CN110272483B - T cell receptor for recognizing SAGE1 antigen short peptide - Google Patents

T cell receptor for recognizing SAGE1 antigen short peptide Download PDF

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CN110272483B
CN110272483B CN201810210760.3A CN201810210760A CN110272483B CN 110272483 B CN110272483 B CN 110272483B CN 201810210760 A CN201810210760 A CN 201810210760A CN 110272483 B CN110272483 B CN 110272483B
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CN110272483A (en
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李懿
陈安安
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Guangzhou Institute of Biomedicine and Health of CAS
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Guangzhou Institute of Biomedicine and Health of CAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Abstract

The present invention provides a T Cell Receptor (TCR) capable of specifically binding to short peptide ATIIHNLREEK derived from SAGE1 antigen, in particular, the antigen short peptide ATIIHNLREEK can form a complex with HLA a1101 and be presented together on the cell surface, and a T Cell Receptor (TCR) capable of specifically binding to ATIIHNLREEK-HLA-a1101 complex. The invention also provides nucleic acid molecule sequences encoding the TCRs and vectors comprising the nucleic acid molecule sequences. In addition, the invention provides cells that transduce a TCR of the invention.

Description

T cell receptor for recognizing SAGE1 antigen short peptide
Technical Field
The invention relates to a T cell receptor for recognizing SAGE1 antigen short peptide, and also relates to SAGE1 specific T cells obtained by transducing the TCR, and application of the T cells in preventing and treating SAGE1 antigen protein related diseases.
Background
SAGE1, an endogenous tumor antigen protein, was degraded into small polypeptides when expressed in cells and presented on the cell surface as complexes with MHC (major histocompatibility Complex) molecules. Studies showed ATIIHNLREEK to be short peptides derived from SAGE1 antigenic protein. SAGE1 antigen was expressed in tumor tissues such as melanoma, bladder Cancer, liver Cancer, epidermoid carcinoma, non-small cell lung Cancer and squamous cell carcinoma, but not in most normal tissues except testis (Martelange V1, De Smet C, De Plaen E, Lurqin C, Boon T. Cancer Res.2000; 60(14): 3848-55; Atanofovic D, et., Cancer biol. Ther.2006; 5(9): 1218-25). For the treatment of the above diseases, chemotherapy, radiotherapy and the like can be used, but both of them cause damages to normal cells themselves.
T cell adoptive immunotherapy is the transfer of reactive T cells specific for a target cell antigen into a patient to act on the target cell. The T Cell Receptor (TCR) is a membrane protein on the surface of T cells that recognizes a corresponding short peptide antigen on the surface of a target cell. In the immune system, the direct physical contact between T cells and Antigen Presenting Cells (APC) is initiated by the binding of antigen short peptide specific TCR and short peptide-major histocompatibility complex (pMHC complex), and then other cell membrane surface molecules of the T cells and APC interact to cause a series of subsequent cell signaling and other physiological reactions, so that T cells with different antigen specificities exert immune effects on their target cells. Therefore, those skilled in the art have focused on isolating a TCR specific for the SAGE1 antigen short peptide to be effective, or transducing the TCR into T cells to obtain T cells specific for the SAGE1 antigen short peptide, thereby making them effective in cellular immunotherapy.
Disclosure of Invention
The invention aims to provide a T cell receptor for recognizing SAGE1 antigen short peptide.
In a first aspect, the invention provides a T Cell Receptor (TCR) capable of binding to the ATIIHNLREEK-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 AESPPDNYGQNFV (SEQ ID NO: 13); and/or the amino acid sequence of CDR3 of the variable domain of the TCR beta chain is ASSPVAGELF (SEQ ID NO: 10).
In another preferred embodiment, the 3 Complementarity Determining Regions (CDRs) of the TCR α chain variable domain are:
αCDR1-DSSSTY(SEQ ID NO:6)
αCDR2-IFSNMDM(SEQ ID NO:7)
alpha CDR3-AESPPDNYGQNFV (SEQ ID NO: 13); and/or
The 3 complementarity determining regions of the TCR β chain variable domain are:
βCDR1-KGHSH(SEQ ID NO:5)
βCDR2-LQKENI(SEQ ID NO:8)
βCDR3-ASSPVAGELF(SEQ ID NO:10)。
in another preferred embodiment, the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, the TCR alpha 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 identical to SEQ ID NO:2 having at least 90% sequence identity.
In another preferred embodiment, the TCR comprises the alpha chain variable domain amino acid sequence SEQ ID NO 1.
In another preferred embodiment, the TCR comprises the beta chain variable domain amino acid sequence SEQ ID NO 2.
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. 15 and/or the beta chain amino acid sequence of the TCR is SEQ ID NO 4, 16.
In another preferred example, the nucleotide sequence of the α chain of the TCR is SEQ ID No. 12, 23 and/or the nucleotide sequence of the β chain of the TCR is SEQ ID No. 14, 24.
In another preferred embodiment, the TCR is soluble.
In another preferred embodiment, the TCR comprises (a) all or part of a TCR α chain, excluding the transmembrane domain; and (b) all or part of the TCR β chain, excluding the transmembrane domain;
and (a) and (b) each comprise a functional variable domain, or comprise a functional variable domain and at least a portion of the TCR chain constant domain.
In another preferred embodiment, the 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 linker peptide sequence.
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 and TRBC1 x 01 of TRAC x 01 exon 1 or Ser57 of TRBC2 x 01 exon 1;
thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser77 of TRBC2 × 01 exon 1;
tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC2 x 01;
thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1;
ser15 and TRBC1 x 01 of TRAC x 01 exon 1 or Glu15 of TRBC2 x 01 exon 1;
arg53 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser54 of TRBC2 × 01 exon 1;
pro89 and TRBC1 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01; and
tyr10 of TRAC × 01 exon 1 and TRBC1 × 01 or TRBC2 × 01 of Glu20 of exon 1.
In another preferred embodiment, the α chain amino acid sequence of the TCR is SEQ ID NO 3, 15 and/or the β chain amino acid sequence of the TCR is SEQ ID NO 4, 16.
In another preferred example, the amino acid sequence of the single-chain TCR α chain is SEQ ID No. 25 and/or the amino acid sequence of the single-chain TCR β chain is SEQ ID No. 27.
In another preferred example, the nucleotide sequence of the single-chain TCR α chain is SEQ ID No. 26 and/or the nucleotide sequence of the single-chain TCR β chain 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 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.
In another preferred embodiment, the therapeutic agent is an anti-CD 3 antibody.
In a second aspect, the invention provides a multivalent TCR complex comprising at least two TCR molecules, at least one of which is a TCR according to the first aspect of the invention.
In a third aspect, the invention provides 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 of SEQ ID NO: 9.
in another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 11.
in another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence encoding the TCR α chain SEQ ID NO: 12. 23 and/or a nucleic acid sequence comprising the nucleotide sequence encoding a TCR β chain SEQ ID NO: 14. 24.
In a fourth aspect, the invention provides 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, the present invention provides an isolated host cell comprising a vector according to the fourth aspect of the present invention or a genome into which has been integrated an exogenous nucleic acid molecule according to the third aspect of the present invention.
In a sixth aspect, the invention provides a cell which transduces a nucleic acid molecule according to the third aspect of the invention or a vector according to the fourth aspect of the invention; preferably, the cell is a T cell or a stem cell.
In a seventh aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to the first aspect of the invention, a TCR complex according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a cell according to the sixth aspect of the invention.
In an eighth aspect, the invention provides the use of a T cell receptor according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a cell according to the sixth aspect of the invention, in the manufacture of a medicament for the treatment of a tumour or an autoimmune disease.
In a ninth aspect, the present invention provides 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 present invention, or a TCR complex according to the second aspect of the present invention, a nucleic acid molecule according to the third aspect of the present invention, a vector according to the fourth aspect of the present invention, or a cell according to the sixth aspect of the present invention, or a pharmaceutical composition according to the seventh aspect of the present invention;
in another preferred embodiment, the disease is a tumor.
In another preferred embodiment, the tumor is selected from the group consisting of: melanoma, gastric cancer, lung cancer (e.g., lung squamous cell carcinoma), esophageal cancer, bladder cancer, head and neck tumors (e.g., head and neck squamous cell carcinoma), prostate cancer, breast cancer, colon cancer, ovarian cancer, renal cell carcinoma, hodgkin's lymphoma, sarcoma, medulloblastoma, leukemia, or a combination thereof.
In another preferred embodiment, the tumor is selected from the group consisting of: melanoma, bladder cancer, liver cancer, epidermoid cancer, non-small cell lung cancer, squamous cell carcinoma, or a combination thereof.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1a, FIG. 1b, FIG. 1c, FIG. 1d, FIG. 1e and FIG. 1f are the TCR α chain variable domain amino acid sequence, TCR α chain variable domain nucleotide sequence, TCR α chain amino acid sequence, TCR α chain nucleotide sequence, TCR α chain amino acid sequence with leader sequence and TCR α chain nucleotide sequence with leader sequence, respectively.
Fig. 2a, fig. 2b, fig. 2c, fig. 2d, fig. 2e and fig. 2f are a TCR β chain variable domain amino acid sequence, a TCR β chain variable domain nucleotide sequence, a TCR β chain amino acid sequence, a TCR β chain nucleotide sequence, a TCR β chain amino acid sequence with a leader sequence and a TCR β chain nucleotide sequence with a leader sequence, respectively.
FIG. 3 shows CD8 of monoclonal cells + And tetramer-PE double positive staining results.
Fig. 4a and 4b are the amino acid and nucleotide sequences, respectively, of a soluble TCR α chain.
