CN117659162A - T cell receptor for recognizing APF and coding sequence and application thereof - Google Patents
T cell receptor for recognizing APF and coding sequence and application thereof Download PDFInfo
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- Peptides Or Proteins (AREA)
Abstract
The present invention provides a T cell receptor capable of specifically binding to a short peptide TSSELMAITR derived from AFP antigen, which antigen 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 T cell receptor and vectors comprising the nucleic acid molecules. In addition, the invention also provides cells transduced with the T cell receptors of the invention.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a T cell receptor for identifying APF, and a coding sequence and application thereof; in particular, it relates to TCR capable of recognizing AFP antigen-derived short peptide and its coding sequence, and also relates to AFP-specific T cell obtained by transducing the above-mentioned TCR and its application in preventing and curing 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
Aiming at the defects of the prior art, the invention aims to provide a T cell receptor for recognizing AFP antigen short peptide.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
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-NSASQS (SEQ ID NO:10)
αCDR2-VYSSGN (SEQ ID NO:11)
alpha CDR3-VVNPGSGNTGKLI (SEQ ID NO: 12); and/or
The 3 complementarity determining regions of the TCR β chain variable domain are:
βCDR1-SGHTA (SEQ ID NO:13)
βCDR2-FQGNSA (SEQ ID NO:14)
βCDR3-ASSLVFGSVWDTQY (SEQ ID NO:15)。
in another preferred embodiment, the TCR comprises a TCR α chain variable domain that is an amino acid sequence having at least 90% (e.g., may be 90%, 92%, 94%, 95%, 96%, 98%, or 100%, etc.) sequence identity to SEQ ID No. 1;
and/or the TCR β chain variable domain is an amino acid sequence having at least 90% (e.g., may be 90%, 92%, 94%, 95%, 96%, 98%, or 100%, etc.) sequence identity to SEQ ID No. 5.
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, the TCR comprising a TCR α chain constant region TRAC 01 and a TCR β chain constant region TRBC 101 or TRBC2 01.
In another preferred embodiment, the alpha 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;
or 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 sets of sites selected from the group consisting of:
amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01;
amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01;
amino acid 46 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01;
or amino acid 47 of TRAV and amino acid 60 of 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 comprises 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 T cell receptor complex comprising at least two T cell receptor molecules, and wherein at least one of the T cell receptor molecules is a T cell receptor according to the first aspect of the invention.
In a third aspect of the invention there is provided a nucleic acid molecule comprising a nucleotide sequence encoding a T cell receptor according to the first aspect of the invention and/or a complement of a nucleotide sequence encoding a T cell receptor according to the first aspect of the invention.
In another preferred embodiment, the nucleic acid molecule is isolated or purified.
In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO. 2 or SEQ ID NO. 33 encoding a TCR alpha chain variable domain.
In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO. 6 or SEQ ID NO. 35 encoding a TCR β chain variable domain.
In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO. 4 encoding a TCR alpha chain and/or comprises the nucleotide sequence SEQ ID NO. 8 encoding a TCR beta chain.
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 having integrated into its genome 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 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 any one or a combination of at least two of the T cell receptor of the first aspect of the invention, the multivalent T cell receptor complex of the second aspect of the invention, the nucleic acid molecule of the third aspect of the invention, the carrier of the fourth aspect of the invention or the cell of the sixth aspect of the invention.
In an eighth aspect of the invention there is provided the use of any one or a combination of at least two of the T cell receptor of the first aspect of the invention, the multivalent T cell receptor complex of the second aspect of the invention or the cell of the sixth aspect of the invention, the use comprising the manufacture of a medicament for the treatment of a tumour or an autoimmune disease.
Preferably, the tumor is an AFP-positive tumor.
In a ninth aspect of the invention there is provided a T cell receptor according to the first aspect of the invention, a multivalent T cell receptor complex according to the second aspect of the invention or a cell according to the sixth aspect of the invention, any one or a combination of at least two thereof, for use as a medicament for 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 of treatment an appropriate amount of any one or a combination of at least two of the T cell receptor of the first aspect of the invention, the multivalent T cell receptor complex of the second aspect of the invention, the cell of the sixth aspect of the invention or the pharmaceutical composition of 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.
