CN115490767A - TCR for identifying AFP antigen and coding sequence thereof - Google Patents

TCR for identifying AFP antigen and coding sequence thereof Download PDF

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CN115490767A
CN115490767A CN202110678571.0A CN202110678571A CN115490767A CN 115490767 A CN115490767 A CN 115490767A CN 202110678571 A CN202110678571 A CN 202110678571A CN 115490767 A CN115490767 A CN 115490767A
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tcr
ser
seq
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杨东雪
钟时
廖有平
郑文静
温桥生
马瑞娟
孙含丽
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Xiangxue Life Science Technology Guangdong Co ltd
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Xiangxue Life Science Technology Guangdong Co ltd
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Priority to PCT/CN2022/099352 priority patent/WO2022262835A1/en
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Abstract

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

Description

TCR for identifying AFP antigen and coding sequence thereof
Technical Field
The present invention relates to a TCR capable of recognizing a short peptide derived from an AFP antigen and the coding sequence thereof, to AFP-specific T cells obtained by transduction of the above TCR, and to their use in the prevention and treatment of AFP-related diseases.
Background
AFP (alpha Fetoprotein), also called alpha Fetoprotein, is a protein expressed during embryonic development and is the main component of embryonic serum. During development, AFP is expressed at relatively high levels in the yolk sac and liver, and is subsequently inhibited. In hepatocellular carcinoma, AFP expression is activated (Butterfield et al.J. Immunol.,2001, apr15 166 (8): 5300-8). AFP is degraded into small molecule polypeptides after intracellular production and binds to MHC (major histocompatibility complex) molecules to form complexes, which are presented on the cell surface. TSSELMAITR (SEQ ID NO: 9) is a short peptide derived from the AFP antigen and is a target for the treatment of AFP-related diseases.
T cell adoptive immunotherapy is the transfer of reactive T cells specific for a target cell antigen into a patient to act on the target cell. The T Cell Receptor (TCR) is a membrane protein on the surface of T cells that recognizes a corresponding short peptide antigen on the surface of a target cell. In the immune system, the direct physical contact between T cells and Antigen Presenting Cells (APC) is initiated by the binding of antigen short peptide specific TCR and short peptide-major histocompatibility complex (pMHC complex), and then other cell membrane surface molecules of the T cells and APC interact to cause a series of subsequent cell signaling and other physiological reactions, so that T cells with different antigen specificities exert immune effects on their target cells. Therefore, those skilled in the art have focused on the isolation of TCRs specific for short AFP antigen peptides and the transduction of the TCRs into T cells to obtain T cells specific for short AFP antigen peptides, thereby enabling them to function in cellular immunotherapy.
Disclosure of Invention
The invention aims to provide a T cell receptor for recognizing AFP antigen short peptide.
In a first aspect of the invention, there is provided a T Cell Receptor (TCR) capable of binding to the TSSELMAITR-HLA a1101 complex.
In another preferred embodiment, the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, the amino acid sequence of CDR3 of the TCR alpha chain variable domain is SGGSNYKLT (SEQ ID NO: 12); and/or the amino acid sequence of CDR3 of the variable domain of the TCR β chain is ASSPGTGVGYT (SEQ ID NO: 15).
In another preferred embodiment, the 3 Complementarity Determining Regions (CDRs) of the TCR α chain variable domain are:
αCDR1-DSVNN (SEQ ID NO:10)
αCDR2-IPSGT (SEQ ID NO:11)
α CDR3-SGGSNYKLT (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-ASSPGTGVGYT (SEQ ID NO:15)。
in another preferred embodiment, the TCR comprises a TCR alpha chain variable domain which is an amino acid sequence having at least 90% sequence identity to SEQ ID No. 1, and a TCR beta chain variable domain; and/or the TCR β chain variable domain is an amino acid sequence having at least 90% 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 beta chain variable domain amino acid sequence SEQ ID NO 5.
In another preferred embodiment, the TCR is an α β heterodimer comprising a TCR α chain constant region TRAC 01 and a TCR β chain constant region TRBC 101 or TRBC2 01.
In another preferred embodiment, the α chain amino acid sequence of the TCR is SEQ ID NO 3 and/or the β chain amino acid sequence of the TCR is SEQ ID NO 7.
In another preferred embodiment, the TCR of the invention is of human origin.
In another preferred embodiment, the TCR is soluble.
In another preferred embodiment, the TCR is single chain.
In another preferred embodiment, the TCR is formed by linking an α chain variable domain to a β chain variable domain via a peptide linker.
In another preferred embodiment, the constant regions of the α and β chains of the TCR are murine constant regions of the α and β chains, respectively.
In another preferred embodiment, the TCR has one or more mutations in amino acid 11, 13, 19, 21, 53, 76, 89, 91, or 94 of the α chain variable region, and/or in the penultimate 3-, 5-, or 7-position of the short peptide amino acid of the α chain J gene; and/or the TCR has one or more mutations in beta chain variable region amino acid 11, 13, 19, 21, 53, 76, 89, 91, or 94 th, and/or beta chain J gene short peptide amino acid penultimate 2, 4 or 6 th, wherein the amino acid position numbering is according to the position numbering listed in IMGT (international immunogenetic information system).
In another preferred embodiment, the α chain variable domain amino acid sequence of the TCR comprises SEQ ID NO 32 and/or the β chain variable domain amino acid sequence of the TCR comprises SEQ ID NO 34.
In another preferred embodiment, the amino acid sequence of the TCR is SEQ ID NO 30.
In another preferred embodiment, the TCR comprises (i) a TCR α chain variable domain and all or part of a TCR α chain constant region other than the transmembrane domain; and (ii) a TCR β chain variable domain and all or part of a TCR β chain constant region other than the transmembrane domain.
In another preferred embodiment, cysteine residues form an artificial disulfide bond between the alpha and beta chain constant domains of the TCR.
In another preferred embodiment, the cysteine residues forming the artificial disulfide bond in the TCR are substituted at one or more groups of sites selected from the group consisting of:
thr48 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser57 of TRBC2 × 01 exon 1;
thr45 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser77 of TRBC2 × 01 exon 1;
tyr10 of TRAC × 01 exon 1 and TRBC1 × 01 or TRBC2 × 01 exon 1 Ser17;
thr45 of TRAC × 01 exon 1 and TRBC1 × 01 or Asp59 of TRBC2 × 01 exon 1;
ser15 of TRAC × 01 exon 1 and TRBC1 × 01 or TRBC2 × 01 exon 1 Glu15;
arg53 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser54 of TRBC2 × 01 exon 1;
pro89 of exon 1 TRAC × 01 and TRBC1 × 01 or TRBC2 × 01 of Ala19 of exon 1; and Tyr10 of TRAC × 01 exon 1 and TRBC1 × 01 or TRBC2 × 01 exon 1 Glu20.
In another preferred embodiment, the α chain amino acid sequence of the TCR is SEQ ID NO 26 and/or the β chain amino acid sequence of the TCR is SEQ ID NO 28.
In another preferred embodiment, the TCR comprises an artificial interchain disulfide bond between the α chain variable region and the β chain constant region.
In another preferred embodiment, the cysteine residues that form the artificial interchain disulfide bond in the TCR are substituted at one or more groups of sites selected from:
amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01;
amino acid 47 of TRAV and equal amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01;
amino acid 46 of TRAV and amino acid 61 of TRBC1 x 01 or TRBC2 x 01 exon 1; or
Amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01.
In another preferred embodiment, the TCR comprises an alpha chain variable domain and a beta chain variable domain and all or part of the beta chain constant domain, excluding the transmembrane domain, but which does not comprise an alpha chain constant domain, the alpha chain variable domain of the TCR forming a heterodimer with the beta chain.
In another preferred embodiment, the TCR has a conjugate attached to the C-or N-terminus of the alpha and/or beta chain.
In another preferred embodiment, the conjugate that binds to the T cell receptor is a detectable label, a therapeutic agent, a PK modifying moiety or a combination of any of these. Preferably, the therapeutic agent is an anti-CD 3 antibody.
In a second aspect of the invention, there is provided a multivalent TCR complex comprising at least two TCR molecules, and wherein at least one of the TCR molecules is a TCR according to the first aspect of the invention.
In a third aspect of the invention, there is provided a nucleic acid molecule comprising a nucleic acid sequence encoding a TCR molecule according to the first aspect of the invention, or a complement thereof.
In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO 2 or SEQ ID NO 33 encoding the variable domain of the TCR alpha chain.
In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO 6 or SEQ ID NO 35 encoding the variable domain of the TCR β chain.
In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO. 4 encoding the TCR alpha chain and/or comprises the nucleotide sequence SEQ ID NO. 8 encoding the 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 a genome into which has been integrated an exogenous nucleic acid molecule according to the third aspect of the invention.
In a sixth aspect of the invention, there is provided a cell 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 according to the first aspect of the invention, a TCR complex according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a cell according to the sixth aspect of the invention.
In an eighth aspect of the invention, there is provided a use of the T cell receptor of the first aspect of the invention, or the TCR complex of the second aspect of the invention, or the cell of the sixth aspect of the invention, for the manufacture of a medicament for the treatment of a tumour or an autoimmune disease, preferably the tumour is liver cancer.
In a ninth aspect, the invention provides 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 for the treatment of a tumour or an autoimmune disease; preferably, the tumor is liver cancer.