Figure 5a and figure 5b are the amino acid and nucleotide sequences, respectively, of a soluble TCR β chain.
Figure 6 is a gel diagram of the soluble TCR obtained after purification. The leftmost lane is reducing gel, the middle lane is molecular weight marker (marker), and the rightmost lane is non-reducing gel.
FIG. 7 is a Biacore kinetic profile of the binding of soluble TCRs of the invention to the ATIIHNLREEK-HLA A1101 complex.
FIG. 8 is a fine detection of primary T transduced by tetramer-stained TCR.
FIG. 9 shows the results of the ELISPOT assay.
FIG. 10 is a graph showing the results of killing of specific target cells by T cells transduced by the TCR of the invention.
Detailed Description
The present inventors have made extensive and intensive studies to find a TCR capable of specifically binding to SAGE1 antigen short peptide ATIIHNLREEK (SEQ ID NO:29), which antigen short peptide ATIIHNLREEK can form a complex with HLA-A1101 and be presented together to the cell surface. The invention also provides nucleic acid molecule sequences encoding the TCRs and vectors comprising the nucleic acid molecule sequences. In addition, the invention provides cells that transduce a TCR of the invention.
Term(s)
MHC molecules are proteins of the immunoglobulin superfamily and may be MHC class I or II molecules. Therefore, it is specific for antigen presentation, different individuals have different MHC, and different short peptides in one protein antigen can be presented on the cell surface of respective APC. Human MHC is commonly referred to as an HLA gene or HLA complex.
The T Cell Receptor (TCR), is the only receptor for a specific antigenic peptide presented on the Major Histocompatibility Complex (MHC). In the immune system, direct physical contact between T cells and Antigen Presenting Cells (APCs) is initiated by the binding of antigen-specific TCRs to pMHC complexes, and then other cell membrane surface molecules of both T cells and APCs interact, which causes a series of subsequent cell signaling and other physiological reactions, thereby allowing T cells of different antigen specificities to exert immune effects on their target cells.
TCRs are cell membrane surface glycoproteins that exist as heterodimers from either the α chain/β chain or the γ chain/δ chain. In 95% of T cells the TCR heterodimer consists of α and β chains, while 5% of T cells have TCRs consisting of γ and δ chains. Native α β heterodimeric TCRs have an α chain and a β chain, which make up the subunits of the 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, in which CDR3 is composed of a variable region and a connecting region, are called hypervariable regions, determine the binding of the TCR to the pMHC complex. The α and β chains of a TCR are generally regarded as having two "domains" each, namely a variable domain and a constant domain, the variable domain being made up of linked variable regions and linking regions. The sequences of TCR constant domains can be found in public databases of the international immunogenetic information system (IMGT), such as the constant domain sequence of the α chain of the TCR molecule is "TRAC 01", the constant domain sequence of the β chain of the TCR molecule is "TRBC 1 01" or "TRBC 2 01". In addition, the α and β chains of the TCR also comprise a transmembrane region and a cytoplasmic region, the cytoplasmic region being very short.
In the present invention, the terms "polypeptide of the invention", "TCR of the invention", "T cell receptor of the invention" are used interchangeably.
Natural interchain disulfide bond and artificial interchain disulfide bond
A set of disulfide bonds, referred to herein as "native interchain disulfide bonds," exist between the C α and C β chains of the membrane proximal region of native TCRs. In the present invention, the artificially introduced interchain covalent disulfide bond, 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 the amino acid sequences of TRAC 01 and TRBC1 × 01 or TRBC2 × 01 are numbered in the order from the N-terminus to the C-terminus, such as in TRBC1 × 01 or TRBC2 × 01, and the 60 th amino acid in the order from the N-terminus to the C-terminus is P (proline), and thus in the present invention it can be described as Pro60 of TRBC1 × 01 or TRBC2 × 01 exon 1, and also as the 60 th amino acid of TRBC1 × 01 or TRBC2 × 01 exon 1, and as in 737bc 3 × 01 or TRBC2 × 01, and the 61 th amino acid in the order from the N-terminus to the C-terminus is Q (glutamine), and thus in the present invention it can be described as TRBC1 × 01 or TRBC 6301 × 01, or TRBC 8501, and similarly as TRBC 8261 or glbc 891. In the present invention, the position numbering of the amino acid sequences of the variable regions TRAV and TRBV follows the position numbering listed in IMGT. If 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
As used herein, the term "SAGE 1 TCR-T" refers to cells that can react against the SAGE1 antigen peptide of the invention, but are non-reactive to other antigen peptides.
As used herein, the terms "Non-SAGE 1TCR transduced T cells", "Non-SAGE 1 TCR-T" are used interchangeably and refer to T cells expressing other TCRs, also understood as TCRs that are directed against the SAGE1 antigen peptide of the invention but do not function well, and which do not respond when added to the SAGE1 antigen peptide of the invention.
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 ATIIHNLREEK-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-DSSSTY(SEQ ID NO:6)
αCDR2-IFSNMDM(SEQ ID NO:7)
alpha CDR3-AESPPDNYGQNFV (SEQ ID NO: 13); and/or
The 3 complementarity determining regions of the TCR β chain variable domain are:
βCDR1-KGHSH(SEQ ID NO:5)
βCDR2-LQKENI(SEQ ID NO:8)
βCDR3-ASSPVAGELF(SEQ ID NO:10)。
chimeric TCRs can be prepared by embedding the above-described amino acid sequences of the CDR regions of the invention into any suitable framework. One skilled in the art can design or synthesize a TCR molecule with the corresponding function based on the CDR regions disclosed herein, so long as the framework structure is compatible with the CDR regions of the TCR of the invention. Thus, the TCR molecules of the invention are those which comprise the above-described α and/or β chain CDR region sequences and any suitable framework structure. The TCR α chain variable domain of the invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity to SEQ ID No. 1; and/or the TCR β chain variable domain of the invention is identical to SEQ ID NO:2, or a variant thereof, and 2 amino acid sequences having at least 90%, preferably 95%, more preferably 98% sequence identity.
In a preferred embodiment of the invention, the TCR molecules of the invention are heterodimers consisting of α and β chains. Specifically, in one aspect the α chain of the heterodimeric TCR molecules comprises a variable domain and a constant domain, the α chain variable domain amino acid sequence comprising the CDR1(SEQ ID NO: 6), CDR2(SEQ ID NO: 7) and CDR3(SEQ ID NO:13) 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:5), CDR2(SEQ ID NO: 8), and CDR3(SEQ ID NO:10) of the above-described β chain. Preferably, the TCR molecule comprises the beta chain variable domain amino acid sequence SEQ ID NO 2. More preferably, the amino acid sequence of the β chain variable domain of the TCR molecule is SEQ ID NO 2.
In a preferred embodiment of the invention, the TCR molecules of the invention are single chain TCR molecules consisting of part or all of the α chain and/or part or all of the β chain. Single chain TCR molecules are described in Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-. From the literature, those skilled in the art are readily able to construct single chain TCR molecules comprising the CDRs regions of the invention. In particular, the single chain TCR molecule comprises V α, V β and C β, preferably linked in order from N-terminus to C-terminus.
The alpha chain variable domain amino acid sequence of the single chain TCR molecule comprises the CDR1(SEQ ID NO: 6), CDR2(SEQ ID NO: 7) and CDR3(SEQ ID NO:13) of the alpha chain described above. Preferably, the single chain TCR molecule comprises an alpha chain variable domain amino acid sequence SEQ ID NO 1. More preferably, the α chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO 1. The amino acid sequence of the beta chain variable domain of the single chain TCR molecule comprises the CDR1(SEQ ID NO:5), CDR2(SEQ ID NO: 8) and CDR3(SEQ ID NO:10) of the above-described beta chain. Preferably, the single chain TCR molecule comprises a beta chain variable domain amino acid sequence SEQ ID NO 2. More preferably, the amino acid sequence of the β chain variable domain of the single chain TCR molecule is SEQ ID NO 2.
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 "TRBC 1 01" or "TRBC 2 01". Arg at position 53 of the amino acid sequence given in TRAC 01 of IMGT, here denoted: TRAC × 01 Arg53 of exon 1, and so on. Preferably, the amino acid sequences of the α chain and/or the β chain of the TCR molecules of the invention are SEQ ID NOs 3, 15 and/or 4, 16.
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 it is desirable to obtain soluble TCR molecules. 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 pmhcs, but also as diagnostic tools for detecting infections or as markers for autoimmune diseases. Similarly, soluble TCRs can be used to deliver therapeutic agents (e.g., cytotoxic or immunostimulatory compounds) to cells presenting a specific antigen, and in addition, soluble TCRs can be conjugated to other molecules (e.g., anti-CD 3 antibodies) to redirect T cells to target them to cells presenting a particular antigen. The invention also obtained a soluble TCR specific for SAGE1 antigen short peptide.