The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.
Compared with the prior art, the invention has the following beneficial effects:
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.
Drawings
FIG. 1 is a CD8 of a monoclonal cell + -APC and tetramer-PE biscationic staining results.
FIG. 2 shows the results of ELISPOT activation function verification of the resulting T cell clones.
FIG. 3a is a gel diagram (reduction gel) of the soluble TCR obtained after purification.
FIG. 3b is a gel diagram (non-reducing gel) of the soluble TCR obtained after purification.
FIG. 4 is a gel diagram of the soluble single chain TCR obtained after purification.
FIG. 5 is a chart of BIAcore kinetics of binding of soluble TCR of the invention to TSSELMAITR-HLA A A1101 complex.
FIG. 6 is a chart of BIAcore kinetics of binding of the soluble single chain TCR of the invention to the TSSELMAITR-HLA A A1101 complex.
FIG. 7 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.
Detailed Description
The 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-HLAA1101 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-NSASQS (SEQ ID NO:10)
αCDR2-VYSSGN (SEQ ID NO:11)
alpha CDR3-VVNPGSGNTGKLI (SEQ ID NO: 12); and/or
The 3 complementarity determining regions of the TCR β chain variable domain are:
βCDR1-SGHTA (SEQ ID NO:13)
βCDR2-FQGNSA (SEQ ID NO:14)
βCDR3-ASSLVFGSVWDTQY (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 an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity to SEQ ID No. 5.
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 (i) 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);
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 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-aacagtgcttctcagtct (SEQ ID NO:16)
CDR2α-gtatactccagtggtaat (SEQ ID NO:17)
CDR3α-gtggtgaaccccgggtctggcaacacaggcaaactaatc (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-tcaggtcatactgcc (SEQ ID NO:19)
CDR2β-ttccaaggcaacagtgca (SEQ ID NO:20)
CDR3β-gccagcagcttagtattcggtagcgtgtgggatacgcagtat (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 a variable domain of a TCR alpha chain of the invention comprises SEG ID NO 33 and/or the nucleotide sequence of the nucleic acid molecule of the invention encoding a variable domain of a TCR beta chain of the invention comprises 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 set forth in the sequence listing 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 short peptide TSSELMAITR-HLA A A1101 complex, including but not limited to tumors, such as liver cancer, lung cancer, fibrosarcoma, breast cancer, colon cancer, prostate cancer.
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. 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.
The amino acid sequence and nucleotide sequence related to the invention are shown as follows:
SEQ ID NO. 1 (TCR. Alpha. Chain variable domain amino acid sequence):
RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNPGSGNTGKLIFGQGTTLQVKP。
SEQ ID NO. 2 (TCR. Alpha. Chain variable domain nucleotide sequence):
cggaaggaggtggagcaggatcctggacccttcaatgttccagagggagccactgtcgctttcaactgtacttacagcaacagtgcttctcagtctttcttctggtacagacaggattgcaggaaagaacctaagttgctgatgtccgtatactccagtggtaatgaagatggaaggtttacagcacagctcaatagagccagccagtatatttccctgctcatcagagactccaagctcagtgattcagccacctacctctgtgtggtgaaccccgggtctggcaacacaggcaaactaatctttgggcaagggacaactttacaagtaaaaccagat。
