaaaga agatggaagg 180

tttacagcac agctcaataa agccagccag tatgtttctc tgctcatcag agactcccag 240

cccagtgatt cagccaccta cctctgtgcc gtgaaggttg gaggatacag caccctcacc 300

tttgggaagg ggactatgct tctagtctct ccagatatcc agaaccctga ccctgccgtt 360

tatcagctgc gtgatagcaa aagcagcgat aaaagcgtgt gcctgttcac cgattttgat 420

agccagacca acgtgagcca gagcaaagat agcgatgtgt acatcaccga taaaaccgtg 480

ctggatatgc gcagcatgga tttcaaaagc aatagcgcgg ttgcgtggag caacaaaagc 540

gattttgcgt gcgcgaacgc gtttaacaac agcatcatcc cggaagatac gttcttctgc 600

agcccagaaa gttcc 615

<210> 28

<211> 241

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 28

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Thr Gly Phe Tyr 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 Ala 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

<210> 29

<211> 723

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

gataccggtg ttagccagga ccccagacac aagatcacaa agaggggaca gaatgtaact 60

ttcaggtgtg atccaatttc tgaacacaac cgcctttatt ggtaccgaca gaccctgggg 120

cagggcccag agtttctgac ttacttccag aatgaagctc aactagaaaa atcaaggctg 180

ctcagtgatc ggttctctgc agagaggcct aagggatctt tctccacctt ggagatccag 240

cgcacagagc agggggactc ggccatgtat ctctgtgcca gcagcccggt gggggagcag 300

tacttcgggc cgggcaccag gctcacggtc acagaggacc tgaaaaacgt gttcccaccc 360

gaggtcgctg tgtttgagcc atcagaatgc gaaattagcc atacccagaa agcgaccctg 420

gtttgtctgg cgaccggttt ttatccggat catgtggaac tgtcttggtg ggtgaacggc 480

aaagaagtgc atagcggtgt ttctaccgat ccgcagccgc tgaaagaaca gccggcgctg 540

aatgatagcc gttatgcgct gtctagccgt ctgcgtgtta gcgcgacctt ttggcaaaat 600

ccgcgtaacc attttcgttg ccaggtgcag ttttatggcc tgagcgaaaa cgatgaatgg 660

acccaggatc gtgcgaagcc ggttacccag attgttagcg cggaagcctg gggccgcgca 720

gat 723

<210> 30

<211> 246

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 30

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

1 5 10 15

Glu Asn Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Lys Val Gly Gly Tyr

85 90 95

Ser Thr Leu Thr Phe Gly Lys Gly Thr Lys Leu Ser Val Ser Pro Gly

100 105 110

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

115 120 125

Gly Ser Glu Gly Gly Thr Gly Asp Thr Gly Val Ser Gln Asp Pro Arg

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Thr Arg Leu Thr Val Thr

245

<210> 31

<211> 738

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 31

caaaaagaag ttgaacagaa tagtggtccg ctgagcgttc cggaaggtga aaatgtgagc 60

attaattgta cctatagtga tcgtggcagc cagagcttct tttggtatcg ccagtatagt 120

ggtaaaagtc cggaactgat tatgtttatc tatagcaatg gcgataagga agatggccgt 180

tttaccgcac agctgaataa ggcaagccag tatgtgagtc tgctgattcg cgatgtgcag 240

ccgagcgata gcgcaaccta cttatgtgca gttaaagttg gcggttatag tactctgacc 300

tttggcaaag gtaccaaact gagcgtgagc ccgggtggtg gtagtgaagg tggtggtagc 360

gaaggcggcg gcagcgaagg tggtggcagt gaaggcggca ccggcgatac cggcgttagt 420

caggaccctc gtcatctgag cgtgaaacgc ggtcagaatg tgaccctgcg ctgcgatccg 480

attagcgaac ataatcgtct gtattggtat cgccaaaccc cgggtcaggg cccggaattt 540

ctgacctatt ttcagaatga agcccagctg gaaaagagcc gcctgctgag cgatcgtttt 600

agtgccgaac gtccgaaagg cagttttagt accctggaaa ttcagcgtgt tgaaccgggc 660

gatagcgcca tgtacctttg tgccagtagt ccggttggcg aacagtattt tggtcctggc 720

acccgtctga ccgttacc 738

<210> 32

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 32

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

1 5 10 15