To obtain a soluble TCR, in one aspect, the inventive TCR may be one in which an artificial disulfide bond is introduced between residues of the constant domains of its alpha and beta chains. Cysteine residues form an artificial interchain disulfide bond between the alpha and beta chain constant domains of the TCR. Cysteine residues may be substituted for other amino acid residues at appropriate positions in native TCRs to form artificial interchain disulfide bonds. For example, a disulfide bond is formed by substituting Thr48 of exon 1 of TRAC × 01 and a cysteine residue of Ser57 of exon 1 of TRBC1 × 01 or TRBC2 × 01. Other sites for introducing cysteine residues to form disulfide bonds may also be: thr45 and TRBC1 x 01 of TRAC x 01 exon 1 or Ser77 of TRBC2 x 01 exon 1; tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC2 x 01; thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1; ser15 and TRBC1 × 01 of TRAC × 01 exon 1 or Glu15 of TRBC2 × 01 exon 1; arg53 and TRBC1 x 01 of TRAC x 01 exon 1 or Ser54 of TRBC2 x 01 exon 1; pro89 and TRBC1 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01; or Tyr10 and TRBC1 and 01 of TRAC 01 exon 1 or Glu20 of TRBC2 and 01 exon 1. I.e., a cysteine residue, in place of any of the above-described alpha and beta chain constant domains. The TCR constant domains of the invention may be truncated at one or more of their C-termini by up to 50, or up to 30, or up to 15, or up to 10, or up to 8 or fewer amino acids, so as not to include a cysteine residue for the purpose of deleting the native disulphide bond, or by mutating the cysteine residue forming the native disulphide bond to another amino acid.
As described above, the TCRs of the invention may comprise artificial disulfide bonds introduced between residues of the constant domains of their alpha and beta chains. It should be noted that the TCRs of the invention may each contain both TRAC constant domain sequences and TRBC1 or TRBC2 constant domain sequences, with or without the artificial disulfide bonds introduced as described above between the constant domains. The TRAC constant domain sequence and TRBC1 or TRBC2 constant domain sequences of the TCR may be linked by the native disulfide bond present in the TCR.
To obtain a soluble TCR, on the other hand, the inventive TCR also comprises a TCR having a mutation in its hydrophobic core region, preferably a mutation that enables an improved stability of the inventive soluble TCR, as described in the patent publication WO 2014/206304. Such TCRs may be mutated at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or positions 3,5,7 from the last amino acid position of the short peptide of the alpha chain J gene (TRAJ), and/or positions 2,4,6 from the last amino acid position of the short peptide of the beta chain J gene (TRBJ), wherein the numbering of the positions of the amino acid sequences is according to the numbering of the positions listed in the International Immunogenetic information System (IMGT). The above-mentioned international system of immunogenetics information is known to the skilled person and the position numbering of the amino acid residues of the different TCRs in IMGT can be derived from this database.
The TCR with the mutated hydrophobic core region of the invention can be a stable soluble single chain TCR formed by connecting the variable domains of the alpha and beta chains of the TCR by a flexible peptide chain. It should be noted that the flexible peptide chain of the present invention can be any peptide chain suitable for linking the TCR α and β chain variable domains.
In addition, for stability, patent document PCT/CN2016/077680 also discloses that the introduction of an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR can significantly improve the stability of the TCR. Thus, the high affinity TCRs of the invention may also contain an artificial interchain disulfide bond between the α chain variable region and the β chain constant region. Specifically, the cysteine residues that form the artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR are substituted for: amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01; amino acid 47 of TRAV and amino acid 61 of TRBC1 x 01 or TRBC2 x 01 exon 1; amino acid 46 of TRAV and amino acid 61 of TRBC1 x 01 or TRBC2 x 01 exon 1; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01. Preferably, such a TCR may comprise (i) all or part of a TCR α chain, excluding its transmembrane domain, and (ii) all or part of a TCR β chain, excluding its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least part of the constant domain of the TCR chain, the α chain forming a heterodimer with the β chain. More preferably, such a TCR may comprise the a chain variable domain and the β chain variable domain and all or part of the β chain constant domain, excluding the transmembrane domain, but which does not comprise the a chain constant domain, the a chain variable domain of the TCR forming a heterodimer with the β chain.
The TCRs of the invention may also be provided in the form of multivalent complexes. Multivalent TCR complexes of the invention comprise polymers formed by association of two, three, four or more TCRs of the invention, such as might be produced as a tetramer using the tetrameric domain of p53, or a complex formed by association of a plurality of TCRs of the invention with another molecule. The TCR complexes of the invention can be used to track or target cells presenting a particular antigen in vitro or in vivo, and can also be used to generate intermediates for other multivalent TCR complexes having such applications.
The TCRs of the invention may be used alone or in covalent or other association, preferably covalently, with a conjugate. The conjugates include a detectable label (for diagnostic purposes, where the TCR is used to detect the presence of cells presenting the ATIIHNLREEK-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 reviews)24, 539); 2. biotoxicity (Chaudhary et al, 1989, Nature 339, 394; Epel et al, 2002, Cancer Immunology and Immunotherapy 51, 565); 3. cytokines such as IL-2 and the like (Gillies et al, 1992, Proc. Natl. Acad. Sci. USA (PNAS)89, 1428; Card et al, 2004, Cancer Immunology and Immunotherapy)53, 345; Halin et al, 2003, Cancer Research 63, 3202); 4. antibody Fc fragment (Mosquera et al, 2005, Journal Of Immunology 174, 4381); 5. antibody scFv fragments (Zhu et al, 1995, International Journal of Cancer 62,319); 6. gold nanoparticles/nanorods (Lapotko et al, 2005, Cancer letters 239, 36; Huang et al, 2006, Journal of the American Chemical Society 128, 2115); 7. viral particles (Peng et al, 2004, Gene therapy)11, 1234); 8. liposomes (Mamot et al, 2005, Cancer research 65, 11631); 9. nano magnetic particles; 10. a prodrug activating enzyme (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, and the like.
In addition, the TCR of the invention may also be a hybrid TCR comprising sequences derived from more than one species. For example, studies have shown that murine TCRs are more efficiently expressed in human T cells than human TCRs. Thus, the inventive TCR may comprise a human variable domain and a murine constant domain. The drawback of this approach is the possibility of eliciting an immune response. Thus, there should be a regulatory regimen to immunosuppresse when it is used for adoptive T cell therapy to allow for the engraftment of murine expressing T cells.
It is understood that the amino acid names herein are expressed in the single english alphabet or the three english alphabets of the international common usage, and the single english alphabet and the three english alphabets of the amino acid names correspond to each other as follows: ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y) and Val (V).
Nucleic acid molecules
A second aspect of the invention provides a nucleic acid molecule encoding a TCR molecule of the first aspect of the invention or a part thereof, which part 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:
αCDR1-gacagctcctccacctac(SEQ ID NO:17)
αCDR2-attttttcaaatatggacatg(SEQ ID NO:18)
αCDR3-gcagagagtccgcctgataactatggtcagaattttgtc(SEQ ID NO:19)。
the nucleotide sequence encoding the CDR regions of the β chain of the TCR molecules of the first aspect of the invention is as follows:
βCDR1-aaaggacacagtcat(SEQ ID NO:20)
βCDR2-ctccagaaagaaaatatc(SEQ ID NO:21)
βCDR3-gccagctcaccggttgccggggagctgttt(SEQ ID NO:22)。
thus, the nucleotide sequence of the nucleic acid molecule of the invention encoding the TCR alpha chain of the invention comprises SEQ ID NO 17, 18 and 19 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 20, 21 and 22.
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 9 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 11. More preferably, the nucleotide sequence of the nucleic acid molecule of the invention comprises SEQ ID NO 12 and/or SEQ ID NO 14.
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. 9.
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 may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. The DNA may be the coding strand or the non-coding strand.
Carrier
The invention also relates to vectors comprising the nucleic acid molecules of the invention, including expression vectors, i.e. constructs capable of expression in vivo or in vitro. Commonly used vectors include bacterial plasmids, bacteriophages and animal and plant viruses.
Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, baculoviral vectors.
Preferably, the vector can transfer the nucleotide of the invention into a cell, such as a T cell, such that the cell expresses a TCR specific for the SAGE1 antigen. Ideally, the vector should be capable of sustained high level expression in T cells.
Cells
The invention also relates to genetically engineered host cells that have been engineered with the vectors or coding sequences of the invention. The host cell comprises a vector of the invention or has integrated into its chromosome a nucleic acid molecule of the invention. The host cell is selected from: prokaryotic and eukaryotic cells, such as E.coli, yeast cells, CHO cells, and the like.
In addition, the invention also includes isolated cells, particularly T cells, that express the TCRs of the invention. The T cell may be derived from a T cell isolated from a subject, or may be part of a mixed population of cells isolated from a subject, such as a population of Peripheral Blood Lymphocytes (PBLs). For example, the cells may be isolated from Peripheral Blood Mononuclear Cells (PBMC), which may be CD4 + Helper T cell or CD8 + Cytotoxic T cells. The cell may be in CD4 + Helper T cell/CD 8 + A mixed population of cytotoxic T cells. Generally, the cells can be activated with an antibody (e.g., an anti-CD 3 or anti-CD 28 antibody) to render them more amenable to transfection, e.g., transfection with a vector comprising a nucleotide sequence encoding a TCR molecule of the invention.
Alternatively, the cell of the invention may also be or be derived from a stem cell, such as a Hematopoietic Stem Cell (HSC). Gene transfer to HSCs does not result in TCR expression on the cell surface, since the CD3 molecule is not expressed on the stem cell surface. However, when stem cells differentiate into lymphoid precursors (lymphoid precursors) that migrate to the thymus, expression of the CD3 molecule will initiate expression of the introduced TCR molecule on the surface of the thymocytes.
There are many methods suitable for T cell transfection using DNA or RNA encoding the TCR of the invention (e.g., Robbins et al, (2008) J.Immunol.180: 6116-. T cells expressing the inventive TCR can be used in adoptive immunotherapy. Those skilled in the art will be able to recognize many suitable methods for adoptive therapy (e.g., Rosenberg et al, (2008) Nat Rev Cancer8 (4): 299-308).