SEQ ID NO. 3 (TCR. Alpha. Chain amino acid sequence):
RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNPGSGNTGKLIFGQGTTLQVKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS。
SEQ ID NO. 4 (TCR. Alpha. Chain nucleotide sequence):
cggaaggaggtggagcaggatcctggacccttcaatgttccagagggagccactgtcgctttcaactgtacttacagcaacagtgcttctcagtctttcttctggtacagacaggattgcaggaaagaacctaagttgctgatgtccgtatactccagtggtaatgaagatggaaggtttacagcacagctcaatagagccagccagtatatttccctgctcatcagagactccaagctcagtgattcagccacctacctctgtgtggtgaaccccgggtctggcaacacaggcaaactaatctttgggcaagggacaactttacaagtaaaaccagatAtccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaagtctgtctgcctattcaccgattttgattctcaaacaaatgtgtcacaaagtaaggattctgatgtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaagagcaacagtgctgtggcctggagcaacaaatctgactttgcatgtgcaaacgccttcaacaacagcattattccagaagacaccttcttccccagcccagaaagttcctgtgatgtcaagctggtcgagaaaagctttgaaacagatacgaacctaaactttcaaaacctgtcagtgattgggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatgacgctgcggctgtggtccagc。
SEQ ID NO. 5 (TCR. Beta. Chain variable domain amino acid sequence):
GAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASSLVFGSVWDTQYFGPGTRLTVL。
SEQ ID NO. 6 (TCR. Beta. Chain variable domain nucleotide sequence):
ggagctggagtctcccagtcccccagtaacaaggtcacagagaagggaaaggatgtagagctcaggtgtgatccaatttcaggtcatactgccctttactggtaccgacagaggctggggcagggcctggagtttttaatttacttccaaggcaacagtgcaccagacaaatcagggctgcccagtgatcgcttctctgcagagaggactggggaatccgtctccactctgacgatccagcgcacacagcaggaggactcggccgtgtatctctgtgccagcagcttagtattcggtagcgtgtgggatacgcagtattttggcccaggcacccggctgacagtgctc。
SEQ ID NO. 7 (TCR. Beta. Chain amino acid sequence):
GAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASSLVFGSVWDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG。
SEQ ID NO 8 (TCR. Beta. Chain nucleotide sequence):
ggagctggagtctcccagtcccccagtaacaaggtcacagagaagggaaaggatgtagagctcaggtgtgatccaatttcaggtcatactgccctttactggtaccgacagaggctggggcagggcctggagtttttaatttacttccaaggcaacagtgcaccagacaaatcagggctgcccagtgatcgcttctctgcagagaggactggggaatccgtctccactctgacgatccagcgcacacagcaggaggactcggccgtgtatctctgtgccagcagcttagtattcggtagcgtgtgggatacgcagtattttggcccaggcacccggctgacagtgctcGaggacctgaaaaacgtgttcccacccgaggtcgctgtgtttgagccatcagaagcagagatctcccacacccaaaaggccacactggtgtgcctggccacaggcttctaccccgaccacgtggagctgagctggtgggtgaatgggaaggaggtgcacagtggggtcagcacagacccgcagcccctcaaggagcagcccgccctcaatgactccagatactgcctgagcagccgcctgagggtctcggccaccttctggcagaacccccgcaaccacttccgctgtcaagtccagttctacgggctctcggagaatgacgagtggacccaggatagggccaaacctgtcacccagatcgtcagcgccgaggcctggggtagagcagactgtggcttcacctccgagtcttaccagcaaggggtcctgtctgccaccatcctctatgagatcttgctagggaaggccaccttgtatgccgtgctggtcagtgccctcgtgctgatggccatggtcaagagaaaggattccagaggc。
SEQ ID NO. 9 (short peptide derived from AFP antigen) TSSELMAITR.
SEQ ID NO 10 (amino acid sequence of alpha CDR 1): nsass qs.
SEQ ID NO. 11 (amino acid sequence of. Alpha. CDR 2): VYSSGN.
SEQ ID NO. 12 (amino acid sequence of. Alpha. CDR 3): VVNPGSGNTGKLI.
SEQ ID NO. 13 (amino acid sequence of. Beta. CDR 1): SGHTA.
SEQ ID NO. 14 (amino acid sequence of. Beta. CDR 2): FQGNSA.
SEQ ID NO. 15 (amino acid sequence of. Beta. CDR 3): ASSLVFGSVWDTQY.