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 liver cancer.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1a, FIG. 1b, FIG. 1c, FIG. 1d, FIG. 1e and FIG. 1f are the amino acid sequence of the TCR α chain variable domain, the nucleotide sequence of the TCR α chain variable domain, the amino acid sequence of the TCR α chain, the nucleotide sequence of the TCR α chain, the amino acid sequence of the TCR α chain with leader sequence and the nucleotide sequence of the TCR α chain with leader sequence, respectively.
Fig. 2a, fig. 2b, fig. 2c, fig. 2d, fig. 2e and fig. 2f are a TCR β chain variable domain amino acid sequence, a TCR β chain variable domain nucleotide sequence, a TCR β chain amino acid sequence, a TCR β chain nucleotide sequence, a TCR β chain amino acid sequence with a leader sequence and a TCR β chain nucleotide sequence with a leader sequence, respectively.
FIG. 3 shows the double positive staining results of monoclonal cells for CD8+ -APC and tetramer-PE.
Fig. 4a and 4b are the amino acid and nucleotide sequences, respectively, of a soluble TCR α chain.
Fig. 5a and 5b are the amino acid and nucleotide sequences, respectively, of a soluble TCR β chain.
Figures 6a and 6b are gel plots of the soluble TCR obtained after purification. In FIGS. 6a and 6b, the lanes on the right side are reducing gel and non-reducing gel, respectively, and the lanes on the left side are molecular weight markers (marker).
FIGS. 7a and 7b show the amino acid and nucleotide sequences, respectively, of a single-chain TCR, with the amino and nucleotide sequences of the linker sequence underlined.
FIGS. 8a and 8b are the amino acid and nucleotide sequences, respectively, of the variable domain of the single chain TCR α chain.
FIGS. 9a and 9b are the amino acid and nucleotide sequences, respectively, of the variable domain of the single-chain TCR β chain.
FIGS. 10a and 10b are gel images of the soluble single chain TCR obtained after purification. In FIGS. 10a and 10b, the lanes on the right hand side are reducing and non-reducing gels, respectively, and the lanes on the left hand side are both molecular weight markers (marker).
FIG. 11 is a BIAcore kinetic profile of binding of soluble TCRs of the invention to the TSSELMAITR-HLA A1101 complex.
FIG. 12 is a BIAcore kinetic profile of binding of soluble single-chain TCRs of the invention to the TSSELMAITR-HLA A1101 complex.
FIG. 13 shows the results of functional verification of the ELISPOT activation of the resulting T cell clones.
Figure 14 is a validation of ELISPOT activation function of effector cells transfected with a TCR of the invention against T2 cells.
FIG. 15 is a graphical representation of the results of functional verification of ELISPOT activation of effector cells transfected with TCRs of the invention against tumor cell lines.
Fig. 16 is a result of verifying the killing function of effector cells transfected with the TCR of the present invention.
Detailed Description
The present inventors have extensively and intensively studied to find a TCR capable of specifically binding to an AFP antigen short peptide TSSELMAITR (SEQ ID NO: 9) which 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 that transduce a TCR of the invention.
Term(s) for
MHC molecules are proteins of the immunoglobulin superfamily and may be MHC class I or class II molecules. Therefore, it is specific for antigen presentation, has different MHC among different individuals, and can present different short peptides of one protein antigen onto the cell surface of respective APC. Human MHC is often referred to as HLA gene or HLA complex.
The T Cell Receptor (TCR), is the only receptor for a specific antigenic peptide presented on the Major Histocompatibility Complex (MHC). In the immune system, direct physical contact between T cells and Antigen Presenting Cells (APCs) is initiated by binding of antigen-specific TCRs to pMHC complexes, and then other cell membrane surface molecules of both T cells and APCs interact, which leads to a series of subsequent cell signaling and other physiological responses, thereby allowing T cells of different antigen specificities to exert an immune effect on their target cells.
TCRs are cell membrane surface glycoproteins that exist as heterodimers from either the α chain/β chain or the γ chain/δ chain. In 95% of T cells the TCR heterodimer consists of α and β chains, while 5% of T cells have TCRs consisting of γ and δ chains. Native α β heterodimeric TCRs have an α chain and a β chain, which constitute subunits of an α β heterodimeric TCR. Broadly, each of the α and β chains comprises a variable region, a linker region and a constant region, and the β chain also typically contains a short diversity region between the variable region and the linker region, but the diversity region is often considered to be part of the linker region. Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, which are chimeric in framework structures (framework regions). The CDR regions determine the binding of the TCR to the pMHC complex, where CDR3 is recombined from variable and linking regions, referred to as hypervariable regions. The α and β chains of a TCR are generally regarded as having two "domains" each, namely a variable domain and a constant domain, the variable domain being made up of linked variable regions and linking regions. The sequences of TCR constant domains can be found in public databases of the international immunogenetic information system (IMGT), e.g. the constant domain sequence of the α chain of the TCR molecule is "TRAC 01", the constant domain sequence of the β chain of the TCR molecule is "TRBC1 01" or "TRBC2 01". In addition, the α and β chains of the TCR also comprise a transmembrane region and a cytoplasmic region, the cytoplasmic region being very short.
In the present invention, the terms "polypeptide of the invention", "TCR of the invention", "T cell receptor of the invention" are used interchangeably.
Natural interchain disulfide bond and artificial interchain disulfide bond
A set of disulfide bonds, referred to herein as "native interchain disulfide bonds", exist between the C α and C β chains in the membrane proximal region of native TCRs. In the present invention, the artificially introduced interchain covalent disulfide bond, which is located differently from the natural interchain disulfide bond, is referred to as an "artificial interchain disulfide bond".
For convenience of description of the positions of disulfide bonds, the positions of TRAC 01 and TRBC1 × 01 or TRBC2 × 01 amino acid sequences are numbered in order from N-terminus to C-terminus, for example, in TRBC1 × 01 or TRBC2 × 01, the 60 th amino acid in order from N-terminus to C-terminus is P (proline), and thus, in the present invention, it may be described as TRBC1 × 01 or TRBC2 × 01 exon 1 Pro60, and also as TRBC1 × 01 or TRBC2 × 01 exon 1 60, for example, in TRBC1 × 01 or TRBC2 × 01, and the 61 st amino acid in order from N-terminus to C-terminus is Q (glutamine), and thus, in the present invention, it may be described as TRBC1 × 01 or TRBC2 × 01 exon 1 Gln61, and also as TRBC1 or TRBC2 × 01, and so on, and further, it may be described as TRBC1 × 01 or TRBC2 × 01. In the present invention, the position numbering of the amino acid sequences of the variable regions TRAV and TRBV follows the position numbering listed in IMGT. If a certain amino acid in TRAV, the position number listed in IMGT is 46, 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.
Detailed Description
TCR molecules
During antigen processing, antigens are degraded intracellularly and then carried to the cell surface through MHC molecules. T cell receptors are capable of recognizing peptide-MHC complexes on the surface of antigen presenting cells. Accordingly, 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 amino acid sequence:
αCDR1-DSVNN (SEQ ID NO:10)
αCDR2-IPSGT (SEQ ID NO:11)
α CDR3-SGGSNYKLT (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-ASSPGTGVGYT (SEQ ID NO:15)。
chimeric TCRs can be prepared by embedding the above-described amino acid sequences of the CDR regions of the invention into any suitable framework. One skilled in the art can design or synthesize a TCR molecule with the corresponding function based on the CDR regions disclosed herein, so long as the framework structure is compatible with the CDR regions of the TCR of the invention. Thus, the TCR molecules of the invention are meant to be TCR molecules comprising the above-described alpha and/or beta chain CDR region sequences and any suitable framework structure. The TCR α chain variable domain of the invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity to SEQ ID No. 1; and/or the TCR β chain variable domain of the invention is 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 α and β chains. In particular, in one aspect the α chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, the α chain variable domain amino acid sequence comprising CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the above-described α chain. Preferably, the TCR molecule comprises an alpha chain variable domain amino acid sequence SEQ ID NO 1. More preferably, the amino acid sequence of the α chain variable domain of the TCR molecule is SEQ ID NO 1. In another aspect, the β chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the β chain variable domain amino acid sequence comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 14), and CDR3 (SEQ ID NO: 15) of the above-described β chain. Preferably, the TCR molecule comprises the beta chain variable domain amino acid sequence SEQ ID NO 5. More preferably, the amino acid sequence of the variable domain of the β chain of the TCR molecule is SEQ ID No. 5.
In a preferred embodiment of the invention, the TCR molecules of the invention are single chain TCR molecules consisting of part or all of the α chain and/or part or all of the β chain. Single chain TCR molecules are described in Chung et al (1994) Proc. Natl. Acad. Sci. USA 91,12654-12658. From the literature, those skilled in the art are readily able to construct single chain TCR molecules comprising the CDRs regions of the invention. In particular, the single chain TCR molecule comprises V α, V β and C β, preferably linked in order from N-terminus to C-terminus.
The alpha chain variable domain amino acid sequence of the single chain TCR molecule comprises CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the alpha chain described above. Preferably, the single chain TCR molecule comprises the alpha chain variable domain amino acid sequence SEQ ID NO 1. More preferably, the α chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO 1. The amino acid sequence of the variable domain of the beta chain of the single-chain TCR molecule comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 14) and CDR3 (SEQ ID NO: 15) of the beta chain. Preferably, the single chain TCR molecule comprises the beta chain variable domain amino acid sequence SEQ ID NO 5. More preferably, the amino acid sequence of the variable domain of the β chain of the single chain TCR molecule is SEQ ID No. 5.
