CN107197625B - T cell receptor for recognizing NY-ESO-1 antigen short peptide - Google Patents

T cell receptor for recognizing NY-ESO-1 antigen short peptide Download PDF

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CN107197625B
CN107197625B CN201680001318.9A CN201680001318A CN107197625B CN 107197625 B CN107197625 B CN 107197625B CN 201680001318 A CN201680001318 A CN 201680001318A CN 107197625 B CN107197625 B CN 107197625B
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leu
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CN107197625A (en
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李懿
区裕升
吴万里
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Xiangxue Life Science Technology (Guangdong) Co.,Ltd.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464484—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/464488—NY-ESO
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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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
    • C12N15/867—Retroviral vectors
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Abstract

The present invention provides a T Cell Receptor (TCR) capable of specifically binding a short peptide SLLMWITQC derived from the NY-ESO-1 antigen, which can form a complex with HLA A0201 and be presented together on the cell surface. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules. In addition, the invention provides cells that transduce a TCR of the invention.

Description

T cell receptor for recognizing NY-ESO-1 antigen short peptide
Technical Field
The invention relates to a TCR capable of recognizing NY-ESO-1 antigen short peptide, NY-ESO-1 specific T cells obtained by transducing the TCR, and application of the T cells in preventing and treating NY-ESO-1 related diseases.
Background
NY-ESO-1 belongs to a tumor-Testis Antigen (CTA) family, can be expressed in Testis and ovary tissues and various different types of tumor tissues, but is not expressed in other normal tissues, and is a tumor Antigen with stronger specificity. NY-ESO-1 is an endogenous antigen that is degraded into small polypeptides upon intracellular production and is presented to the cell surface in a complex with MHC (major histocompatibility Complex) molecules. SLLMWITQC (157-165) is a short peptide derived from the NY-ESO-1 antigen, a target for the treatment of NY-ESO-1 related diseases. NY-ESO-1 was shown to be expressed in a variety of tumor tissues, with very high expression in neuroblastoma (Rodolfo M, et al, Cancer Res,2003,63(20): 6948-. 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 made an effort to isolate a TCR specific for the NY-ESO-1 antigen short peptide and to transduce the TCR into T cells to obtain T cells specific for the NY-ESO-1 antigen short peptide, thereby allowing them to play a role in cellular immunotherapy.
Disclosure of Invention
The invention aims to provide a T cell receptor for identifying NY-ESO-1 antigen short peptide and application thereof.
In a first aspect of the invention, there is provided a T Cell Receptor (TCR) capable of binding specifically to the SLLMWITQC-HLA complex comprising a TCR a chain variable domain and a TCR β chain variable domain, and the TCR a chain variable domain is an amino acid sequence having at least 90% (preferably at least 95%, more preferably at least 98%, most preferably at least 99%) sequence identity to SEQ ID NO: 1.
In another preferred embodiment, the TCR β chain variable domain is a variant of SEQ ID NO:5 an amino acid sequence having at least 90% sequence identity.
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 ALTLNNAGNMLT (SEQ ID NO: 12); and/or the amino acid sequence of CDR3 of the variable domain of the TCR beta chain is ASLDPRAGTDTQY (SEQ ID NO: 15).
In another preferred embodiment, the 3 Complementarity Determining Regions (CDRs) of the TCR α chain variable domain are:
α CDR1-ATGYPS(SEQ ID NO:10)；
alpha CDR2-ATKADDK (SEQ ID NO: 11); and
alpha CDR3-ALTLNNAGNMLT (SEQ ID NO: 12); and/or
The 3 complementarity determining regions of the TCR β chain variable domain are:
β CDR1-SNHLY(SEQ ID NO:13)；
beta CDR2-FYNNEI (SEQ ID NO: 14); and
β CDR3-ASLDPRAGTDTQY(SEQ ID NO:15)。
in another preferred embodiment, the TCR comprises the alpha chain variable domain amino acid sequence SEQ ID NO 1.
In another preferred embodiment, the TCR comprises the beta chain variable domain amino acid sequence SEQ ID NO 5.
In another preferred embodiment, the TCR is an α β heterodimer comprising a TCR α chain constant region TRAC 01 and a TCR β chain constant region TRBC1 01 or TRBC2 01.
In another preferred embodiment, the α chain amino acid sequence of the TCR is SEQ ID NO:3 and/or the beta chain amino acid sequence of the TCR is SEQ ID NO 7.
In another preferred embodiment, the TCR is soluble.
In another preferred embodiment, the TCR is single chain.
In another preferred embodiment, the TCR is formed by linking an α chain variable domain to a β chain variable domain via a peptide linker.