SEQ ID NO. 16 (nucleotide sequence of alpha CDR 1): aacagtgcttctcagtct.
SEQ ID NO:17 (nucleotide sequence of alpha CDR 2): gtatactccagtggtaat.
SEQ ID NO. 18 (nucleotide sequence of alpha CDR 3): gtggtgaaccccgggtctggcaacacaggcaaactaatc.
SEQ ID NO. 19 (nucleotide sequence of. Beta. CDR 1): tcaggtcatactgcc.
SEQ ID NO. 20 (nucleotide sequence of. Beta. CDR 2): ttccaaggcaacagtgca.
SEQ ID NO. 21 (nucleotide sequence of beta CDR 3): gccagcagcttagtattcggtagcgtgtgggatacgcagtat.
SEQ ID NO. 22 (TCR alpha chain amino acid sequence with leader sequence)
ISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNPGSGNTGKLIFGQGTTLQVKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS。
SEQ ID NO. 23 (TCR. Alpha. Chain nucleotide sequence with leader):
atatccttgagagttttactggtgatcctgtggcttcagttaagctgggtttggagccaacggaaggaggtggagcaggatcctggacccttcaatgttccagagggagccactgtcgctttcaactgtacttacagcaacagtgcttctcagtctttcttctggtacagacaggattgcaggaaagaacctaagttgctgatgtccgtatactccagtggtaatgaagatggaaggtttacagcacagctcaatagagccagccagtatatttccctgctcatcagagactccaagctcagtgattcagccacctacctctgtgtggtgaaccccgggtctggcaacacaggcaaactaatctttgggcaagggacaactttacaagtaaaaccagatAtccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaagtctgtctgcctattcaccgattttgattctcaaacaaatgtgtcacaaagtaaggattctgatgtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaagagcaacagtgctgtggcctggagcaacaaatctgactttgcatgtgcaaacgccttcaacaacagcattattccagaagacaccttcttccccagcccagaaagttcctgtgatgtcaagctggtcgagaaaagctttgaaacagatacgaacctaaactttcaaaacctgtcagtgattgggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatgacgctgcggctgtggtccagc。
SEQ ID NO. 24 (TCR. Beta. Chain amino acid sequence with leader):
GTRLLFWVAFCLLGAYHTGAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASSLVFGSVWDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG。
SEQ ID NO. 25 (TCR. Beta. Chain nucleotide sequence with leader):
ggcaccaggctcctcttctgggtggccttctgtctcctgggggcatatcacacaggagctggagtctcccagtcccccagtaacaaggtcacagagaagggaaaggatgtagagctcaggtgtgatccaatttcaggtcatactgccctttactggtaccgacagaggctggggcagggcctggagtttttaatttacttccaaggcaacagtgcaccagacaaatcagggctgcccagtgatcgcttctctgcagagaggactggggaatccgtctccactctgacgatccagcgcacacagcaggaggactcggccgtgtatctctgtgccagcagcttagtattcggtagcgtgtgggatacgcagtattttggcccaggcacccggctgacagtgctcgaggacctgaaaaacgtgttcccacccgaggtcgctgtgtttgagccatcagaagcagagatctcccacacccaaaaggccacactggtgtgcctggccacaggcttctaccccgaccacgtggagctgagctggtgggtgaatgggaaggaggtgcacagtggggtcagcacagacccgcagcccctcaaggagcagcccgccctcaatgactccagatactgcctgagcagccgcctgagggtctcggccaccttctggcagaacccccgcaaccacttccgctgtcaagtccagttctacgggctctcggagaatgacgagtggacccaggatagggccaaacctgtcacccagatcgtcagcgccgaggcctggggtagagcagactgtggcttcacctccgagtcttaccagcaaggggtcctgtctgccaccatcctctatgagatcttgctagggaaggccaccttgtatgccgtgctggtcagtgccctcgtgctgatggccatggtcaagagaaaggattccagaggc。
SEQ ID NO. 26 (amino acid sequence of the alpha chain of soluble TCR):
RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNPGSGNTGKLIFGQGTTLQVKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFCSPESS。