In a preferred embodiment of the invention, the constant domain of the TCR molecules of the invention is a human constant domain. The person skilled in the art knows or can obtain the human constant domain amino acid sequence by consulting public databases of relevant books or IMGT (international immunogenetic information system). For example, the constant domain sequence of the α chain of the TCR molecules of the invention can be "TRAC 01", and the constant domain sequence of the β chain of the TCR molecules can be "TRBC1 01" or "TRBC2 01". Arg at position 53 of the amino acid sequence given in TRAC 01 of IMGT, here denoted: TRAC × 01 Arg53 of exon 1, and so on. Preferably, the amino acid sequence of the α chain of the TCR molecules of the invention is SEQ ID No. 3, and/or the amino acid sequence of the β chain is SEQ ID No. 7.
Naturally occurring TCRs are membrane proteins that are stabilized by their transmembrane regions. Like immunoglobulins (antibodies) as antigen recognition molecules, TCRs can also be developed for diagnostic and therapeutic applications, where soluble TCR molecules are required. Soluble TCR molecules do not include their transmembrane regions. Soluble TCRs have a wide range of uses, not only for studying the interaction of TCRs with pmhcs, but also as diagnostic tools for detecting infections or as markers for autoimmune diseases. Similarly, soluble TCRs can be used to deliver therapeutic agents (e.g., cytotoxic or immunostimulatory compounds) to cells presenting a specific antigen, and in addition, soluble TCRs can be conjugated to other molecules (e.g., anti-CD 3 antibodies) to redirect T cells to target them to cells presenting a particular antigen. The present invention also provides soluble TCRs with specificity for AFP antigen short peptides.
To obtain a soluble TCR, in one aspect, the inventive TCR may be one in which an artificial disulfide bond is introduced between residues of the constant domains of its alpha and beta chains. Cysteine residues form an artificial interchain disulfide bond between the alpha and beta chain constant domains of the TCR. Cysteine residues may be substituted for other amino acid residues at appropriate positions in native TCRs to form artificial interchain disulfide bonds. For example, a disulfide bond is formed by substituting Thr48 of exon 1 of TRAC × 01 and a cysteine residue of Ser57 of exon 1 of TRBC1 × 01 or TRBC2 × 01. Other sites for introducing cysteine residues to form disulfide bonds may also be: thr45 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser77 of TRBC2 × 01 exon 1; tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC2 x 01; thr45 of TRAC × 01 exon 1 and TRBC1 × 01 or Asp59 of TRBC2 × 01 exon 1; ser15 of TRAC × 01 exon 1 and TRBC1 × 01 or TRBC2 × 01 exon 1 Glu15; arg53 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser54 of TRBC2 × 01 exon 1; pro89 of exon 1 TRAC × 01 and TRBC1 × 01 or TRBC2 × 01 of Ala19 of exon 1; or Tyr10 of exon 1 of TRAC × 01 and TRBC1 × 01 or TRBC2 × 01, glu20 of exon 1. I.e., a cysteine residue, in place of any of the above-described alpha and beta chain constant domains. The TCR constant domains of the invention may be truncated at one or more of their C-termini by up to 50, or up to 30, or up to 15, or up to 10, or up to 8 or fewer amino acids, so as not to include a cysteine residue for the purpose of deleting the native disulphide bond, or by mutating the cysteine residue forming the native disulphide bond to another amino acid.
As described above, the TCRs of the invention may comprise artificial disulfide bonds introduced between residues of the constant domains of their alpha and beta chains. It should be noted that the TCRs of the invention may each contain both TRAC constant domain sequences and TRBC1 or TRBC2 constant domain sequences, with or without the artificial disulfide bonds introduced as described above between the constant domains. The TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the TCR may be linked by the native disulfide bond present in the TCR.
To obtain a soluble TCR, the inventive TCR may, on the other hand, also comprise a TCR having a mutation in its hydrophobic core region, preferably a mutation that results in an improved stability of the inventive soluble TCR, as described in the patent publication WO 2014/206304. Such TCRs may be mutated at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or positions 3, 5, 7 of the reciprocal amino acid position of the short peptide of the alpha chain J gene (TRAJ), and/or positions 2, 4, 6 of the reciprocal amino acid position of the short peptide of the beta chain J gene (TRBJ), wherein the position numbering of the amino acid sequence is according to the position numbering listed in the International immunogenetic information System (IMGT). The above-mentioned international system of immunogenetics information is known to the skilled person and the position numbering of the amino acid residues of the different TCRs in IMGT can be derived from this database.
The TCR with the mutated hydrophobic core region of the present invention may be a stable soluble single chain TCR consisting of a flexible peptide chain connecting the variable domains of the α and β chains of the TCR. It should be noted that the flexible peptide chain of the present invention can be any peptide chain suitable for linking the TCR α and β chain variable domains. The single-chain soluble TCR constructed as in example 4 of the invention has an alpha chain variable domain amino acid sequence of SEQ ID NO. 32 and an encoded nucleotide sequence of SEQ ID NO. 33; the amino acid sequence of the beta chain variable domain is SEQ ID NO. 34, and the coded nucleotide sequence is SEQ ID NO. 35.
In addition, for stability, patent document 201680003540.2 also discloses that 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 TCR of the invention may also comprise an artificial interchain disulfide bond between the α chain variable region and the β chain constant region. Specifically, the cysteine residues that form the artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR are substituted for: amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01; amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01; amino acid 46 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01. Preferably, such a TCR may comprise (i) all or part of a TCR α chain, excluding its transmembrane domain, and (ii) all or part of a TCR β chain, excluding its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least part of the constant domain of the TCR chain, the α chain forming a heterodimer with the β chain. More preferably, such a TCR may comprise the a chain variable domain and the β chain variable domain and all or part of the β chain constant domain, excluding the transmembrane domain, but which does not comprise the a chain constant domain, the a chain variable domain of the TCR forming a heterodimer with the β chain.
The TCRs of the invention may also be provided in the form of multivalent complexes. Multivalent TCR complexes of the invention comprise polymers formed by association of two, three, four or more TCRs of the invention, such as might be formed by tetramer formation with the tetrameric domain of p53, or complexes formed by association of a plurality of TCRs of the invention with another molecule. The TCR complexes of the invention can be used to track or target cells presenting a particular antigen in vitro or in vivo, and also to generate intermediates for other multivalent TCR complexes having such applications.
The TCRs of the invention may be used alone or in covalent or other association, preferably covalently, with a conjugate. The conjugates include a detectable label (for diagnostic purposes, where the TCR is used to detect the presence of cells presenting the TSSELMAITR-HLA a1101 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.
Therapeutic agents that may be associated or conjugated with the TCRs of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, cancer metastasis reviews (Cancer metastasis) 24, 539); 2. biotoxics (Chaudhary et al, 1989, nature 339, 394, epel et al, 2002, cancer Immunology and Immunotherapy) 51, 565); 3. cytokines such as IL-2 etc (Gillies et al, 1992, journal of the national academy of sciences (PNAS) 89, 1428, card et al, 2004, cancer Immunology and Immunotherapy) 53, 345, hain et al, 2003, cancer Research (Cancer Research) 63, 3202); 4. antibody Fc fragment (Mosquera et al, 2005, journal Of Immunology 174, 4381); 5. antibody scFv fragments (Zhu et al, 1995, international Journal of Cancer 62, 319); 6. gold nanoparticles/nanorods (Lapotko et al, 2005, cancer communications (Cancer letters) 239, 36, huang et al, 2006, journal of the American Chemical Society 128, 2115); 7. viral particles (Peng et al, 2004, gene therapy 11, 1234); 8. liposomes (Mamot et al, 2005, cancer research 65, 11631); 9. nano magnetic particles; 10. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, and the like.
In addition, the TCR of the invention may also be a hybrid TCR comprising sequences derived from more than one species. For example, studies have shown that murine TCRs are more efficiently expressed in human T cells than human TCRs. Thus, the inventive TCR may comprise a human variable domain and a murine constant domain. The drawback of this approach is the possibility of eliciting an immune response. Thus, there should be a regulatory regimen to immunosuppresse when it is used for adoptive T cell therapy to allow for the engraftment of murine expressing T cells.
It should be understood that the amino acid names herein are expressed in terms of international single-letter or three-letter english letters, and the single-letter english letter and three-letter english letters of the amino acid names correspond to the following relationships: ala (A), arg (R), asn (N), asp (D), cys (C), gln (Q), glu (E), gly (G), his (H), ile (I), leu (L), lys (K), met (M), phe (F), pro (P), ser (S), thr (T), trp (W), tyr (Y), val (V).
Nucleic acid molecules
A second aspect of the invention provides a nucleic acid molecule encoding a TCR molecule of the first aspect of the invention or a portion thereof, which may be one or more CDRs, variable domains of the alpha and/or beta chains, and the alpha and/or beta chains.
The nucleotide sequence encoding the α chain CDR regions of the TCR molecules of the first aspect of the invention is as follows:
CDR1α-gactctgtgaacaat(SEQ ID NO:16)
CDR2α-attccctcagggaca(SEQ ID NO:17)
CDR3α-agtggaggtagcaactataaactgaca(SEQ ID NO:18)
the nucleotide sequence encoding the β chain CDR regions of the TCR molecules of the first aspect of the invention is as follows:
CDR1β-tctgaacacaaccgc(SEQ ID NO:19)
CDR2β-ttccagaatgaagctcaa(SEQ ID NO:20)
CDR3β-gccagcagccccgggacaggggttggctacacc(SEQ ID NO:21)
thus, the nucleotide sequence of the nucleic acid molecule of the invention encoding the TCR alpha chain of the invention comprises SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 18 and/or the nucleotide sequence of the nucleic acid molecule of the invention encoding the TCR beta chain of the invention comprises SEQ ID NO 19, SEQ ID NO 20 and SEQ ID NO 21.