Glu Asn Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Lys Val Gly Gly Tyr

85 90 95

Ser Thr Leu Thr Phe Gly Lys Gly Thr Lys Leu Ser Val Ser Pro

100 105 110

<210> 33

<211> 333

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 33

caaaaagaag ttgaacagaa tagtggtccg ctgagcgttc cggaaggtga aaatgtgagc 60

attaattgta cctatagtga tcgtggcagc cagagcttct tttggtatcg ccagtatagt 120

ggtaaaagtc cggaactgat tatgtttatc tatagcaatg gcgataagga agatggccgt 180

tttaccgcac agctgaataa ggcaagccag tatgtgagtc tgctgattcg cgatgtgcag 240

ccgagcgata gcgcaaccta cttatgtgca gttaaagttg gcggttatag tactctgacc 300

tttggcaaag gtaccaaact gagcgtgagc ccg 333

<210> 34

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 34

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 35

<211> 333

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 35

gataccggcg ttagtcagga ccctcgtcat ctgagcgtga aacgcggtca gaatgtgacc 60

ctgcgctgcg atccgattag cgaacataat cgtctgtatt ggtatcgcca aaccccgggt 120

cagggcccgg aatttctgac ctattttcag aatgaagccc agctggaaaa gagccgcctg 180

ctgagcgatc gttttagtgc cgaacgtccg aaaggcagtt ttagtaccct ggaaattcag 240

cgtgttgaac cgggcgatag cgccatgtac ctttgtgcca gtagtccggt tggcgaacag 300

tattttggtc ctggcacccg tctgaccgtt acc 333

Claims (10)

1. A T Cell Receptor (TCR) comprising a TCR a chain variable domain and a TCR β chain variable domain, wherein the TCR is capable of binding to the TSSELMAITR-HLA a1101 complex, and wherein the 3 Complementarity Determining Regions (CDRs) of the TCR a chain variable domain are:

αCDR1-DRGSQS(SEQ ID NO:10)

αCDR2-IYSNGD(SEQ ID NO:11)

Alpha CDR3-AVKVGGYSTLT (SEQ ID NO: 12); and/or

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

βCDR1-SEHNR(SEQ ID NO:13)

βCDR2-FQNEAQ(SEQ ID NO:14)

βCDR3-ASSPVGEQY(SEQ ID NO:15)。

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

3. A TCR as claimed in claim 1 wherein the C-or N-terminus of the α and/or β chain of the TCR is associated with a conjugate; preferably, 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.

4. A multivalent TCR complex comprising at least two TCR molecules, and wherein at least one TCR molecule is a TCR as claimed in any preceding claim.

5. A nucleic acid molecule comprising a nucleic acid sequence encoding a TCR molecule according to any preceding claim or a complement thereof;

preferably, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:2 or SEQ ID No. 33 and/or said nucleic acid molecule comprises the nucleotide sequence SEQ ID NO:6 or SEQ ID NO. 35.

6. A vector comprising the nucleic acid molecule of claim 5; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector.

7. An isolated host cell comprising the vector of claim 6 or the nucleic acid molecule of claim 5 integrated into a chromosome.

8. A cell, wherein the cell transduces the nucleic acid molecule of claim 5 or the vector of claim 6; preferably, the cells are T cells or stem cells.

9. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR as claimed in any one of claims 1 to 3, a TCR complex as claimed in claim 4, a nucleic acid molecule as claimed in claim 5 or a cell as claimed in claim 8.

10. Use of a T cell receptor according to any one of claims 1 to 3, or a TCR complex as claimed in claim 4, or a cell as claimed in claim 8, for the manufacture of a medicament for the treatment of a tumour or an autoimmune disease.

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