SEQ ID NO 27 (nucleotide sequence of the alpha chain of soluble TCR):
cgcaaagaagtggaacaggatcctggacccttcaatgttccagagggagccactgtcgctttcaactgtacttacagcaacagtgcttctcagtctttcttctggtacagacaggattgcaggaaagaacctaagttgctgatgtccgtatactccagtggtaatgaagatggaaggtttacagcacagctcaatagagccagccagtatatttccctgctcatcagagactccaagctcagtgattcagccacctacctctgtgtggtgaaccccgggtctggcaacacaggcaaactaatctttgggcaagggacaactttacaagtaaaaccagatatccagaaccctgaccctgccgtttatcagctgcgtgatagcaaaagcagcgataaaagcgtgtgcctgttcaccgattttgatagccagaccaacgtgagccagagcaaagatagcgatgtgtacatcaccgataaaaccgtgctggatatgcgcagcatggatttcaaaagcaatagcgcggttgcgtggagcaacaaaagcgattttgcgtgcgcgaacgcgtttaacaacagcatcatcccggaagatacgttcttctgcagcccagaaagttcc。
SEQ ID NO. 28 (amino acid sequence of the β chain of soluble TCR):
GAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASSLVFGSVWDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSECEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD。
SEQ ID NO. 29 (nucleotide sequence of the β chain of soluble TCR):
ggcgcgggcgtgagccagtcccccagtaacaaggtcacagagaagggaaaggatgtagagctcaggtgtgatccaatttcaggtcatactgccctttactggtaccgacagaggctggggcagggcctggagtttttaatttacttccaaggcaacagtgcaccagacaaatcagggctgcccagtgatcgcttctctgcagagaggactggggaatccgtctccactctgacgatccagcgcacacagcaggaggactcggccgtgtatctctgtgccagcagcttagtattcggtagcgtgtgggatacgcagtattttggcccaggcacccggctgacagtgctcgaggacctgaaaaacgtgttcccacccgaggtcgctgtgtttgagccatcagaatgcgaaattagccatacccagaaagcgaccctggtttgtctggcgaccggtttttatccggatcatgtggaactgtcttggtgggtgaacggcaaagaagtgcatagcggtgtttctaccgatccgcagccgctgaaagaacagccggcgctgaatgatagccgttatgcgctgtctagccgtctgcgtgttagcgcgaccttttggcaaaatccgcgtaaccattttcgttgccaggtgcagttttatggcctgagcgaaaacgatgaatggacccaggatcgtgcgaagccggttacccagattgttagcgcggaagcctggggccgcgcagat。
SEQ ID NO. 30 (amino acid sequence of single chain TCR molecule):
RKEVEQDPGPLNVPEGETVAINCTYSNSASQSFFWYRQDPGKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDVKPSDSATYFCVVNPGSGNTGKLIFGQGTTLQVKPGGGSEGGGSEGGGSEGGGSEGGTGGAGVSQSPSNLSVEKGKDVELRCDPISGHTALYWYRQRPGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRVQPEDSAVYFCASSLVFGSVWDTQYFGPGTRLTVD。
SEQ ID NO. 31 (nucleotide sequence of single chain TCR molecule):
cgtaaagaagttgaacaggaccctggcccgctgaatgttccggaaggtgaaaccgttgcaattaattgtacctatagcaatagcgcaagtcagagcttcttttggtatcgccaggaccctggtaaagaaccgaaactgctgatgagtgtgtatagtagcggcaatgaagatggtcgctttaccgcacagctgaatcgcgcaagccagtatattagcctgctgattcgcgatgtgaaaccgagtgatagtgccacctatttctgtgttgttaatcctggtagtggcaataccggtaaactgatcttcggtcagggtaccactctgcaggttaaaccgggtggcggcagtgaaggtggcggtagtgaaggtggt ggtagtgaaggcggtggcagcgaaggtggcaccggcggtgcaggtgttagtcagagcccgagtaatctgagcgtggaaaagggcaaagatgttgaactgcgttgcgatccgattagtggccataccgcactgtattggtatcgtcagcgtccgggtcagggcctggaatttctgatctattttcagggcaatagcgccccggataaaagcggcctgccgagcgatcgttttagcgccgaacgcaccggtgaaagcgttagtaccctgaccattcagcgtgttcagccggaagatagtgccgtgtatttctgtgcaagtagtctggtgtttggcagtgtgtgggatacccagtattttggtcctggtactcgtctgaccgttgat。