The nucleotide sequence of the nucleic acid molecule of the invention may be single-stranded or double-stranded, the nucleic acid molecule may be RNA or DNA, and may or may not contain an intron. Preferably, the nucleotide sequence of the nucleic acid molecule of the invention does not comprise an intron but is capable of encoding a polypeptide of the invention, e.g. the nucleotide sequence of the nucleic acid molecule of the invention encoding a TCR alpha chain variable domain of the invention comprises SEQ ID NO 2 and/or the nucleotide sequence of the nucleic acid molecule of the invention encoding a TCR beta chain variable domain of the invention comprises SEQ ID NO 6. Alternatively, the nucleotide sequence of a nucleic acid molecule of the invention encoding a TCR α chain variable domain of the invention comprises SEQ ID NO 33 and/or the nucleotide sequence of a nucleic acid molecule of the invention encoding a TCR β chain variable domain of the invention comprises SEQ ID NO 35. More preferably, the nucleotide sequence of the nucleic acid molecule of the invention comprises SEQ ID NO. 4 and/or SEQ ID NO. 8. Alternatively, the nucleotide sequence of the nucleic acid molecule of the invention is SEQ ID NO. 31.
It will be appreciated that, due to the degeneracy of the genetic code, different nucleotide sequences may encode the same polypeptide. Thus, the nucleic acid sequence encoding the TCR of the present invention may be identical to or a degenerate variant of the nucleic acid sequences shown in the figures of the present invention. As illustrated by one of the examples herein, a "degenerate variant" refers to a nucleic acid sequence that encodes a protein sequence having SEQ ID NO. 1, but differs from the sequence of SEQ ID NO. 2.
The nucleotide sequence may be codon optimized. Different cells differ in the utilization of specific codons, and the expression level can be increased by changing the codons in the sequence according to the type of the cell. Codon usage tables for mammalian cells as well as for a variety of other organisms are well known to those skilled in the art.
The full-length sequence of the nucleic acid molecule of the present invention or a fragment thereof can be obtained by, but not limited to, PCR amplification, recombination, or artificial synthesis. At present, DNA sequences encoding the TCRs of the invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or e.g., vectors) and cells known in the art. The DNA may be the coding strand or the non-coding strand.
Carrier
The invention also relates to vectors comprising the nucleic acid molecules of the invention, including expression vectors, i.e. constructs capable of expression in vivo or in vitro. Commonly used vectors include bacterial plasmids, bacteriophages and animal and plant viruses.
Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, baculovirus vectors.
Preferably, the vector can transfer the nucleotide of the invention into a cell, such as a T cell, such that the cell expresses a TCR specific for an AFP antigen. Ideally, the vector should be capable of sustained high level expression in T cells.
Cells
The invention also relates to genetically engineered host cells that have been engineered with the vectors or coding sequences of the invention. The host cell comprises the vector of the invention or has the nucleic acid molecule of the invention integrated into the chromosome. The host cell is selected from: prokaryotic and eukaryotic cells, such as E.coli, yeast cells, CHO cells, and the like.
In addition, the invention also includes isolated cells expressing the TCR of the invention, which may be, but is not limited to, T cells, NK cells, NKT cells, stem cells, particularly T cells. The T cell may be derived from a T cell isolated from a subject, or may be part of a mixed population of cells isolated from a subject, such as a population of Peripheral Blood Lymphocytes (PBLs). For example, the cells may be isolated from Peripheral Blood Mononuclear Cells (PBMCs), and may be CD4+ helper T cells or CD8+ cytotoxic T cells. The cells may be in a mixed population of CD4+ helper T cells/CD 8+ cytotoxic T cells. Generally, the cells can be activated with an antibody (e.g., an anti-CD 3 or anti-CD 28 antibody) to make them more amenable to transfection, e.g., transfection with a vector comprising a nucleotide sequence encoding a TCR molecule of the invention.
Alternatively, the cell of the invention may also be or be derived from a stem cell, such as a Hematopoietic Stem Cell (HSC). Gene transfer to HSCs does not result in TCR expression on the cell surface, since the CD3 molecule is not expressed on the stem cell surface. However, when stem cells differentiate into lymphoid precursors (lymphoid precursors) that migrate to the thymus, expression of the CD3 molecule will initiate expression of the introduced TCR molecule on the surface of the thymocytes.
There are many methods suitable for T cell transfection using DNA or RNA encoding the TCR of the invention (e.g., robbins et al, (2008) J.Immunol.180: 6116-6131). T cells expressing the inventive TCR can be used in adoptive immunotherapy. One skilled in the art will be aware of many suitable methods for adoptive therapy (e.g., rosenberg et al, (2008) Nat Rev Cancer8 (4): 299-308).
AFP antigen associated diseases
The present invention also relates to a method for the treatment and/or prevention of a disease associated with AFP in a subject, comprising the step of adoptive transfer of AFP-specific T cells to the subject. The AFP-specific T cells recognize the TSSELMAITR-HLA A1101 complex.
The AFP-specific T cells of the invention can be used for treating any AFP-related disease presenting AFP antigen short peptide TSSELMAITR-HLA A1101 complex, including but not limited to tumors such as liver cancer and the like.
Method of treatment
Treatment may be effected by isolating T cells from patients or volunteers suffering from a disease associated with the AFP antigen and introducing the TCR of the invention into such T cells, followed by reinfusion of these genetically engineered cells into the patient. Accordingly, the present invention provides a method of treating an AFP-related disorder comprising infusing into a patient an isolated T cell expressing a TCR of the invention, preferably the T cell is derived from the patient per se. Generally, this involves (1) isolating T cells from the patient, (2) transducing T cells in vitro with a nucleic acid molecule of the invention or a nucleic acid molecule capable of encoding a TCR molecule of the invention, and (3) infusing the genetically modified T cells into the patient. The number of cells isolated, transfected and transfused can be determined by a physician.
The main advantages of the invention are:
(1) The inventive TCR is capable of specifically binding to the AFP antigen short peptide complex TSSELMAITR-HLA A1101, and the effector cells transduced with the inventive TCR are capable of being specifically activated.
(2) Effector cells transduced with the inventive TCR are capable of specifically killing AFP-positive target cells.
The following specific examples further illustrate the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example as described in Sambrook and Russell et al, molecular Cloning: A Laboratory Manual (third edition) (2001) CSHL Press, or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.
Example 1 cloned AFP antigen short peptide specific T cells
Peripheral Blood Lymphocytes (PBLs) from healthy volunteers of genotype HLA-A1101 were stimulated using the synthetic short peptide TSSELMAITR (SEQ ID NO:9; kingstony Biotech, inc.). The TSSELMAITR short peptide is renatured with HLA-A1101 with a biotin marker to prepare a pMHC haploid. These haploids were combined with streptavidin labeled with PE (BD Co.) to form PE-labeled tetramers, which were sorted from anti-CD 8-APC double positive cells. The sorted cells were expanded and subjected to secondary sorting as described above, followed by single cloning by limiting dilution. Monoclonal cells were stained with tetramer and double positive clones were selected as shown in FIG. 3. The double positive clones obtained by screening layer by layer also need to meet the requirement of further function test.
IFN- γ is a potent immunomodulatory factor produced by activated T lymphocytes, and therefore this example examines the IFN- γ numbers by ELISPOT assays well known to those skilled in the art to verify the activation function and antigen specificity of cells transfected with the TCR of the invention. The function and specificity of the T cell clone were further tested by ELISPOT assay. The effector cells used in the IFN-. Gamma.ELISPOT experiment of this example were T cell clones obtained in the present invention, the target cells were T2-A11 (T2 cells transfected with HLA-A1101) loaded with TSSELMAITR short peptides, SK-MEL-28-AFP (AFP overexpression), and the control group were T2 cells loaded with other antigen short peptides and SK-MEL-28.
Firstly, preparing an ELISPOT plate, wherein the ELISPOT experiment steps are as follows: the components of the assay were added to the ELISPOT plate in the following order: 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 to give a final concentration of 10-5M, and 20. Mu.l of the medium (test medium) was added to the blank group, and 2 wells were set. Then incubated overnight (37 ℃,5% CO) 2 ). The plate was then washed and subjected to secondary detection and color development, the plate was dried for 1 hour, and spots formed on the membrane were counted using an immuno spot plate READER (ELISPOT READER system; AID Co.). As shown in FIG. 13, the obtained T cell clones showed high IFN-. Gamma.release from T2-A11 and SK-MEL-28-AFP loaded with TSSELMAITR short peptides, but did not substantially respond to T2-A11 and SK-MEL-28 loaded with other antigen short peptides.
Example 2 construction of TCR Gene and vector for obtaining AFP antigen short peptide-specific T cell clone
Using Quick-RNA TM Total RNA from the T cell clone specific to the antigen short peptide TSSELMAITR and restricted by HLA-A1101 selected in example 1 was extracted by MiniPrep (ZYMO research). The cDNA was synthesized using a SMART RACE cDNA amplification kit from clontech, using primers designed in the C-terminal conserved region of the human TCR gene. The sequences were cloned into the T vector (TAKARA) and sequenced. It should be noted that the sequence is complementary and does not include introns. The chain and chain sequence structures of the TCR expressed by the double positive clone are shown in fig. 1 and fig. 2, respectively, fig. 1a, fig. 1b, fig. 1c, fig. 1d, fig. 1e and fig. 1f are the 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 leader sequence and TCR alpha chain nucleotide sequence with leader sequence, respectively, after sequencing; FIG. 2a, FIG. 2b, FIG. 2c, FIG. 2d, FIG. 2e and FIG. 2f are the amino acid sequence of the TCR β chain variable domain and the nucleotide sequence of the TCR β chain variable domain, respectivelySequences, TCR β chain amino acid sequences, TCR β chain nucleotide sequences, TCR β chain amino acid sequences with leader sequences and TCR β chain nucleotide sequences with leader sequences.