SAGE1 antigen-related diseases
The present invention also relates to a method for treating and/or preventing a disease associated with SAGE1 antigen protein in a subject, comprising the step of adoptively transferring SAGE 1-specific T cells to the subject. The SAGE 1-specific T cells recognized the ATIIHNLREEK-HLA-A1101 complex.
SAGE1 specific T cells of the invention can be used to treat any SAGE1 antigen protein related disease presenting SAGE1 antigen short peptide ATIIHNLREEK-HLA-A1101 complex, including but not limited to tumors such as melanoma, and other solid tumors such as gastric cancer, lung cancer, esophageal cancer, bladder cancer, head and neck squamous cell carcinoma, prostate cancer, breast cancer, colon cancer, ovarian cancer, etc.
Method of treatment
Treatment may be carried out by isolating T cells from patients or volunteers with a disease associated with SAGE1 antigen and introducing the TCR of the invention into such T cells, followed by reinfusion of these genetically engineered cells into the patient. Accordingly, the present invention provides a method of treating a disease associated with SAGE1 comprising infusing into a patient isolated T cells expressing a TCR according to the invention, preferably the T cells are derived from the patient themselves. 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 present invention include:
(1) the TCR provided by the invention can be specifically bound with SAGE1 antigen short peptide complex ATIIHNLREEK-HLA-A1101, and cells transduced with the TCR provided by the invention can be specifically activated and have strong specific killing effect on target cells.
The following specific examples further illustrate the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example as described in Sambrook and Russell et al, Molecular Cloning: A Laboratory Manual (third edition) (2001) CSHL Press, or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.
Example 1 cloning SAGE1 antigen short peptide specific T cells
Peripheral Blood Lymphocytes (PBLs) from healthy volunteers of HLA-A1101 were stimulated with the synthetic short peptide SAGE1388-398ATIIHNLREEK (Nanjing Kingskan Biotech Co., Ltd.). SAGE1388-398ATIIHNLREEK short peptide and HLA-A1101 with biotin label are renatured to prepare pHLA haploid. These haploids were combined with streptavidin labeled with PE (BD Co.) to form PE-labeled tetramers, which were sorted for double positive anti-CD 8-APC cells. The sorted cells were expanded and subjected to secondary sorting as described above, followed by monoclonal culture by limiting dilution. Monoclonal cells were stained with tetramer and double positive clones were selected as shown in FIG. 3.
Example 2 construction of TCR Gene and vector for obtaining SAGE1 antigen short peptide specific T cell clone
Using Quick-RNA TM Total RNA from SAGE 1388-398-specific, HLA-A1101-restricted T cell clone selected in example 1 was extracted by MiniPrep (ZYMO research). cDNA was synthesized using SMART RACE cDNA amplification kit from clontech, using primers designed to be conserved at the C-terminus of the human TCR gene. The sequences were cloned into the T vector (TAKARA) and sequenced. The alpha chain and beta chain sequence structures of the TCR expressed by the double positive clone are shown in figure 1 and figure 2 respectively after sequencing, and figure 1a, figure 1b, figure 1c and figure 1d are the TCR alpha chain variable domain amino acid sequence, the TCR alpha chain variable domain nucleotide sequence, the TCR alpha chain amino acid sequence and the TCR alpha chain nucleotide sequence respectively; fig. 2a, fig. 2b, fig. 2c and fig. 2d are a TCR β chain variable domain amino acid sequence, a TCR β chain variable domain nucleotide sequence, a TCR β chain amino acid sequence and a TCR β chain nucleotide sequence, respectively.
The alpha chain was identified to comprise CDRs with the following amino acid sequences:
αCDR1-DSSSTY(SEQ ID NO:6)
αCDR2-IFSNMDM(SEQ ID NO:7)
αCDR3-AESPPDNYGQNFV(SEQ ID NO:13)
the beta chain comprises CDRs having the following amino acid sequences:
βCDR1-KGHSH(SEQ ID NO:5)
βCDR2-LQKENI(SEQ ID NO:8)
βCDR3-ASSPVAGELF(SEQ ID NO:10)。
the full-length genes of TCR α and β chains were cloned into the lentiviral expression vector, plenti (adddge), by overlap (overlap) PCR, respectively. The method specifically comprises the following steps: the TCR alpha chain and the TCR beta chain are connected by overlap PCR to obtain the TCR alpha-2A-TCR beta segment. And (3) carrying out enzyme digestion and connection on the lentivirus expression vector and the TCR alpha-2A-TCR beta to obtain a pLenti-SAGE1TRA-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 antigenic short peptides of the invention
To obtain soluble TCR molecules, the α and β chains of the TCR molecules of the invention may comprise only the variable and part of the constant domains thereof, respectively, and a cysteine residue has been introduced into the constant domains of the α and β chains, respectively, to form artificial interchain disulfide bonds, at the positions Thr48 of exon 1 TRAC × 01 and Ser57 of exon 1 TRBC2 × 01, respectively; the amino acid sequence and nucleotide sequence of the alpha chain are shown in FIGS. 4a and 4b, respectively, and the amino acid sequence and nucleotide sequence of the beta chain are shown in FIGS. 5a and 5b, respectively, and the introduced cysteine residues are shown in bold and underlined letters. The above-mentioned gene sequences of interest for the TCR alpha and beta chains were synthesized and inserted into the expression vector pET28a + (Novagene) by standard methods described in Molecular Cloning A Laboratory Manual (third edition, Sambrook and Russell), and the upstream and downstream Cloning sites were NcoI and NotI, respectively. The insert was confirmed by sequencing without error.
The expression vectors for the TCR α and β chains were transformed into the expression bacteria BL21(DE3) by chemical transformation, respectively, the bacteria were grown in LB medium and induced with a final concentration of 0.5mM IPTG at an OD600 of 0.6, and the inclusion bodies formed after the α and β chains of the TCR were expressed were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution several times, and finally the inclusion bodies were dissolved in 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT),10mM ethylenediaminetetraacetic acid (EDTA),20mM tris (pH 8.1).
The TCR α and β chains after lysis were separated by 1: 1 was rapidly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1),3.7mM cystamine,6.6mM beta-mericapoethylamine (4 ℃) to a final concentration of 60 mg/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 FIG. 6.
Example 4 binding characterization
BIAcore analysis
This example demonstrates that soluble TCR molecules of the invention are capable of specifically binding to the ATIIHNLREEK-HLA a1101 complex.
The binding activity of the TCR molecules obtained in example 3 to the ATIIHNLREEK-HLA A1101 complex was measured using a BIAcore T200 real-time assay system. Anti-streptavidin antibody (GenScript) was added to coupling buffer (10mM sodium acetate buffer, pH 4.77), and then the antibody was passed through CM5 chip previously activated with EDC and NHS to immobilize the antibody on the chip surface, and finally the unreacted activated surface was blocked with ethanolamine hydrochloric acid solution to complete the coupling process at a coupling level of about 15,000 RU.
The low concentration of streptavidin was flowed over the antibody coated chip surface, then ATIIHNLREEK-HLA A1101 complex was flowed over the detection channel, the other channel served as a reference channel, and 0.05mM biotin was flowed over the chip at a flow rate of 10. mu.L/min for 2min to block the remaining binding sites of streptavidin.
The ATIIHNLREEK-HLA A1101 complex is prepared as follows:
a. purification of
Collecting 100ml of E.coli bacterial liquid for inducing expression of heavy chain or light chain, centrifuging at 8000g at 4 ℃ for 10min, washing the bacterial cells once with 10ml of PBS, then resuspending the bacterial cells by vigorous shaking with 5ml of BugBuster Master Mix Extraction Reagents (Merck), carrying out rotary incubation at room temperature for 20min, centrifuging at 6000g at 4 ℃ for 15min, discarding supernatant, and collecting inclusion bodies.
Resuspending the inclusion body in 5ml of BugBuster Master Mix, and rotary incubating at room temperature for 5 min; adding 30ml of BugBuster diluted by 10 times, mixing uniformly, and centrifuging at 6000g at 4 ℃ for 15 min; 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 to resuspend the inclusion bodies at pH 8.0, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, finally dissolving the inclusion bodies by using 20mM Tris-HCl 8M urea, detecting the purity of the inclusion bodies by SDS-PAGE, and measuring the concentration by using a BCA kit.
b. Renaturation
The synthesized short peptide ATIIHNLREEK (Nanjing Kingskan Biotech Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg/ml. Inclusion bodies of light and heavy chains were 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. ATIIHNLREEK peptide was added to 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 on a HiTrap Q HP (GE general electric) anion exchange column (5ml bed volume). The protein was eluted using a 0-400mM NaCl linear gradient prepared using an Akta purifier (GE general electric company), 20mM Tris pH 8.0, pMHC was eluted at about 250mM NaCl, the peak fractions were collected, and the purity was checked by SDS-PAGE.
d. Biotinylation of the peptide
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
Biotinylated-labeled pMHC molecules were concentrated to 1ml using Millipore ultrafiltration tubes, biotinylated pMHC was purified by gel filtration chromatography, HiPrepTM 16/60S200HR 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 single peak elution at approximately 55 ml. The fractions containing the protein were pooled, concentrated using Millipore ultrafiltration tubes, protein concentration was determined by BCA (Thermo), and biotinylated pMHC molecules were stored in aliquots at-80 ℃ by addition of the protease inhibitor cocktail (Roche). Kinetic parameters were calculated using BIAcore Evaluation software, and the kinetic profile of the soluble TCR molecules and complex binding obtained is shown in FIG. 7.