SEQ ID NO. 32 (amino acid sequence of the alpha chain variable domain of a single chain TCR molecule):
RKEVEQDPGPLNVPEGETVAINCTYSNSASQSFFWYRQDPGKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDVKPSDSATYFCVVNPGSGNTGKLIFGQGTTLQVKP。
SEQ ID NO. 33 (nucleotide sequence of the alpha chain variable domain of a single chain TCR molecule):
cgtaaagaagttgaacaggaccctggcccgctgaatgttccggaaggtgaaaccgttgcaattaattgtacctatagcaatagcgcaagtcagagcttcttttggtatcgccaggaccctggtaaagaaccgaaactgctgatgagtgtgtatagtagcggcaatgaagatggtcgctttaccgcacagctgaatcgcgcaagccagtatattagcctgctgattcgcgatgtgaaaccgagtgatagtgccacctatttctgtgttgttaatcctggtagtggcaataccggtaaactgatcttcggtcagggtaccactctgcaggttaaaccg。
SEQ ID NO 34 (amino acid sequence of the β chain variable domain of a single chain TCR molecule):
GAGVSQSPSNLSVEKGKDVELRCDPISGHTALYWYRQRPGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRVQPEDSAVYFCASSLVFGSVWDTQYFGPGTRLTVD。
SEQ ID NO. 35 (nucleotide sequence of the β chain variable domain of a single chain TCR molecule):
ggtgcaggtgttagtcagagcccgagtaatctgagcgtggaaaagggcaaagatgttgaactgcgttgcgatccgattagtggccataccgcactgtattggtatcgtcagcgtccgggtcagggcctggaatttctgatctattttcagggcaatagcgccccggataaaagcggcctgccgagcgatcgttttagcgccgaacgcaccggtgaaagcgttagtaccctgaccattcagcgtgttcagccggaagatagtgccgtgtatttctgtgcaagtagtctggtgtttggcagtgtgtgggatacccagtattttggtcctggtactcgtctgaccgttgat。
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. 1. 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, the plates were dried for 1h, and spots formed on the membrane were counted using an immunoblotter plate reader (ELISPOT READER system; AID company). As shown in FIG. 2, 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. cDNA synthesis uses a clontech SMART RACE cDNA amplification kit, and the primers usedThe design is in the C-terminal conservation region of 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.
Sequencing, the biscationic clone expressed TCR:
the TCR alpha chain variable domain amino acid sequence is shown as SEQ ID NO. 1, the TCR alpha chain variable domain nucleotide sequence is shown as SEQ ID NO. 2, the TCR alpha chain amino acid sequence is shown as SEQ ID NO. 3, the TCR alpha chain nucleotide sequence is shown as SEQ ID NO. 4, the TCR alpha chain amino acid sequence with the leader sequence is shown as SEQ ID NO. 22, and the TCR alpha chain nucleotide sequence with the leader sequence is shown as SEQ ID NO. 23.
The TCR beta chain variable domain amino acid sequence is shown as SEQ ID NO. 5, the TCR beta chain variable domain nucleotide sequence is shown as SEQ ID NO. 6, the TCR beta chain amino acid sequence is shown as SEQ ID NO. 7, the TCR beta chain nucleotide sequence is shown as SEQ ID NO. 8, the TCR beta chain amino acid sequence with the leader sequence is shown as SEQ ID NO. 24, and the TCR beta chain nucleotide sequence with the leader sequence is shown as SEQ ID NO. 25.