The alpha chain was identified to comprise CDRs having the amino acid sequences:
αCDR1-DSVNN (SEQ ID NO:10)
αCDR2-IPSGT (SEQ ID NO:11)
αCDR3-SGGSNYKLT (SEQ ID NO:12)
the beta chain comprises CDRs having the following amino acid sequences:
βCDR1-SEHNR (SEQ ID NO:13)
βCDR2-FQNEAQ (SEQ ID NO:14)
βCDR3-ASSPGTGVGYT (SEQ ID NO:15)。
the full-length genes of the TCR α and β chains were cloned into the lentiviral expression vector pllenti (addendum) by overlap (overlap) PCR, respectively. The method comprises the following specific steps: the TCR alpha chain and the TCR beta chain are connected by overlap PCR to obtain the TCR alpha-2A-TCR beta segment. The lentivirus expression vector and the TCR alpha-2A-TCR beta are connected by enzyme digestion to obtain a pLenti-TRA-2A-TRB-IRES-NGFR plasmid. As a control, a lentiviral vector pLenti-eGFP expressing eGFP was also constructed. The pseudovirus was then packaged again at 293T/17.
Example 3 expression, refolding and purification of soluble TCR specific for short peptide AFP antigen
To obtain soluble TCR molecules, the α and β chains of the TCR molecules of the invention may comprise only the variable and part of the constant domains thereof, respectively, and one cysteine residue has been introduced into the constant domains of the α and β chains, respectively, to form an artificial interchain disulfide bond, the amino acid and nucleotide sequences of the α chain being as shown in figures 4a and 4b, respectively, and the amino acid and nucleotide sequences of the β chain being as shown in figures 5a and 5b, respectively. The above-mentioned desired gene sequences for the TCR alpha and beta chains were synthesized and inserted into the expression vector pET28a + (Novagene) by standard methods described in Molecular Cloning A Laboratory Manual (third edition, sambrook and Russell), and the upstream and downstream Cloning sites were NcoI and NotI, respectively. The insert was confirmed by sequencing without error.
The expression vectors of TCR alpha and beta chains are respectively transformed into expression bacteria BL21 (DE 3) by a chemical transformation method, and the bacteria grow in LB culture solution and grow on OD 600 At 0.6 induction with final concentration of 0.5mM IPTG, inclusion bodies formed after expression of the α and β chains of the TCR were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution several times, and finally dissolved in 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA), 20mM Tris (pH 8.1).
The solubilized TCR α and β chains were rapidly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine,6.6mM β -mercapoethylamine (4 ℃) at a mass ratio of 1 to a final concentration of 60mg/mL. After mixing, the solution was dialyzed against 10 times the volume of deionized water (4 ℃ C.), and after 12 hours, the deionized water was changed to a buffer (20mM Tris, pH 8.0) and dialysis was continued at 4 ℃ for 12 hours. The solution after completion of dialysis was filtered through a 0.45. Mu.M filter and then purified by an anion exchange column (HiTrap Q HP,5ml, GE Healthcare). The TCR eluted with peaks containing successfully renatured α and β dimers was confirmed by SDS-PAGE gel. The TCR was subsequently further purified by gel filtration chromatography (HiPrep 16/60, sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was greater than 90% as determined by SDS-PAGE and the concentration was determined by BCA. The SDS-PAGE gel of the soluble TCR of the invention is shown in FIGS. 6a and 6 b.
Example 4 Generation of soluble Single chain TCR specific for short peptides of AFP antigen
The variable domains of TCR α and β chains in example 2 were constructed as a stable soluble single-chain TCR molecule linked by a flexible short peptide (linker) using site-directed mutagenesis as described in WO 2014/206304. The amino acid sequence and nucleotide sequence of the single-chain TCR molecule are shown in fig. 7a and 7b, respectively, and the amino acid sequence and nucleotide sequence of the linker are 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 beta chain variable domain are shown in FIG. 9a and FIG. 9b, respectively.
The target gene was digested simultaneously with Nco I and Not I, and ligated to pET28a vector digested simultaneously with Nco I and Not I. The ligation product was transformed into e.coli DH5 α, spread on LB plates containing kanamycin, cultured at 37 ℃ for overnight inversion, positive clones were selected for PCR screening, positive recombinants were sequenced, and after the correct sequence was determined, recombinant plasmids were extracted and transformed into e.coli BL21 (DE 3) for expression.
Example 5 expression, renaturation and purification of soluble Single chain TCR specific for AFP antigen short peptides
The entire BL21 (DE 3) colony containing the recombinant plasmid pET28 a-template strand prepared in example 4 was inoculated into LB medium containing kanamycin and cultured at 37 ℃ to OD 600 At 0.6-0.8, IPTG was added to a final concentration of 0.5mM and incubation continued for 4h at 37 ℃. The cell pellet was harvested by centrifugation at 5000rpm for 15min, the 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, centrifuged at 6000rpm for 15min, and the inclusion bodies were collected. The inclusion bodies were dissolved in buffer (20 mM Tris-HCl pH 8.0,8M urea), the insoluble material was removed by high speed centrifugation, the supernatant was quantified by BCA method and split charged, and stored at-80 ℃ for further use.
To 5mg of solubilized single-chain TCR inclusion body protein, 2.5mL of buffer (6M Gua-HCl,50mM Tris-HCl pH 8.1, 100mM NaCl,10mM EDTA) was added, DTT was added to a final concentration of 10mM, and treatment was carried out at 37 ℃ for 30min. The treated single-chain TCR was added dropwise to 125mL of renaturation buffer (100 mM Tris-HCl pH 8.1,0.4M L-arginine, 5M urea, 2mM EDTA,6.5mM beta-mercaptoethylamine, 1.87mM Cystamine) with a syringe, stirred at 4 ℃ for 10min, and then the renaturation solution was filled into a cellulose membrane dialysis bag with a cut-off of 4kDa, and the dialysis bag was placed in 1L of precooled water and stirred slowly at 4 ℃ overnight. After 17 hours, the dialysate was changed to 1L of pre-chilled buffer (20 mM Tris-HCl pH 8.0), dialysis was continued at 4 ℃ for 8h, and then dialysis was continued overnight with the same fresh buffer. After 17 hours, the sample was filtered through a 0.45 μ M filter, vacuum degassed, passed through an anion exchange column (HiTrap Q HP, GE Healthcare), protein purified by a linear gradient of 0-1M NaCl in 20mM Tris-HCl pH 8.0, the collected fractions were analyzed by SDS-PAGE, fractions containing single-chain TCR were concentrated and further purified by a gel filtration column (Superdex 7510/300, GE Healthcare), and the target fraction was also analyzed by SDS-PAGE.
The eluted fractions for BIAcore analysis were further tested for purity using gel filtration. The conditions are as follows: the chromatography column Agilent Bio SEC-3 (300A,
Figure BDA0003121861830000181
) The mobile phase is 150mM phosphate buffer solution, the flow rate is 0.5mL/min, the column temperature is 25 ℃, and the ultraviolet detection wavelength is 214nm.
The SDS-PAGE gel of the soluble single-chain TCR obtained by the invention is shown in FIGS. 10a and 10 b.
Example 6 binding characterization
This example demonstrates, by BIAcore analysis, that soluble TCR molecules of the invention are capable of specifically binding to the TSSELMAITR-HLA a1101 complex.
The binding activity of the TCR molecules obtained in examples 3 and 5 to the TSSELMAITR-HLA a1101 complex was tested using a BIAcore T200 real-time assay system. Anti-streptavidin antibody (GenScript) was added to coupling buffer (10 mM sodium acetate buffer, pH 4.77), the antibody was then passed through a CM5 chip previously activated with EDC and NHS to immobilize the antibody on the chip surface, and finally the unreacted activated surface was blocked with ethanolamine hydrochloride solution to complete the coupling process at a coupling level of about 15,000 RU.
And (2) enabling low-concentration streptavidin to flow through the surface of the chip coated with the antibody, then enabling the TSSELMAITR-HLA A1101 complex to flow through a detection channel, enabling the other channel to serve as a reference channel, and enabling 0.05mM biotin to flow through the chip at the flow rate of 10 mu L/min for 2min to block the remaining binding sites of the streptavidin.
The TSSELMAITR-HLA A1101 complex is prepared by the following steps:
a. purification of
Collecting 100ml E.coli liquid for inducing expression of heavy chain or light chain, centrifuging at 4 ℃ for 10min at 8000g, washing the thalli once with 10ml PBS, then resuspending the thalli with 5ml BugBuster Master Mix Extraction Reagents (Merck) by vigorous shaking, rotatably incubating at room temperature for 20min, centrifuging at 4 ℃ for 15min at 6000g, discarding supernatant, and collecting inclusion body.