Example 5SAGE 1-specific T cell receptor Lentiviral packaging and Primary T cell transduction SAGE1TCR
(a) Production of lentiviruses by Rapid-mediated transient transfection of 293T/17 cells
A third generation lentiviral packaging system was used to package lentiviruses containing the gene encoding the desired TCR. 293T/17 cells were transfected with 4 plasmids (a lentiviral vector containing pLenti-SAGE1TRA-2A-TRB-IRES-NGFR as described In example 2, and 3 plasmids containing other components necessary for the construction of infectious but non-replicating lentiviral particles) using rapid-mediated transient transfection (Express-In-mediated transfection) (Open Biosystems).
For transfection, cells were seeded at day 0 on a 15 cm petri dish at 1.7X 10 7 293T/17 cells, which were distributed evenly on the culture dish with a degree of confluence slightly higher than 50%. Transfecting plasmids on day 1, packaging pLenti-SAGE1TRA-2A-TRB-IRES-NGFR and pLenti-eGFP pseudoviruses, mixing the expression plasmids with packaging plasmids pMDLg/pRRE, pRSV-REV and pMD.2G uniformly, and using the following dosage in a 15 cm diameter plate: 15 microgram: 5 microgram, 5 microgram: 5 micrograms. The ratio of the transfection reagent PEI-MAX to the plasmid was 2:1 and the amount used was 60 micrograms per plate. The specific operation is as follows: adding the expression plasmid and the packaging plasmid into 1800 microliters of OPTI-MEM (Gibbo, catalog No. 31985- 2 And (5) culturing. Transfection was carried out for 5-7 hours, the transfection medium was removed, and the medium was changed to DMEM (Gibbco, Cat. C11995500bt) complete medium containing 10% fetal bovine serum at 37 ℃ under 5% CO 2 And (5) culturing. Culture supernatants containing packaged lentiviruses were collected on days 3 and 4. To harvest the packaged lentivirus, the collected culture supernatant was centrifuged at 3000g for 15min to remove cell debris, filtered through a 0.22 micron filter (Merck Millipore, catalog # SLGP033RB), and finally concentrated using a 50KD cut-off concentration tube (Merck Millipore, catalog # UFC905096), to remove most of the supernatant, and finally concentrated to 1ml, and aliquots were frozen at-80 ℃. Pseudovirus samples were taken for virus titer determination, procedures were referenced to p24ELISA (Clontech, cat # 632200) kitAnd (4) instructions. As a control, a pseudovirus transformed with pLenti-eGFP was also included.
(b) Transduction of primary T cells with lentiviruses containing SAGE 1-specific T cell receptor genes
CD8 isolated from blood of healthy volunteers + T cells, then transduced with the packaged lentivirus. These cells were counted in 48-well plates in 1X 10 medium 1640 (Gibbo, Cat. No. C11875500bt) with 10% FBS (Gibbo, Gibco, Cat. No. C10010500BT) containing 50IU/ml IL-2 and 10ng/ml IL-7 6 Cells/ml (0.5 ml/well) were incubated with pre-washed anti-CD 3/CD28 antibody-coated beads (T cell amplicons, life technologies, cat No. 11452D) overnight for stimulation, cells: bead 2: 1.
after overnight stimulation, the concentrated SAGE 1-specific T cell receptor gene lentivirus was added at an MOI of 10 according to the virus titer measured by the p24ELISA kit, and centrifuged at 900g at 32 ℃ for 1 hour. After infection, the lentivirus infection solution was removed and the cells were resuspended in 1640 medium containing 10% FBS with 50IU/ml IL-2 and 10ng/ml IL-7 at 37 ℃/5% CO 2 The cells were cultured for 3 days. Cells were counted 3 days after transduction and diluted to 0.5X 10 6 Individual cells/ml. Cells were counted every two days, replaced or supplemented with fresh medium containing 50IU/ml IL-2 and 10ng/ml IL-7, maintaining the cells at 0.5X 10 6 -1×10 6 Individual cells/ml. Cells were analyzed by flow cytometry starting on day 3 and starting on day 5 for functional assays (e.g., ELISPOT for IFN- γ release and non-radioactive cytotoxicity assays). Cryopreserving aliquots of cells, at least 4X 10, from day 10 or as the cells slow division and become smaller in size 6 Individual cell/tube (1X 10) 7 Individual cells/ml, 90% FBS/10% DMSO).
(c) Tetramer staining of TCR transduced primary T cells
SAGE1388-398ATIIHNLREEK short peptide and HLA-A1101 with biotin labels are renatured to prepare pHLA haploid. These haplotypes were combined with PE-labeled streptavidin (BD) into a PE-labeled tetramer called SAGE 1-tetramer-PE. This tetramer was able to label T cells expressing the SAGE 1-specific T cell receptor gene as positive cells. Incubating the transfected T cell sample of (b) with SAGE1-tetramer-PE on ice for 30 min, then adding anti-mouse beta chain-APC (biolegend) antibody, and continuing the incubation on ice for 15 min. Samples were washed 2 times with PBS containing 2% FBS and then T cells that were double positive for SAGE1-tetramer-PE and CD8 expressing the SAGE1 specific T cell receptor gene were detected OR sorted using BD Calibur OR BD Arial and data analysis was analyzed using CellQuest software (BD) OR FlowJo software (Tree Star Inc, Ashland, OR).
As a result of detection analysis, as shown in FIG. 8, after staining with SAGE1-tetramer-PE and anti-mouse beta chain antibody, the T cells of the blank control group without TCR lentivirus infection were few SAGE1-tetramer-PE and anti-mouse beta chain-APC double positive cells, while the T cells infected with TCR lentivirus appeared SAGE1-tetramer-PE and anti-mouse beta chain-APC double positive cells expressing TCR, and when staining with other tetramer-PE than SAGE1-tetramer-PE, there were few non-specific double positive cells.
Example 6 validation of SAGE 1-specific TCR function
4.1ELISPOT protocol
The following assays were performed to demonstrate the activation of TCR-transduced T cells in response specifically to target cells. IFN-. gamma.production as measured by the ELISPOT assay was used as a readout for T cell activation.
Reagent
Test medium: 10% FBS (Gibbo, catalog number 16000-
Washing buffer solution: 0.01M PBS/0.05% Tween 20
PBS (Gibbo Co., catalog number C10010500BT)
PVDF ELISPOT 96-well plate (Merck Millipore, Cat. No. MSIPS4510)
Human IFN-. gamma.ELISPOT PVDF-enzyme kit (BD) contains all other reagents required (capture and detection antibodies, streptavidin-alkaline phosphatase and BCIP/NBT solution)
Method
Target cell preparation
The target cells of this example were Epstein-Barr virus (EBV) transformed immortalized Lymphoblastoid Cell Lines (LCLs). B95-8 cells were induced to produce EBV-containing culture supernatants by phorbol myristate acetate (TPA), centrifuged at 4 deg.C/600 g for 10min to remove impurities, filtered through 0.22 μm filter, and aliquoted for-70 deg.C storage. From Peripheral Blood Lymphocytes (PBLs) of healthy volunteers of the genotype HLA-A11/A02/A24 (both homozygote and heterozygote), 10ml of 2X 10-concentration blood was taken 7 One ml PBL suspension in 25 cm square culture flask, adding cyclosporine at 37 deg.C/CO 2 Incubate for 1 hour in incubator, thaw one EBV portion quickly, add to the cells at 1/10 dilution, shake gently and place the flask upright at 37 deg.C/CO 2 Culturing in an incubator. After 12 days of culture, the culture was continued by adding 10ml of a medium, and after about 30 days, the culture was further expanded and subjected to flow assay, in which CD19 was present + CD23 hi CD58 + Is an immortalized Lymphoblastoid Cell Line (LCL). The ELISPOT test uses HLA-A11/02 as the target cell.
Effector cell preparation
The effector cells (T cells) of this assay were CD8 expressing SAGE 1-specific TCR analyzed by flow cytometry in example 3 + T cells, and CD8 of the same volunteer + T served as negative control effector cells. T cells were stimulated with anti-CD 3/CD28 coated beads (T cell amplicons, life technologies), transduced with lentivirus carrying SAGE1 specific TCR gene (according to example 3), expanded in 1640 medium containing 10% FBS with 50IU/ml IL-2 and 10ng/ml IL-7 until 9-12 days after transduction, then placed in test medium and washed by centrifugation at 300g for 10min at RT. The cells were then resuspended in the test medium at 2 × the desired final concentration. Negative control effector cells were treated as well.
ELISPOT
The well plates were prepared as follows according to the manufacturer's instructions: 10ml of sterile PBS per plate 1: anti-human IFN-. gamma.capture antibody was diluted at 200, and 100. mu.l of the diluted capture antibody was aliquoted into each well. The plates were incubated overnight at 4 ℃. After incubation, the well plates were washed to remove excess capture antibody. 100 μ l/well of RPMI1640 medium containing 10% FBS was added and the well plates were incubated at room temperature for 2 hours to close the well plates. The media was then washed from the well plate, and any residual wash buffer was removed by flicking and tapping the ELISPOT well plate on paper.
SAGE1TCR-T cells (SAGE1TCR transduced T cells, effector cells), Non-SAGE1TCR transduced T cells (Non-SAGE1TCR-T, effector cells expressing other TCR) and LCLs-A11/02 (target cells) were prepared as described in example 3 and the corresponding short peptide was added to the respective experimental groups, where P is P SAGE1 Is SAGE1388-398ATIIHNLREEK short peptide, P A02 And P A11 Is a non-SAGE1TCR specific binding short peptide.
The components of the assay were then added to ELISPOT well plates in the following order:
130 microliter target cells 154000 cells/ml (total of about 20000 target cells/well is obtained).
50 microliter of effector cells (2000 SAGE1TCR-T cells).