In another preferred embodiment, the TCR has one or more mutations in amino acid 11, 13, 19, 21, 53, 76, 89, 91, or 94 of the α chain variable region, and/or in the penultimate 3-, 5-, or 7-position of the short peptide amino acid of the α chain J gene; and/or the TCR has one or more mutations in beta chain variable region amino acid 11, 13, 19, 21, 53, 76, 89, 91, or 94 th, and/or beta chain J gene short peptide amino acid penultimate 2,4 or 6 th, wherein the amino acid position numbering is according to the position numbering listed in IMGT (international immunogenetic information system).
In another preferred embodiment, the α chain variable domain amino acid sequence of the TCR comprises SEQ ID NO 32 and/or the β chain variable domain amino acid sequence of the TCR comprises SEQ ID NO 34.
In another preferred embodiment, the amino acid sequence of the TCR is SEQ ID NO 30.
In another preferred embodiment, the TCR comprises (a) all or part of a TCR α chain, excluding the transmembrane domain; and (b) all or part of a TCR β chain, excluding the transmembrane domain;
and (a) and (b) each comprise a functional variable domain, or comprise a functional variable domain and at least a portion of the TCR chain constant domain.
In another preferred embodiment, the cysteine residues form an artificial disulfide bond between the alpha and beta chain constant domains of the TCR.
In another preferred embodiment, the cysteine residues forming the artificial disulfide bond in the TCR are substituted at one or more groups of sites selected from the group consisting of:
thr48 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser57 of TRBC2 × 01 exon 1;
thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser77 of TRBC2 × 01 exon 1;
tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC 2x 01;
thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1;
ser15 and TRBC1 × 01 of TRAC × 01 exon 1 or Glu15 of TRBC2 × 01 exon 1;
arg53 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser54 of TRBC2 × 01 exon 1;
pro89 and TRBC1 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01; and
tyr10 and TRBC1 × 01 of exon 1 of TRAC × 01 or Glu20 of exon 1 of TRBC2 × 01.
In another preferred embodiment, the α chain amino acid sequence of the TCR is SEQ ID NO 26 and/or the β chain amino acid sequence of the TCR is SEQ ID NO 28.
In another preferred embodiment, the TCR 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 peptide, a detectable label, a therapeutic agent, a PK modifying moiety or a combination of any of these. Preferably, the therapeutic agent is an anti-CD 3 antibody.
In a second aspect of the invention, there is provided a multivalent TCR complex comprising at least two TCR molecules, and wherein at least one of the TCR molecules is a TCR according to the first aspect of the invention.
In a third aspect of the invention, there is provided a nucleic acid molecule comprising a nucleic acid sequence encoding a TCR molecule according to the first aspect of the invention, or the complement thereof.
In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence encoding the variable domain of the TCR α chain SEQ ID NO:2 or SEQ ID NO: 33.
In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:6 or SEQ ID NO 35.
In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence encoding the TCR α chain SEQ ID NO:4 and/or comprises the nucleotide sequence encoding the TCR β chain SEQ ID NO: 8.
in a fourth aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the third aspect of the invention; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector.
In a fifth aspect of the invention, there is provided an isolated host cell comprising a vector according to the fourth aspect of the invention or a genome into which has been integrated an exogenous nucleic acid molecule according to the third aspect of the invention.
In a sixth aspect of the invention, there is provided a cell which transduces a nucleic acid molecule according to the third aspect of the invention or a vector according to the fourth aspect of the invention; preferably, the cell is a T cell or a stem cell.
In a seventh aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to the first aspect of the invention, a TCR complex according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, or a cell according to the sixth aspect of the invention.
In an eighth aspect, the invention provides the use of a T cell receptor according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a vector according to the fourth aspect of the invention or a cell according to the sixth aspect of the invention, for the manufacture of a medicament for the treatment of a tumour or an autoimmune disease.
In a ninth aspect of the invention, there is provided a method of treating a disease comprising administering to a subject in need thereof an amount of a TCR according to the first aspect of the invention, a TCR complex according to the second aspect of the invention, a cell according to the sixth aspect of the invention or a pharmaceutical composition according to the seventh aspect of the invention;