Resuspending the inclusion body in 5ml of BugBuster Master Mix, and rotary incubating at room temperature for 5min; adding 30ml of 10-fold diluted BugBuster, uniformly mixing, and centrifuging at 4 ℃ at 6000g for 15min; discarding supernatant, adding 30ml of 10-fold diluted BugBuster to resuspend the inclusion bodies, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, repeating twice, adding 30ml of 20mM Tris-HCl with pH of 8.0 to resuspend the inclusion bodies, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, finally dissolving the inclusion bodies by using 20mM Tris-HCl8M urea, detecting the purity of the inclusion bodies by SDS-PAGE, and detecting the concentration by using a BCA kit.
b. Renaturation
The synthetic short peptide TSSELMAITR was dissolved in DMSO to a concentration of 20 mg/ml. Inclusion of light and heavy chains was solubilized using 8M urea, 20mM Tris pH 8.0, 10mM DTT, and further denatured by addition of 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA prior to renaturation. TSSELMAITR peptide was added to a renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidative glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4 ℃) at 25mg/L (final concentration), followed by the addition of 20mg/L of light chain and 90mg/L of heavy chain in sequence (final concentration, heavy chain was added in three portions, 8 h/time), and renaturation was carried out at 4 ℃ for at least 3 days until completion, and SDS-PAGE was checked for success or failure.
c. Purification after renaturation
The renaturation buffer was replaced by dialysis against 10 volumes of 20mM Tris pH 8.0, at least twice to reduce the ionic strength of the solution sufficiently. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and then loaded onto a HiTrap Q HP (GE general electric) anion exchange column (5 ml bed volume). The protein was eluted using a linear gradient of 0-400mM NaCl prepared using an Akta purifier (GE general electric) at 20mM Tris pH 8.0, pMHC was eluted at about 250mM NaCl, and the peak fractions were collected and subjected to purity detection using SDS-PAGE.
d. Biotinylation of the compound
The purified pMHC molecules were concentrated using Millipore ultrafiltration tubes while the buffer was replaced with 20mM Tris pH 8.0, followed by addition of biotinylation reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50. Mu.M D-Biotin, 100. Mu.g/ml BirA enzyme (GST-BirA), incubation of the mixture overnight at room temperature, and SDS-PAGE to determine the completion of biotinylation.
e. Purification of the biotinylated Complex
The biotinylated pMHC molecules were concentrated to 1ml using Millipore ultrafiltration tubes, the biotinylated pMHC was purified by gel filtration chromatography, the HiPrepTM 16/60S200 HR column (GE general electric) was pre-equilibrated with filtered PBS using an Akta purifier (GE general electric), loaded with 1ml of concentrated biotinylated pMHC molecules and then eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules appeared as a unimodal elution at approximately 55 ml. The fractions containing the protein were pooled, concentrated using Millipore ultrafiltration tubes, protein concentration was determined by BCA (Thermo), and biotinylated pMHC molecules were stored in aliquots at-80 ℃ by addition of the protease inhibitor cocktail (Roche).
Kinetic parameters are calculated by using BIAcore Evaluation software, and kinetic maps of the soluble TCR molecule and the soluble single-chain TCR molecule combined with the TSSELMAITR-HLA A1101 complex are obtained and are respectively shown in FIG. 11 and FIG. 12. The mapping shows that the soluble TCR molecules and the soluble single-chain TCR molecules obtained by the invention can be combined with the TSSELMAITR-HLA A1101 complex. Meanwhile, the method is utilized to detect the binding activity of the soluble TCR molecule and the short peptides of other irrelevant antigens and the HLA compound, and the result shows that the TCR molecule is not bound with other irrelevant antigens.
Example 7 Effector cell activation assay for short peptide-loaded T2 cells transfected with a TCR of the invention
The effector cells used in this experiment were CD3+ T cells expressing the TCR of the invention, and CD3+ T cells transfected with other TCRs (A6) from the same volunteer were used as a control. The target cells used were T2-A11 loaded with AFP antigen short peptide TSSELMAITR, and unloaded T2-A11 loaded with other antigen short peptides was used as control. The components of the assay were added to ELISPOT well plates: target cells 1 × 104 target cells/well, effector cells 2 × 103 cells/well (calculated as transfection positivity), and two duplicate wells were set. TSSELMAITR short peptides were then added to the corresponding wells to give a final concentration of 10-6M in the ELISPOT well plates.
The well plates were prepared as follows according to the manufacturer's instructions: 10ml of sterile PBS per plate 1: anti-human IFN-. Gamma.capture antibody was diluted at 200, and 100. Mu.l of the diluted capture antibody was aliquoted into each well. The plates were incubated overnight at 4 ℃. After incubation, the well plates were washed to remove excess capture antibody. Add 100. Mu.l/well of RPMI 1640 medium containing 10% FBS and incubate the well plates at room temperature for 2 hours to close the well plates. The media was then washed from the well plate, and any residual wash buffer was removed by flicking and tapping the ELISPOT well plate on paper.
The plates were then incubated overnight (37 ℃/5% CO) 2 ) The next day, the medium was discarded, the well plate was washed 2 times with double distilled water and 3 times with wash buffer, and tapped on a paper towel to remove residual wash buffer. The detection antibody was then diluted with 10% FBS-containing PBS at 1. The well plate was incubated at room temperature for 2 hours, washed 3 times with wash buffer and the well plate was tapped on a paper towel to remove excess wash buffer. The streptavidin-alkaline phosphatase was diluted with 10% fbs-containing PBS as 1. The plates were then washed 2 times with 4 washes of PBS and tapped on a paper towel to remove excess wash buffer and PBS. After washing, 100 microliter of BCIP/NBT solution provided by the kit is added for development. And covering the well plate with tinfoil paper in the developing period, keeping the well plate in the dark, and standing for 5-15 minutes. Spots on the developing plate were routinely detected during this period to determine the optimum time for terminating the reaction. The BCIP/NBT solution was removed and the well plate was rinsed with double steam to stop the development reaction, spun dry, then the bottom of the well plate was removed, the well plate was dried at room temperature until each well was completely dried, and the spots formed in the bottom membrane of the well plate were counted using an immunopot plate counter (CTL, cellular Technology Limited). The number of ELSPOT spots observed in each well was plotted using a graphpad prism 6.
The experimental results are shown in fig. 14, for T2 cells loaded with TSSELMAITR short peptide, the target cells of T cells transfected with the TCR of the present invention have obvious activating effect, while T cells transfected with other TCRs have no substantial response; meanwhile, T cells transfected with the TCR of the invention are inactive against T2 cells loaded with other antigenic short peptides or unloaded.
Example 8 Effect of transfection of TCR of the invention on tumor cell lines
This example also examined the function and specificity of the inventive TCR in cells by ELISPOT assay. The effector cells used were CD3+ T cells expressing the TCR specific for the AFP antigen oligopeptide of the invention, and CD3+ T cells transfected by the same volunteer with other TCR (A6) and free-stained (NC) served as a control. The target cell is tumor cell line, and the positive tumor cell line is HepG2-A11-B2M (HLA A1101 and beta 2M overexpression) and SK-MEL-28-AFP; the negative tumor cell lines used were HepG2, SK-MEL-28, SNU423 and HUCCT1 as controls.
First, an ELISPOT plate was prepared. ELISPOT plate ethanol activation coating, 4 degrees C overnight. On day 1 of the experiment, the coating was removed, washed and blocked, incubated for two hours at room temperature, the blocking solution removed, and the components of the assay added to an ELISPOT plate: the target cell is 2X 10 4 2X 10 effector cells per well 3 One well (calculated as positive transfection) and two duplicate wells were set. Incubation overnight (37 ℃,5% CO) 2 ). On day 2 of the experiment, the plates were washed and subjected to secondary detection and color development, dried, and the spots formed on the membrane were counted using an immuno-spot plate READER (ELISPOT READER system; AID20 Co.).
The results of the experiment are shown in fig. 15, the effector cells transfected with the TCR of the invention are specifically activated for the positive tumor cell line, and the null transfected T cells transfected with other TCRs are substantially inactivated; whereas, for negative tumor cell lines, effector cells transfected with the inventive TCR were inactive.
Example 9 killing function assay of Effector cells transfected with TCR of the invention
This example also demonstrates the killing function of cells transfected with a TCR of the invention by measuring LDH release by non-radioactive cytotoxicity assays well known to those skilled in the art. This example LDH assay uses CD3+ T cells isolated from blood of healthy volunteers to transfect a TCR of the inventionEffector cells and CD3+ T cells transfected with other TCRs (A6) or free-standing (NC) from the same volunteer were used as negative controls. The target cell is tumor cell line, and the positive tumor cell lines are HepG2-A11-B2M (HLA A1101 and beta 2M overexpression) and SK-MEL-28-AFP; the negative tumor cell lines used were HepG2, SK-MEL-28, SNU423 and HUCCT1 as controls. LDH plates were first prepared and the individual components of the assay were added to the plates in the following order: target cell 3X 10 4 Single cell/well, effector cell 3X 10 4 Individual cells/well (calculated as transfection positivity) were added to the corresponding wells and three replicate wells were set. Meanwhile, an effector cell spontaneous hole, a target cell maximum hole, a volume correction control hole and a culture medium background control hole are arranged. Incubation overnight (37 ℃,5% CO) 2 ). On day 2 of the experiment, color development was detected, and after termination of the reaction, the absorbance was recorded at 490nm using a microplate reader (Bioteck).
The results of the experiments are shown in fig. 16, for AFP positive tumor cell lines, effector cells transfected with the TCR of the invention showed strong killing efficacy, while effector cells transfected with other TCRs or free-staining did not kill; meanwhile, T cells transfected with the TCR of the invention have no killing activity on negative tumor cell lines.