20 microliter 10 -4 Mol/l SAGE1PX 251388-398 ATIIHNLREEK/P A02 /P A11 Short peptide solution (final concentration of 10) -6 Moles/liter).
All wells were made in triplicate for addition.
The well plates were then incubated overnight (37 ℃/5% CO2) for the next day, the medium was discarded, the well plates were washed 2 times with double distilled water, then 3 times with wash buffer, and tapped on paper towels to remove residual wash buffer. Primary antibody was then detected by dilution with PBS containing 10% FBS and added to each well at 100. mu.l/well. The well plates were incubated at room temperature for 2 hours, washed 3 times with wash buffer, and the well plates were tapped on paper towels to remove excess wash buffer.
PBS containing 10% FBS was used at 1: streptavidin-alkaline phosphatase was diluted 10000, 100 microliters of diluted streptavidin-alkaline phosphatase was added to each well and the wells were incubated at room temperature for 1 hour. The plate was then washed 3 times with wash buffer and 2 times with PBS, and the excess wash buffer and PBS was removed by tapping the plate on a paper towel. 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. During this period, spots on the developing well plate were routinely detected, and the optimal time for terminating the reaction was determined. The BCIP/NBT solution was removed and the well plate was rinsed with double-distilled water to stop the development reaction, spun-dried, then the bottom of the well plate was removed, the well plate was dried at room temperature until each well was completely dried, and then the spots formed in the bottom film of the well plate were counted using an immune spot plate counter (CTL, cell Technology Limited).
Results
SAGE1TCR transduced T cells were tested by ELISPOT assay (described above) for IFN- γ release in response to SAGE1388-398ATIIHNLREEK short peptide loaded target cells and non-specific short peptide loaded target cells. The number of ELSPOT spots observed in each well was plotted using Graphpad prism 6.
As shown in FIG. 9, SAGE1TCR-T cells (effector cells) or LCL cells (target cells) alone added the corresponding short peptide without IFN-. gamma.release.
SAGE1TCR-T cells (effector cells) capable of interacting with a load P SAGE1 The LCLs-A11/02 cells react to release more IFN-gamma.
SAGE1TCR-T cell (Effector cell) pair Loading P A11 Or P A02 The LCLs-A11/02 cells released little IFN-gamma.
Non-SAGE1TCR transduced T cells (Non-SAGE1TCR-T, effector cells expressing other TCR) versus P-loaded SAGE1 The release of IFN-gamma from LCLs-A11/02 cells was minimal, i.e., the non-SAGE1TCR had no recognition effect on SAGE1388-398ATIIHNLREEK short peptides.
4.2 non-Radioactive cytotoxicity assay protocol
The test is 51 Colorimetric substitution test for Cr release cytotoxicity test Lactate Dehydrogenase (LDH) released after cell lysis was quantitatively determined. LDH released in the medium was detected using a 30 min coupled enzymatic reaction in which LDH converted a tetrazolium salt (INT) to red formazan (formazan). The amount of red product produced is proportional to the number of cells lysed. 490nm visible absorbance data can be collected using a standard 96-well plate reader.
Material
CytoTox
Figure BDA0001597193560000241
The non-radioactive cytotoxicity assay (Promega, G1780) contained a substrate mixture, assay buffer, lysis solution and stop buffer.
Test medium: 10% FBS (heat inactivated, Gibbo, Cat. 16000-.
Microwell round bottom 96 well tissue culture plates (Nunc, Cat. No. 163320)
96-well immunoplate Maxisorb (Nunc, Cat. No. 442404)
Method
Target cell preparation
The target cells LCLs used in this assay were prepared as described above for the ELISPOT protocol. Target cells were prepared in assay medium: the concentration of the target cells is adjusted to 3X 10 5 One/ml, 50. mu.l/well to obtain 1.5X 10 4 Individual cells/well.
Effector cell preparation
The effector cells (T cells) of this experiment were CD8 expressing SAGE1 specific TCR analyzed by flow cytometry in example 3 + T cells (SAGE1 TCR-T). Other TCRs specific for short peptides expressing non-SAGE1 were used as controls.
Ratio of effector cells to target cells 1: 1 and 2:1, wherein the target cells are quantitative (3X 10) 5 One/ml, 50. mu.l/well to obtain 1.5X 10 4 Individual cells/well), effector cells vary according to the effective target ratio.
Preparation of short peptide solution
SAGE1388-398ATIIHNLREEK (or P) X1 And P X2 Two non-specific short peptides) were diluted to 10-fold with 5% FBS-containing RPMI1640 medium by 10-fold dilution method -6 M working solution, the final concentration of which is 10 after the working solution is added into an experimental group -6 M。
(a) Detection of killing capability of effector cells by loading target cells with short peptides at different concentrations
Preparation of the test
The components of the assay were added to a microwell round bottom 96 well tissue culture plate in the following order:
80ul of target cells (prepared as described above) were added to each well
100ul of effector cells (prepared as described above) were added to each well
20ul of the short peptide solution was added to each well (20 ul of medium was directly supplemented to the experimental group without short peptide loading).
A control group was prepared as follows:
experimental group without short peptide loading: contains 100ul effector cells and 100ul target cells.
The effector cells release spontaneously: there were only 100ul of effector cells.
Target cells release: there are only 100ul of target cells.
Maximum release of target cells: there are only 100ul target cells.
Reagent medium control: there were only 200ul of medium.
All wells were prepared in triplicate and the final volume was 200ul (insufficient media make up).
Incubate at 37 ℃ for 24 hours. Before collecting supernatants from all wells, target cell maximum release control wells were placed at-70 ℃ for approximately 30 minutes and thawed at 37 ℃ for 15 minutes to allow total lysis of target cells.
The plate was centrifuged at 250g for 4 min. 50ul of supernatant from each well of the assay plate was transferred to the corresponding well of a 96-well immunoplate Maxisorb plate. The substrate mixture was reconstituted with assay buffer (12ml) and 50ul was added to each well of the plate. The plate was covered and incubated at room temperature in the dark for 30 minutes. 50ul of stop solution was added to each well of the plate to stop the reaction. The absorbance at 490nm was recorded counted over 1 hour after addition of the stop solution.
The result of the calculation
The absorbance values of the medium background were subtracted from all the absorbance values of the experimental, target cell spontaneous release and effector cell spontaneous release groups.
The corrected values obtained above were substituted into the following formula to calculate the percent cytotoxicity resulting from each effect-to-target ratio.
% cytotoxicity 100 × (experiment-effector cell spontaneous-target cell spontaneous)/(target cell maximal-target cell spontaneous)
Results
SAGE1TCR-T cells were tested for LDH release in response to SAGE1388-398ATIIHNLREEK short peptide and non-specific short peptide loaded target cells by non-radioactive cytotoxicity assays (as described above). The absorbance of 490nm visible light in each well was plotted using Graphpad prism 6.
The statistical results of the experimental data are shown in FIG. 10, load 10 -6 Under the concentration of the short peptide of M, the killing effect of SAGE1TCR-T cells on target cells LCLs-A11/02 is gradually enhanced along with the increase of the proportion of effector cells and target cells; for non-loaded short peptide or loaded P X1 /P X2 The target cells LCLs-A11/02 of the nonspecific short peptide have no killing effect.
Control group of Non-SAGE1 TCR-transduced T cells (Non-SAGE1TCR-T, effector cells expressing other TCR) versus P-loaded SAGE1 The LCLs-A11/02 cells of (1) had no lytic effect, and it can be seen that the non-SAGE1TCR cells had no recognition effect on SAGE1388-398ATIIHNLREEK short peptides.