The alpha chain was identified to comprise CDRs with the following amino acid sequences:
αCDR1-NSASQS (SEQ ID NO:10)
αCDR2-VYSSGN (SEQ ID NO:11)
αCDR3-VVNPGSGNTGKLI (SEQ ID NO:12)
the β chain comprises CDRs having the following amino acid sequences:
βCDR1-SGHTA (SEQ ID NO:13)
βCDR2-FQGNSA (SEQ ID NO:14)
βCDR3-ASSLVFGSVWDTQY (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 molecule of this example may each comprise only its variable domain and a portion of its constant domain, and a cysteine residue is introduced into each of the constant domains of the α and β chains to form an artificial interchain disulfide bond, the α chain having the amino acid sequence shown in SEQ ID NO:26, the α chain having the nucleotide sequence shown in SEQ ID NO:27, the β chain having the amino acid sequence shown in SEQ ID NO:28, and the β chain having the nucleotide sequence shown in SEQ ID NO: 29. 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), respectively, and cloning sites upstream and downstream were Nco I and Not I, 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 dissolved in a solution containing 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 a final concentration of 60mg/mL in a solution containing 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine,6.6mM. Beta. -mercaptoethylamine (4 ℃). 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. The SDS-PAGE gel of the soluble TCR obtained by the invention is shown in fig. 3a and 3b, wherein the right lanes of fig. 3a and 3b are respectively a reducing gel and a non-reducing gel, and the left lanes are molecular weight markers.
EXAMPLE 4 production of AFP antigen short peptide specific soluble Single chain TCR
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 of the single-chain TCR molecule is shown as SEQ ID NO. 30, the nucleotide sequence of the single-chain TCR molecule is shown as SEQ ID NO. 31, and the amino acid sequence and the nucleotide sequence of the linker are underlined. The amino acid sequence of the alpha chain variable domain is shown as SEQ ID NO. 32, the nucleotide sequence of the alpha chain variable domain is shown as SEQ ID NO. 33, the amino acid sequence of the beta chain variable domain is shown as SEQ ID NO. 34, and the nucleotide sequence of the beta chain variable domain is shown as SEQ ID NO. 35.
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 containing the recombinant plasmid pET28 a-template strand prepared in example 4 were all inoculated into LB medium containing kanamycin, and cultured at 37℃to OD 600 IPTG was added to a final concentration of 0.5mM at 0.6-0.8 and incubation was continued for 4h at 37 ℃. 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,50mM 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 17h, the dialysate was changed to 1L of pre-chilled buffer (20 mM Tris-HCl pH 8.0), dialysis was continued for 8h at 4℃and then the dialysate was changed to the same fresh buffer and dialysis continued overnight. After 17h, 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 pH 8.0, 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 7.8X100 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 the obtained soluble single chain TCR is shown in FIG. 4, wherein the left lane is a non-reducing gel, the right lane is a reducing gel, and the middle lane is a molecular weight marker.
Example 6 characterization in combination
This example demonstrates by BIAcore analysis that soluble TCR molecules 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 an anti-streptavidin antibody (GenScript) to a coupling buffer (10 mM sodium acetate buffer, pH 4.77), then flowing the antibody through a CM5 chip previously activated with EDC and NHS to immobilize the antibody on the chip surface, and finally blocking the unreacted activated surface with an ethanolamine-HCl solution at a coupling level of about 15000RU.
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 0.05mM 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 of E.coli bacterial liquid for inducing expression of heavy chains or light chains, centrifuging at 8000g at 4 ℃ for 10min, washing the bacterial body once by using 10mL of PBS, then severely shaking and re-suspending the bacterial body by using 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 of BugBuster diluted 10 times, mixing well, and centrifuging at 4 ℃ for 15min at 6000 g; the supernatant was discarded, the inclusion bodies were resuspended by adding 30mL of 10-fold diluted BugBuster, mixing, centrifuging at 4℃for 15min, repeating twice, adding 30mL of 20mM Tris-HCl pH 8.0, mixing, centrifuging at 4℃for 15min, finally dissolving the inclusion bodies with 20mM Tris-HCl 8M urea, detecting the purity of the inclusion bodies by SDS-PAGE, and measuring the 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 a solution containing 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, hiPrepTM16/60s200 HR column (GE general electric company) was pre-equilibrated with filtered PBS using Akta purifier (GE general electric company), 1mL of concentrated biotinylated pMHC molecules were loaded, and then 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).