All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> snow-fragrance Life sciences technology (Guangdong) Co., ltd
<120> TCR for identifying AFP antigen and coding sequence thereof
<130> P2021-1578
<160> 35
<170> PatentIn version 3.5
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gccacgactg tcgctacgga acgctacagc ttattgtaca tttcctcttc ccagaccaca 240
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<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 15
Ala Ser Ser Pro Gly Thr Gly Val Gly Tyr Thr
1 5 10
<210> 16
<211> 15
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 16
gactctgtga acaat 15
<210> 17
<211> 15
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 17
attccctcag ggaca 15
<210> 18
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 18
agtggaggta gcaactataa actgaca 27
<210> 19
<211> 15
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 19
tctgaacaca accgc 15
<210> 20
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 20
ttccagaatg aagctcaa 18
<210> 21
<211> 33
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 21
gccagcagcc ccgggacagg ggttggctac acc 33
<210> 22
<211> 268
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 22
Met Lys Arg Ile Leu Gly Ala Leu Leu Gly Leu Leu Ser Ala Gln Val
1 5 10 15
Cys Cys Val Arg Gly Ile Gln Val Glu Gln Ser Pro Pro Asp Leu Ile
20 25 30
Leu Gln Glu Gly Ala Asn Ser Thr Leu Arg Cys Asn Phe Ser Asp Ser
35 40 45
Val Asn Asn Leu Gln Trp Phe His Gln Asn Pro Trp Gly Gln Leu Ile
50 55 60
Asn Leu Phe Tyr Ile Pro Ser Gly Thr Lys Gln Asn Gly Arg Leu Ser
65 70 75 80
Ala Thr Thr Val Ala Thr Glu Arg Tyr Ser Leu Leu Tyr Ile Ser Ser
85 90 95
Ser Gln Thr Thr Asp Ser Gly Val Tyr Phe Cys Ser Gly Gly Ser Asn
100 105 110
Tyr Lys Leu Thr Phe Gly Lys Gly Thr Leu Leu Thr Val Asn Pro Asn
115 120 125
Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser
130 135 140
Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn
145 150 155 160
Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val
165 170 175
Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp
180 185 190
Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile
195 200 205
Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val
210 215 220
Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln
225 230 235 240
Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly
245 250 255
Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
260 265
<210> 23
<211> 804
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 23
atgaagagga tattgggagc tctgctgggg ctcttgagtg cccaggtttg ctgtgtgaga 60
ggaatacaag tggagcagag tcctccagac ctgattctcc aggagggagc caattccacg 120
ctgcggtgca atttttctga ctctgtgaac aatttgcagt ggtttcatca aaacccttgg 180
ggacagctca tcaacctgtt ttacattccc tcagggacaa aacagaatgg aagattaagc 240
gccacgactg tcgctacgga acgctacagc ttattgtaca tttcctcttc ccagaccaca 300
gactcaggcg tttatttctg cagtggaggt agcaactata aactgacatt tggaaaagga 360
actctcttaa ccgtgaatcc aaatatccag aaccctgacc ctgccgtgta ccagctgaga 420
gactctaaat ccagtgacaa gtctgtctgc ctattcaccg attttgattc tcaaacaaat 480
gtgtcacaaa gtaaggattc tgatgtgtat atcacagaca aaactgtgct agacatgagg 540
tctatggact tcaagagcaa cagtgctgtg gcctggagca acaaatctga ctttgcatgt 600
gcaaacgcct tcaacaacag cattattcca gaagacacct tcttccccag cccagaaagt 660
tcctgtgatg tcaagctggt cgagaaaagc tttgaaacag atacgaacct aaactttcaa 720
aacctgtcag tgattgggtt ccgaatcctc ctcctgaaag tggccgggtt taatctgctc 780
atgacgctgc ggctgtggtc cagc 804
<210> 24
<211> 309
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 24
Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala
1 5 10 15
Asp His Ala Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr
20 25 30
Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His
35 40 45
Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe
50 55 60
Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu
65 70 75 80
Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu
85 90 95
Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala
100 105 110
Ser Ser Pro Gly Thr Gly Val Gly Tyr Thr Phe Gly Ser Gly Thr Arg
115 120 125
Leu Thr Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala
130 135 140
Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr
145 150 155 160
Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser
165 170 175
Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro
180 185 190
Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu
195 200 205
Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn
210 215 220
His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu
225 230 235 240
Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu
245 250 255
Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln
260 265 270
Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala
275 280 285
Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val
290 295 300
Lys Arg Lys Asp Phe
305
<210> 25
<211> 927
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 25
atgggcacca gcctcctctg ctggatggcc ctgtgtctcc tgggggcaga tcacgcagat 60
actggagtct cccaggaccc cagacacaag atcacaaaga ggggacagaa tgtaactttc 120
aggtgtgatc caatttctga acacaaccgc ctttattggt accgacagac cctggggcag 180
ggcccagagt ttctgactta cttccagaat gaagctcaac tagaaaaatc aaggctgctc 240
agtgatcggt tctctgcaga gaggcctaag ggatctttct ccaccttgga gatccagcgc 300
acagagcagg gggactcggc catgtatctc tgtgccagca gccccgggac aggggttggc 360
tacaccttcg gttcggggac caggttaacc gttgtagagg acctgaacaa ggtgttccca 420
cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 480
ctggtgtgcc tggccacagg cttcttcccc gaccacgtgg agctgagctg gtgggtgaat 540
gggaaggagg tgcacagtgg ggtcagcacg gacccgcagc ccctcaagga gcagcccgcc 600
ctcaatgact ccagatactg cctgagcagc cgcctgaggg tctcggccac cttctggcag 660
aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 720
tggacccagg atagggccaa acccgtcacc cagatcgtca gcgccgaggc ctggggtaga 780
gcagactgtg gctttacctc ggtgtcctac cagcaagggg tcctgtctgc caccatcctc 840
tatgagatcc tgctagggaa ggccaccctg tatgctgtgc tggtcagcgc ccttgtgttg 900
atggccatgg tcaagagaaa ggatttc 927
<210> 26
<211> 202
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 26
Met Gly Ile Gln Val Glu Gln Ser Pro Pro Asp Leu Ile Leu Gln Glu
1 5 10 15
Gly Ala Asn Ser Thr Leu Arg Cys Asn Phe Ser Asp Ser Val Asn Asn
20 25 30
Leu Gln Trp Phe His Gln Asn Pro Trp Gly Gln Leu Ile Asn Leu Phe
35 40 45
Tyr Ile Pro Ser Gly Thr Lys Gln Asn Gly Arg Leu Ser Ala Thr Thr
50 55 60
Val Ala Thr Glu Arg Tyr Ser Leu Leu Tyr Ile Ser Ser Ser Gln Thr
65 70 75 80
Thr Asp Ser Gly Val Tyr Phe Cys Ser Gly Gly Ser Asn Tyr Lys Leu
85 90 95
Thr Phe Gly Lys Gly Thr Leu Leu Thr Val Asn Pro Asn Ile Gln Asn
100 105 110
Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys
115 120 125
Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln
130 135 140
Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met
145 150 155 160
Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys
165 170 175
Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu
180 185 190
Asp Thr Phe Phe Cys Ser Pro Glu Ser Ser
195 200
<210> 27
<211> 606
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 27
atgggcattc aggtggaaca gagtcctcca gacctgattc tccaggaggg agccaattcc 60
acgctgcggt gcaatttttc tgactctgtg aacaatttgc agtggtttca tcaaaaccct 120
tggggacagc tcatcaacct gttttacatt ccctcaggga caaaacagaa tggaagatta 180
agcgccacga ctgtcgctac ggaacgctac agcttattgt acatttcctc ttcccagacc 240
acagactcag gcgtttattt ctgcagtgga ggtagcaact ataaactgac atttggaaaa 300
ggaactctct taaccgtgaa tccaaatatc cagaaccctg accctgccgt ttatcagctg 360
cgtgatagca aaagcagcga taaaagcgtg tgcctgttca ccgattttga tagccagacc 420
aacgtgagcc agagcaaaga tagcgatgtg tacatcaccg ataaaaccgt gctggatatg 480
cgcagcatgg atttcaaaag caatagcgcg gttgcgtgga gcaacaaaag cgattttgcg 540
tgcgcgaacg cgtttaacaa cagcatcatc ccggaagata cgttcttctg cagcccagaa 600
agttcc 606
<210> 28
<211> 244
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 28
Met Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg
1 5 10 15
Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg
20 25 30
Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr
35 40 45
Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp
50 55 60
Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile
65 70 75 80
Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser
85 90 95
Pro Gly Thr Gly Val Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr
100 105 110
Val Val Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe
115 120 125
Glu Pro Ser Glu Cys Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val
130 135 140
Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp
145 150 155 160
Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro
165 170 175
Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser
180 185 190
Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe
195 200 205
Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr
210 215 220
Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp
225 230 235 240
Gly Arg Ala Asp
<210> 29
<211> 732
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 29
atggataccg gcgtgagcca ggaccccaga cacaagatca caaagagggg acagaatgta 60
actttcaggt gtgatccaat ttctgaacac aaccgccttt attggtaccg acagaccctg 120
gggcagggcc cagagtttct gacttacttc cagaatgaag ctcaactaga aaaatcaagg 180
ctgctcagtg atcggttctc tgcagagagg cctaagggat ctttctccac cttggagatc 240
cagcgcacag agcaggggga ctcggccatg tatctctgtg ccagcagccc cgggacaggg 300
gttggctaca ccttcggttc ggggaccagg ttaaccgttg tagaggacct gaaaaacgtg 360
ttcccacccg aggtcgctgt gtttgagcca tcagaatgcg aaattagcca tacccagaaa 420
gcgaccctgg tttgtctggc gaccggtttt tatccggatc atgtggaact gtcttggtgg 480
gtgaacggca aagaagtgca tagcggtgtt tctaccgatc cgcagccgct gaaagaacag 540
ccggcgctga atgatagccg ttatgcgctg tctagccgtc tgcgtgttag cgcgaccttt 600
tggcaaaatc cgcgtaacca ttttcgttgc caggtgcagt tttatggcct gagcgaaaac 660
gatgaatgga cccaggatcg tgcgaagccg gttacccaga ttgttagcgc ggaagcctgg 720
ggccgcgcag at 732
<210> 30
<211> 245
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 30
Met Gly Ile Gln Val Glu Gln Ser Pro Pro Asp Leu Asn Val Gln Glu
1 5 10 15
Gly Glu Asn Val Thr Ile Arg Cys Asn Phe Ser Asp Ser Val Asn Asn
20 25 30
Leu Gln Trp Phe His Gln Asn Pro Gly Gly Gln Leu Ile Asn Leu Phe
35 40 45
Tyr Ile Pro Ser Gly Thr Lys Gln Asn Gly Arg Leu Ser Ala Thr Thr
50 55 60
Val Ala Thr Glu Arg Tyr Ser Leu Leu Tyr Ile Ser Ser Val Gln Pro
65 70 75 80
Thr Asp Ser Gly Val Tyr Phe Cys Ser Gly Gly Ser Asn Tyr Lys Leu
85 90 95
Thr Phe Gly Lys Gly Thr Lys Leu Thr Val Asn Pro Gly Gly Gly Ser
100 105 110
Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu
115 120 125
Gly Gly Thr Gly Asp Thr Gly Val Ser Gln Asp Pro Arg His Leu Ser
130 135 140
Val Lys Arg Gly Gln Asn Val Thr Leu Arg Cys Asp Pro Ile Ser Glu
145 150 155 160
His Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Pro Gly Gln Gly Pro Glu
165 170 175
Phe Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu
180 185 190
Leu Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr
195 200 205
Leu Glu Ile Gln Arg Val Glu Pro Gly Asp Ser Ala Met Tyr Phe Cys
210 215 220
Ala Ser Ser Pro Gly Thr Gly Val Gly Tyr Thr Phe Gly Ser Gly Thr
225 230 235 240
Arg Leu Thr Val Asp
245
<210> 31
<211> 735
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 31
atgggtattc aagttgaaca gagtccgccg gatctgaatg ttcaggaagg cgaaaatgtg 60
accattcgct gcaattttag cgatagcgtg aataatctgc agtggtttca tcagaatccg 120
ggcggtcagc tgattaatct gttttatatt ccgagtggta ccaaacagaa tggccgtctg 180
agcgcaacca ccgttgccac cgaacgctat agtctgctgt atattagcag tgttcagccg 240
accgatagtg gcgtttattt ctgtagcggt ggcagcaatt ataaactgac ctttggtaaa 300
ggtaccaaac tgaccgttaa tccgggcggc ggtagtgaag gtggtggcag cgaaggtggt 360
ggtagtgaag gcggcggttc agaaggtggc accggtgaca ccggcgttag ccaggaccct 420
cgtcatctga gtgtgaaacg tggtcagaat gttaccctgc gttgcgatcc gattagcgaa 480
cataatcgtc tgtattggta tcgtcagacc ccgggccagg gtccggaatt tctgacctat 540
tttcagaatg aagcccagct ggaaaagagt cgtctgctga gtgatcgttt tagtgcagaa 600
cgtccgaaag gcagctttag caccctggaa attcagcgtg ttgaaccagg cgatagtgca 660
atgtatttct gtgcaagctc tcctggcacc ggtgttggct atacctttgg cagtggcact 720
cgtctgaccg ttgat 735
<210> 32
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 32
Gly Ile Gln Val Glu Gln Ser Pro Pro Asp Leu Asn Val Gln Glu Gly
1 5 10 15
Glu Asn Val Thr Ile Arg Cys Asn Phe Ser Asp Ser Val Asn Asn Leu
20 25 30
Gln Trp Phe His Gln Asn Pro Gly Gly Gln Leu Ile Asn Leu Phe Tyr
35 40 45
Ile Pro Ser Gly Thr Lys Gln Asn Gly Arg Leu Ser Ala Thr Thr Val
50 55 60
Ala Thr Glu Arg Tyr Ser Leu Leu Tyr Ile Ser Ser Val Gln Pro Thr
65 70 75 80
Asp Ser Gly Val Tyr Phe Cys Ser Gly Gly Ser Asn Tyr Lys Leu Thr
85 90 95
Phe Gly Lys Gly Thr Lys Leu Thr Val Asn Pro
100 105
<210> 33
<211> 321
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 33
ggtattcaag ttgaacagag tccgccggat ctgaatgttc aggaaggcga aaatgtgacc 60
attcgctgca attttagcga tagcgtgaat aatctgcagt ggtttcatca gaatccgggc 120
ggtcagctga ttaatctgtt ttatattccg agtggtacca aacagaatgg ccgtctgagc 180
gcaaccaccg ttgccaccga acgctatagt ctgctgtata ttagcagtgt tcagccgacc 240
gatagtggcg tttatttctg tagcggtggc agcaattata aactgacctt tggtaaaggt 300
accaaactga ccgttaatcc g 321
<210> 34
<211> 113
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<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 Phe Cys Ala Ser Ser Pro
85 90 95
Gly Thr Gly Val Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Asp
<210> 35
<211> 339
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 35
gacaccggcg ttagccagga ccctcgtcat ctgagtgtga aacgtggtca gaatgttacc 60
ctgcgttgcg atccgattag cgaacataat cgtctgtatt ggtatcgtca gaccccgggc 120
cagggtccgg aatttctgac ctattttcag aatgaagccc agctggaaaa gagtcgtctg 180
ctgagtgatc gttttagtgc agaacgtccg aaaggcagct ttagcaccct ggaaattcag 240
cgtgttgaac caggcgatag tgcaatgtat ttctgtgcaa gctctcctgg caccggtgtt 300
ggctatacct ttggcagtgg cactcgtctg accgttgat 339

Claims (10)

1. A T Cell Receptor (TCR) capable of binding to the TSSELMAITR-HLA a1101 complex, the TCR comprising a TCR a chain variable domain and a TCR β chain variable domain, wherein the 3 Complementarity Determining Regions (CDRs) of the TCR a chain variable domain are:
αCDR1-DSVNN (SEQ ID NO:10)
αCDR2-IPSGT (SEQ ID NO:11)
α CDR3-SGGSNYKLT (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-ASSPGTGVGYT (SEQ ID NO:15)。
2. a TCR as claimed in claim 1 which comprises a TCR α chain variable domain which is an amino acid sequence having at least 90% sequence identity to SEQ ID No. 1 and a TCR β chain variable domain; and/or the TCR β chain variable domain is an amino acid sequence having at least 90% sequence identity to SEQ ID No. 5.
3. A TCR as claimed in claim 1 wherein a conjugate is attached to the C-or N-terminus of the α and/or β chains of the TCR; 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, at least one of which is a TCR as claimed in any one of the preceding claims.
5. A nucleic acid molecule comprising a nucleic acid sequence encoding a TCR molecule according to any preceding claim, or the complement thereof;
preferably, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO 2 or SEQ ID NO 33 encoding the variable domain of the TCR alpha chain; and/or
The nucleic acid molecule comprises the nucleotide sequence SEQ ID NO 6 or SEQ ID NO 35 encoding the variable domain of the TCR beta chain.
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 a nucleic acid molecule of claim 5 integrated into the chromosome.
8. A cell which transduces the nucleic acid molecule of claim 5 or the vector of claim 6; preferably, the cell is a T cell or a stem cell.
9. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to any one of claims 1-3, a TCR complex according to claim 4, a nucleic acid molecule according to claim 5, or a cell according to claim 8.
10. Use of a T cell receptor according to any one of claims 1-3, or a TCR complex according to claim 4 or a cell according to claim 8, for the preparation of a medicament for the treatment of a tumour or an autoimmune disease.
CN202110678571.0A 2021-06-18 2021-06-18 TCR for identifying AFP antigen and coding sequence thereof Pending CN115490767A (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202110678571.0A CN115490767A (en) 2021-06-18 2021-06-18 TCR for identifying AFP antigen and coding sequence thereof
PCT/CN2022/099352 WO2022262835A1 (en) 2021-06-18 2022-06-17 Tcr for identifying afp antigen and coding sequence thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110678571.0A CN115490767A (en) 2021-06-18 2021-06-18 TCR for identifying AFP antigen and coding sequence thereof

Publications (1)

Publication Number Publication Date
CN115490767A true CN115490767A (en) 2022-12-20

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Country Link
CN (1) CN115490767A (en)
WO (1) WO2022262835A1 (en)

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110343166B (en) * 2018-04-03 2022-10-28 香雪生命科学技术（广东）有限公司 T cell receptor recognizing AFP antigen short peptides
CN111171137A (en) * 2018-11-13 2020-05-19 中国科学院广州生物医药与健康研究院 T cell receptor for identifying AFP antigen short peptide and its coding sequence
CN111171156A (en) * 2018-11-13 2020-05-19 中国科学院广州生物医药与健康研究院 T cell receptor for identifying AFP antigen short peptide and its coding sequence
WO2021022447A1 (en) * 2019-08-05 2021-02-11 广东香雪精准医疗技术有限公司 T cell receptor capable of recognizing afp antigen-derived short peptide

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