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> Guangzhou biomedical and health research institute of Chinese academy of sciences
<120> T cell receptor recognizing SAGE1 antigen short peptide
<130> P2017-2276
<160> 29
<170> PatentIn version 3.5
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Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser Val Arg Glu Gly Asp
1 5 10 15
Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser Ser Ser Thr Tyr Leu
20 25 30
Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu Gln Leu Leu Thr Tyr
35 40 45
Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln Arg Leu Thr Val Leu
50 55 60
Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg Ile Ala Asp Thr Gln
65 70 75 80
Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu Ser Pro Pro Asp Asn
85 90 95
Tyr Gly Gln Asn Phe Val Phe Gly Pro Gly Thr Arg Leu Ser Val Leu
100 105 110
Pro Tyr
<210> 2
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<212> PRT
<213> Artificial sequence (artificial sequence)
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Asn Ala Gly Val Met Gln Asn Pro Arg His Leu Val Arg Arg Arg Gly
1 5 10 15
Gln Glu Ala Arg Leu Arg Cys Ser Pro Met Lys Gly His Ser His Val
20 25 30
Tyr Trp Tyr Arg Gln Leu Pro Glu Glu Gly Leu Lys Phe Met Val Tyr
35 40 45
Leu Gln Lys Glu Asn Ile Ile Asp Glu Ser Gly Met Pro Lys Glu Arg
50 55 60
Phe Ser Ala Glu Phe Pro Lys Glu Gly Pro Ser Ile Leu Arg Ile Gln
65 70 75 80
Gln Val Val Arg Gly Asp Ser Ala Ala Tyr Phe Cys Ala Ser Ser Pro
85 90 95
Val Ala Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu
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<210> 3
<211> 254
<212> PRT
<213> Artificial sequence (artificial sequence)
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Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser Val Arg Glu Gly Asp
1 5 10 15
Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser Ser Ser Thr Tyr Leu
20 25 30
Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu Gln Leu Leu Thr Tyr
35 40 45
Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln Arg Leu Thr Val Leu
50 55 60
Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg Ile Ala Asp Thr Gln
65 70 75 80
Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu Ser Pro Pro Asp Asn
85 90 95
Tyr Gly Gln Asn Phe Val Phe Gly Pro Gly Thr Arg Leu Ser Val Leu
100 105 110
Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser
115 120 125
Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln
130 135 140
Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys
145 150 155 160
Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val
165 170 175
Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn
180 185 190
Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys
195 200 205
Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn
210 215 220
Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val
225 230 235 240
Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
245 250
<210> 4
<211> 291
<212> PRT
<213> Artificial sequence (artificial sequence)
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Asn Ala Gly Val Met Gln Asn Pro Arg His Leu Val Arg Arg Arg Gly
1 5 10 15
Gln Glu Ala Arg Leu Arg Cys Ser Pro Met Lys Gly His Ser His Val
20 25 30
Tyr Trp Tyr Arg Gln Leu Pro Glu Glu Gly Leu Lys Phe Met Val Tyr
35 40 45
Leu Gln Lys Glu Asn Ile Ile Asp Glu Ser Gly Met Pro Lys Glu Arg
50 55 60
Phe Ser Ala Glu Phe Pro Lys Glu Gly Pro Ser Ile Leu Arg Ile Gln
65 70 75 80
Gln Val Val Arg Gly Asp Ser Ala Ala Tyr Phe Cys Ala Ser Ser Pro
85 90 95
Val Ala Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu
100 105 110
Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro
115 120 125
Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu
130 135 140
Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn
145 150 155 160
Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys
165 170 175
Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu
180 185 190
Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys
195 200 205
Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp
210 215 220
Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg
225 230 235 240
Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser
245 250 255
Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala
260 265 270
Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp
275 280 285
Ser Arg Gly
290
<210> 5
<211> 5
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 5
Lys Gly His Ser His
1 5
<210> 6
<211> 6
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 6
Asp Ser Ser Ser Thr Tyr
1 5
<210> 7
<211> 7
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 7
Ile Phe Ser Asn Met Asp Met
1 5
<210> 8
<211> 6
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 8
Leu Gln Lys Glu Asn Ile
1 5
<210> 9
<211> 342
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 9
ggagaggatg tggagcagag tcttttcctg agtgtccgag agggagacag ctccgttata 60
aactgcactt acacagacag ctcctccacc tacttatact ggtataagca agaacctgga 120
gcaggtctcc agttgctgac gtatattttt tcaaatatgg acatgaaaca agaccaaaga 180
ctcactgttc tattgaataa aaaggataaa catctgtctc tgcgcattgc agacacccag 240
actggggact cagctatcta cttctgtgca gagagtccgc ctgataacta tggtcagaat 300
tttgtctttg gtcccggaac cagattgtcc gtgctgccct at 342
<210> 10
<211> 10
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 10
Ala Ser Ser Pro Val Ala Gly Glu Leu Phe
1 5 10
<210> 11
<211> 336
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 11
aatgccggcg tcatgcagaa cccaagacac ctggtcagga ggaggggaca ggaggcaaga 60
ctgagatgca gcccaatgaa aggacacagt catgtttact ggtatcggca gctcccagag 120
gaaggtctga aattcatggt ttatctccag aaagaaaata tcatagatga gtcaggaatg 180
ccaaaggaac gattttctgc tgaatttccc aaagagggcc ccagcatcct gaggatccag 240
caggtagtgc gaggagattc ggcagcttat ttctgtgcca gctcaccggt tgccggggag 300
ctgttttttg gagaaggctc taggctgacc gtactg 336
<210> 12
<211> 762
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 12
ggagaggatg tggagcagag tcttttcctg agtgtccgag agggagacag ctccgttata 60
aactgcactt acacagacag ctcctccacc tacttatact ggtataagca agaacctgga 120
gcaggtctcc agttgctgac gtatattttt tcaaatatgg acatgaaaca agaccaaaga 180
ctcactgttc tattgaataa aaaggataaa catctgtctc tgcgcattgc agacacccag 240
actggggact cagctatcta cttctgtgca gagagtccgc ctgataacta tggtcagaat 300
tttgtctttg gtcccggaac cagattgtcc gtgctgccct atatccagaa ccctgaccct 360
gccgtgtacc agctgagaga ctctaaatcc agtgacaagt ctgtctgcct attcaccgat 420
tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 480
actgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 540
aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 600
ttccccagcc cagaaagttc ctgtgatgtc aagctggtcg agaaaagctt tgaaacagat 660
acgaacctaa actttcaaaa cctgtcagtg attgggttcc gaatcctcct cctgaaagtg 720
gccgggttta atctgctcat gacgctgcgg ctgtggtcca gc 762
<210> 13
<211> 13
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 13
Ala Glu Ser Pro Pro Asp Asn Tyr Gly Gln Asn Phe Val
1 5 10
<210> 14
<211> 873
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 14
aatgccggcg tcatgcagaa cccaagacac ctggtcagga ggaggggaca ggaggcaaga 60
ctgagatgca gcccaatgaa aggacacagt catgtttact ggtatcggca gctcccagag 120
gaaggtctga aattcatggt ttatctccag aaagaaaata tcatagatga gtcaggaatg 180
ccaaaggaac gattttctgc tgaatttccc aaagagggcc ccagcatcct gaggatccag 240
caggtagtgc gaggagattc ggcagcttat ttctgtgcca gctcaccggt tgccggggag 300
ctgttttttg gagaaggctc taggctgacc gtactggagg acctgaaaaa cgtgttccca 360
cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 420
ctggtgtgcc tggccacagg cttctacccc gaccacgtgg agctgagctg gtgggtgaat 480
gggaaggagg tgcacagtgg ggtcagcaca gacccgcagc ccctcaagga gcagcccgcc 540
ctcaatgact ccagatactg cctgagcagc cgcctgaggg tctcggccac cttctggcag 600
aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 660
tggacccagg atagggccaa acctgtcacc cagatcgtca gcgccgaggc ctggggtaga 720
gcagactgtg gcttcacctc cgagtcttac cagcaagggg tcctgtctgc caccatcctc 780
tatgagatct tgctagggaa ggccaccttg tatgccgtgc tggtcagtgc cctcgtgctg 840
atggccatgg tcaagagaaa ggattccaga ggc 873
<210> 15
<211> 275
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 15
Met Lys Thr Phe Ala Gly Phe Ser Phe Leu Phe Leu Trp Leu Gln Leu
1 5 10 15
Asp Cys Met Ser Arg Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser
20 25 30
Val Arg Glu Gly Asp Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser
35 40 45
Ser Ser Thr Tyr Leu Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu
50 55 60
Gln Leu Leu Thr Tyr Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln
65 70 75 80
Arg Leu Thr Val Leu Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg
85 90 95
Ile Ala Asp Thr Gln Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu
100 105 110
Ser Pro Pro Asp Asn Tyr Gly Gln Asn Phe Val Phe Gly Pro Gly Thr
115 120 125
Arg Leu Ser Val Leu Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr
130 135 140
Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr
145 150 155 160
Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val
165 170 175
Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys
180 185 190
Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala
195 200 205
Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser
210 215 220
Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr
225 230 235 240
Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile
245 250 255
Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu
260 265 270
Trp Ser Ser
275
<210> 16
<211> 310
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 16
Met Asp Thr Arg Leu Leu Cys Cys Ala Val Ile Cys Leu Leu Gly Ala
1 5 10 15
Gly Leu Ser Asn Ala Gly Val Met Gln Asn Pro Arg His Leu Val Arg
20 25 30
Arg Arg Gly Gln Glu Ala Arg Leu Arg Cys Ser Pro Met Lys Gly His
35 40 45
Ser His Val Tyr Trp Tyr Arg Gln Leu Pro Glu Glu Gly Leu Lys Phe
50 55 60
Met Val Tyr Leu Gln Lys Glu Asn Ile Ile Asp Glu Ser Gly Met Pro
65 70 75 80
Lys Glu Arg Phe Ser Ala Glu Phe Pro Lys Glu Gly Pro Ser Ile Leu
85 90 95
Arg Ile Gln Gln Val Val Arg Gly Asp Ser Ala Ala Tyr Phe Cys Ala
100 105 110
Ser Ser Pro Val Ala Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu
115 120 125
Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val
130 135 140
Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu
145 150 155 160
Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp
165 170 175
Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln
180 185 190
Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser
195 200 205
Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His