The kinetic parameters were calculated by BIAcore Evaluation software, and the kinetic profiles of the soluble TCR molecule of the invention and the binding of the soluble single-chain TCR molecule to the TSSELMAITR-HLA A1101 complex are shown in FIG. 5 and FIG. 6, respectively, wherein FIG. 5 is a BIAcore kinetic profile of the binding of the soluble TCR of the invention to the TSSELMAITR-HLA A1101 complex, and FIG. 6 is a BIAcore kinetic profile of the binding of the soluble single-chain TCR of the invention to the TSSELMAITR-HLA A1101 complex. 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 example are 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 cell used was T2-A11 loaded with AFP antigen short peptide TSSELMAITR.
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 be 10 in turn -6 M to 10 -11 A total of 6 gradient concentrations. Incubation 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. 7, T2-A11 loaded with TSSELMAITR short peptide had a significant activating effect on T cells transfected with TCR of the invention, while T cells transfected with other TCR remained inactive at the highest peptide concentration.
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.
The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.
Claims (10)
1. A T cell receptor comprising a TCR a chain variable domain and a TCR β chain variable domain, the T cell receptor being capable of binding to the TSSELMAITR-HLA a1101 complex, and the 3 complementarity determining regions of the TCR a chain variable domain being:
αCDR1-NSASQS (SEQ ID NO:10)
αCDR2-VYSSGN (SEQ ID NO:11)
alpha CDR3-VVNPGSGNTGKLI (SEQ ID NO: 12); and/or
The 3 complementarity determining regions of the TCR β chain variable domain are:
βCDR1-SGHTA (SEQ ID NO:13)
βCDR2-FQGNSA (SEQ ID NO:14)
βCDR3-ASSLVFGSVWDTQY (SEQ ID NO:15)。
2. the T cell receptor of claim 1, wherein the T cell receptor comprises a TCR a chain variable domain and a TCR β chain variable domain, the TCR a chain variable domain being an amino acid sequence having at least 90% sequence identity to SEQ ID No. 1;
and/or the TCR β chain variable domain is an amino acid sequence having at least 90% sequence identity to SEQ ID No. 5.
3. The T cell receptor of claim 1, wherein the C-or N-terminus of the α -chain and/or β -chain of the T cell receptor is conjugated to a conjugate;
preferably, the conjugate that binds to the T cell receptor comprises 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 T cell receptor complex comprising at least two T cell receptor molecules, wherein at least one of the T cell receptor molecules is the T cell receptor of any one of claims 1-3.
5. A nucleic acid molecule comprising a nucleotide sequence encoding the T cell receptor of any one of claims 1-3, and/or a complement of the T cell receptor nucleotide sequence of any one of claims 1-3;
preferably, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO. 2 or SEQ ID NO. 33 encoding the TCR alpha chain variable domain and/or the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO. 6 or SEQ ID NO. 35 encoding the TCR beta chain variable domain.
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 having incorporated into its chromosome an exogenous nucleic acid molecule of claim 5.
8. A cell transduced with 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 any one or a combination of at least two of the T cell receptor of any one of claims 1-3, the multivalent T cell receptor complex of claim 4, the nucleic acid molecule of claim 5, or the cell of claim 8;
Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
10. Use of the T cell receptor of any one of claims 1-3, the multivalent T cell receptor complex of claim 4, or the cell of claim 8, or a combination of at least two, wherein the use comprises for the manufacture of a medicament for the treatment of a tumor or an autoimmune disease.
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