210 215 220
Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp
225 230 235 240
Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala
245 250 255
Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly
260 265 270
Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr
275 280 285
Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys
290 295 300
Arg Lys Asp Ser Arg Gly
305 310
<210> 17
<211> 18
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 17
gacagctcct ccacctac 18
<210> 18
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 18
attttttcaa atatggacat g 21
<210> 19
<211> 39
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 19
gcagagagtc cgcctgataa ctatggtcag aattttgtc 39
<210> 20
<211> 15
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 20
aaaggacaca gtcat 15
<210> 21
<211> 18
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 21
ctccagaaag aaaatatc 18
<210> 22
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 22
gccagctcac cggttgccgg ggagctgttt 30
<210> 23
<211> 825
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 23
atgaagacat ttgctggatt ttcgttcctg tttttgtggc tgcagctgga ctgtatgagt 60
agaggagagg atgtggagca gagtcttttc ctgagtgtcc gagagggaga cagctccgtt 120
ataaactgca cttacacaga cagctcctcc acctacttat actggtataa gcaagaacct 180
ggagcaggtc tccagttgct gacgtatatt ttttcaaata tggacatgaa acaagaccaa 240
agactcactg ttctattgaa taaaaaggat aaacatctgt ctctgcgcat tgcagacacc 300
cagactgggg actcagctat ctacttctgt gcagagagtc cgcctgataa ctatggtcag 360
aattttgtct ttggtcccgg aaccagattg tccgtgctgc cctatatcca gaaccctgac 420
cctgccgtgt accagctgag agactctaaa tccagtgaca agtctgtctg cctattcacc 480
gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 540
aaaactgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 600
aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 660
ttcttcccca gcccagaaag ttcctgtgat gtcaagctgg tcgagaaaag ctttgaaaca 720
gatacgaacc taaactttca aaacctgtca gtgattgggt tccgaatcct cctcctgaaa 780
gtggccgggt ttaatctgct catgacgctg cggctgtggt ccagc 825
<210> 24
<211> 930
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 24
atggacacca gactactctg ctgtgcggtc atctgtcttc tgggggcagg tctctcaaat 60
gccggcgtca tgcagaaccc aagacacctg gtcaggagga ggggacagga ggcaagactg 120
agatgcagcc caatgaaagg acacagtcat gtttactggt atcggcagct cccagaggaa 180
ggtctgaaat tcatggttta tctccagaaa gaaaatatca tagatgagtc aggaatgcca 240
aaggaacgat tttctgctga atttcccaaa gagggcccca gcatcctgag gatccagcag 300
gtagtgcgag gagattcggc agcttatttc tgtgccagct caccggttgc cggggagctg 360
ttttttggag aaggctctag gctgaccgta ctggaggacc tgaaaaacgt gttcccaccc 420
gaggtcgctg tgtttgagcc atcagaagca gagatctccc acacccaaaa ggccacactg 480
gtgtgcctgg ccacaggctt ctaccccgac cacgtggagc tgagctggtg ggtgaatggg 540
aaggaggtgc acagtggggt cagcacagac ccgcagcccc tcaaggagca gcccgccctc 600
aatgactcca gatactgcct gagcagccgc ctgagggtct cggccacctt ctggcagaac 660
ccccgcaacc acttccgctg tcaagtccag ttctacgggc tctcggagaa tgacgagtgg 720
acccaggata gggccaaacc tgtcacccag atcgtcagcg ccgaggcctg gggtagagca 780
gactgtggct tcacctccga gtcttaccag caaggggtcc tgtctgccac catcctctat 840
gagatcttgc tagggaaggc caccttgtat gccgtgctgg tcagtgccct cgtgctgatg 900
gccatggtca agagaaagga ttccagaggc 930
<210> 25
<211> 208
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 25
Met Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser Val Arg Glu Gly
1 5 10 15
Asp Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser Ser Ser Thr Tyr
20 25 30
Leu Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu Gln Leu Leu Thr
35 40 45
Tyr Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln Arg Leu Thr Val
50 55 60
Leu Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg Ile Ala Asp Thr
65 70 75 80
Gln Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu Ser Pro Pro Asp
85 90 95
Asn Tyr Gly Gln Asn Phe Val Phe Gly Pro Gly Thr Arg Leu Ser Val
100 105 110
Leu Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp
115 120 125
Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser
130 135 140
Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp
145 150 155 160
Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala
165 170 175
Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn
180 185 190
Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser
195 200 205
<210> 26
<211> 624
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 26
atgggtgaag atgttgaaca gagtcttttc ctgagtgtcc gagagggaga cagctccgtt 60
ataaactgca cttacacaga cagctcctcc acctacttat actggtataa gcaagaacct 120
ggagcaggtc tccagttgct gacgtatatt ttttcaaata tggacatgaa acaagaccaa 180
agactcactg ttctattgaa taaaaaggat aaacatctgt ctctgcgcat tgcagacacc 240
cagactgggg actcagctat ctacttctgt gcagagagtc cgcctgataa ctatggtcag 300
aattttgtct ttggtcccgg aaccagattg tccgtgctgc cctatatcca gaaccctgac 360
cctgccgtgt accagctgag agactctaag tcgagtgaca agtctgtctg cctattcacc 420
gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 480
aaatgtgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 540
aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 600
ttcttcccca gcccagaaag ttcc 624
<210> 27
<211> 243
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 27
Met Asn Ala Gly Val Met Gln Asn Pro Arg His Leu Val Arg Arg Arg
1 5 10 15
Gly Gln Glu Ala Arg Leu Arg Cys Ser Pro Met Lys Gly His Ser His
20 25 30
Val Tyr Trp Tyr Arg Gln Leu Pro Glu Glu Gly Leu Lys Phe Met Val
35 40 45
Tyr Leu Gln Lys Glu Asn Ile Ile Asp Glu Ser Gly Met Pro Lys Glu
50 55 60
Arg Phe Ser Ala Glu Phe Pro Lys Glu Gly Pro Ser Ile Leu Arg Ile
65 70 75 80
Gln Gln Val Val Arg Gly Asp Ser Ala Ala Tyr Phe Cys Ala Ser Ser
85 90 95
Pro Val Ala Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val
100 105 110
Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu
115 120 125
Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys
130 135 140
Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val
145 150 155 160
Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu
165 170 175
Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser Arg
180 185 190
Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His Phe Arg
195 200 205
Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln
210 215 220
Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly
225 230 235 240
Arg Ala Asp
<210> 28
<211> 729
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 28
atgaatgcag gtgttatgca gaatccaaga cacctggtca ggaggagggg acaggaggca 60
agactgagat gcagcccaat gaaaggacac agtcatgttt actggtatcg gcagctccca 120
gaggaaggtc tgaaattcat ggtttatctc cagaaagaaa atatcataga tgagtcagga 180
atgccaaagg aacgattttc tgctgaattt cccaaagagg gccccagcat cctgaggatc 240
cagcaggtag tgcgaggaga ttcggcagct tatttctgtg ccagctcacc ggttgccggg 300
gagctgtttt ttggagaagg ctctaggctg accgtactgg aggacctgaa aaacgtgttc 360
ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 420
acactggtgt gcctggccac cggtttctac cccgaccacg tggagctgag ctggtgggtg 480
aatgggaagg aggtgcacag tggggtctgc acagacccgc agcccctcaa ggagcagccc 540
gccctcaatg actccagata cgctctgagc agccgcctga gggtctcggc caccttctgg 600
caggaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 660
gagtggaccc aggatagggc caaacccgtc acccagatcg tcagcgccga ggcctggggt 720
agagcagac 729
<210> 29
<211> 11
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 29
Ala Thr Ile Ile His Asn Leu Arg Glu Glu Lys
1 5 10

Claims (11)

1. A T Cell Receptor (TCR) capable of binding to the ATIIHNLREEK-HLA-a1101 complex, the TCR comprising a TCR α chain variable domain and a TCR β chain variable domain, wherein the 3 complementarity determining regions of the TCR α chain variable domain are:
αCDR1-DSSSTY (SEQ ID NO:6)
αCDR2-IFSNMDM (SEQ ID NO:7)
alpha CDR3-AESPPDNYGQNFV (SEQ ID NO: 13); and
the 3 complementarity determining regions of the TCR β chain variable domain are:
βCDR1-KGHSH (SEQ ID NO:5)
βCDR2-LQKENI (SEQ ID NO:8)
βCDR3-ASSPVAGELF (SEQ ID NO:10)。
2. the T cell receptor of claim 1, wherein the TCR comprises the alpha chain variable domain amino acid sequence SEQ ID NO 1.
3. The T cell receptor of claim 1, wherein the TCR comprises the β chain variable domain amino acid sequence SEQ ID No. 2.
4. The T cell receptor of claim 1, wherein the α chain amino acid sequence of the TCR is SEQ ID NO: 3. 15 and the beta chain amino acid sequences of the TCR are SEQ ID NO 4, 16.
5. A multivalent TCR complex which comprises at least two TCR molecules, and wherein at least one TCR molecule is a TCR as claimed in claim 1.
6. A nucleic acid molecule comprising a nucleic acid sequence encoding a TCR molecule of claim 1, or the complement thereof.
7. A vector comprising the nucleic acid molecule of claim 6.
8. An isolated host cell comprising the vector of claim 7 or having integrated into its genome the exogenous nucleic acid molecule of claim 6.
9. A cell that transduces the nucleic acid molecule of claim 6 or the vector of claim 7.
10. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to claim 1, a TCR complex according to claim 5, a nucleic acid molecule according to claim 6, a vector according to claim 7, or a cell according to claim 9.
11. Use of the T cell receptor of claim 1, or the TCR complex of claim 5, the nucleic acid molecule of claim 6, the vector of claim 7, or the cell of claim 9, for the preparation of a medicament for the treatment of a tumor or an autoimmune disease.
CN201810210760.3A 2018-03-14 2018-03-14 T cell receptor for recognizing SAGE1 antigen short peptide Active CN110272483B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106478797A (en) * 2015-12-10 2017-03-08 广州市香雪制药股份有限公司 Stem from the tumor antigen small peptide of SAGE1
WO2017203526A1 (en) * 2016-05-23 2017-11-30 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Methods of diagnosing cancer using cancer testis antigens

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106478797A (en) * 2015-12-10 2017-03-08 广州市香雪制药股份有限公司 Stem from the tumor antigen small peptide of SAGE1
WO2017203526A1 (en) * 2016-05-23 2017-11-30 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Methods of diagnosing cancer using cancer testis antigens

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Cancer-testis antigen expression in digestive tract carcinomas: frequent expression in esophageal squamous cell carcinoma and its precursor lesions;Yao-Tseng Chen 等;《Cancer Immunol Res》;20140531;第2卷(第5期);全文 *
Lack of ADAM2, CALR3 and SAGE1 Cancer/Testis Antigen Expression in Lung and Breast Cancer;Emeaga Maheswaran 等;《PLoS One》;20150807;第7卷(第10期);全文 *

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