preferably, the disease is neuroblastoma, sarcoma, malignant melanoma, prostate cancer, bladder cancer, breast cancer, multiple myeloma, hepatocellular carcinoma, oral squamous carcinoma, and esophageal cancer.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1a, FIG. 1b, FIG. 1c, FIG. 1d, FIG. 1e and FIG. 1f are the amino acid sequence of the TCR α chain variable domain, the nucleotide sequence of the TCR α chain variable domain, the amino acid sequence of the TCR α chain, the nucleotide sequence of the TCR α chain, the amino acid sequence of the TCR α chain with leader sequence and the nucleotide sequence of the TCR α chain with leader sequence, respectively.
Fig. 2a, fig. 2b, fig. 2c, fig. 2d, fig. 2e and fig. 2f are a TCR β chain variable domain amino acid sequence, a TCR β chain variable domain nucleotide sequence, a TCR β chain amino acid sequence, a TCR β chain nucleotide sequence, a TCR β chain amino acid sequence with a leader sequence and a TCR β chain nucleotide sequence with a leader sequence, respectively.
FIG. 3 is 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.
Fig. 5a and 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.
FIGS. 7a and 7b are the amino acid and nucleotide sequences, respectively, of a single-chain TCR.
FIGS. 8a and 8b are the amino acid and nucleotide sequences, respectively, of a single chain TCR α chain.
Fig. 9a and 9b are the amino acid and nucleotide sequences, respectively, of a single chain TCR β chain.
FIGS. 10a and 10b are the amino acid and nucleotide sequences, respectively, of a single-chain TCR linker sequence (linker).
FIG. 11 is a BIAcore kinetic profile of binding of soluble TCRs of the invention to SLLMWITQC-HLA A0201 complex.
FIGS. 12a and 12b show the results of an ELISPOT assay for the functional and specific detection of TCRs of the invention.
Detailed Description
The present inventors have made extensive and intensive studies to find a TCR capable of specifically binding to the NY-ESO-1 antigen short peptide SLLMWITQC (157-165) (SEQ ID NO:9) which can form a complex with HLA A0201 and be presented together on the cell surface. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules. In addition, the invention provides cells that transduce a TCR of the invention.
Term(s) for
MHC molecules are proteins of the immunoglobulin superfamily, which may be MHC class I or class II molecules. Therefore, it is specific for antigen presentation, different individuals have different MHC, and different short peptides in one protein antigen can be presented on the cell surface of respective APC. Human MHC is commonly referred to as an HLA gene or HLA complex.
The T Cell Receptor (TCR), is the only receptor for a specific antigenic peptide presented on the Major Histocompatibility Complex (MHC). In the immune system, direct physical contact between T cells and Antigen Presenting Cells (APCs) is initiated by the binding of antigen-specific TCRs to pMHC complexes, and then other cell membrane surface molecules of both T cells and APCs interact, which causes a series of subsequent cell signaling and other physiological reactions, thereby allowing T cells of different antigen specificities to exert immune effects on their target cells.
TCRs are cell membrane surface glycoproteins that exist as heterodimers from either the α chain/β chain or the γ chain/δ chain. In 95% of T cells the TCR heterodimer consists of α and β chains, while 5% of T cells have TCRs consisting of γ and δ chains. Native α β heterodimeric TCRs have an α chain and a β chain, which constitute subunits of an α β heterodimeric TCR. Broadly, each of the α and β chains comprises a variable region, a linker region and a constant region, and the β chain also typically contains a short diversity region between the variable region and the linker region, but the diversity region is often considered to be part of the linker region. Each variable region comprises 3 CDRs (complementarity determining regions) CDR1, CDR2 and CDR3, which are chimeric in framework structures (framework regions). The CDR regions determine the binding of the TCR to the pMHC complex, where CDR3 is recombined from variable and connecting regions, referred to as hypervariable regions. The α and β chains of a TCR are generally regarded as having two "domains" each, namely a variable domain and a constant domain, the variable domain being made up of linked variable regions and linking regions. The sequences of TCR constant domains can be found in public databases of the international immunogenetic information system (IMGT), such as the constant domain sequence of the α chain of the TCR molecule is "TRAC 01", the constant domain sequence of the β chain of the TCR molecule is "TRBC 1 01" or "TRBC 2 01". In addition, the α and β chains of the TCR also comprise a transmembrane region and a cytoplasmic region, the cytoplasmic region being very short.
In the present invention, the terms "polypeptide of the invention", "TCR of the invention", "T cell receptor of the invention" are used interchangeably.
Detailed Description
TCR molecules
During antigen processing, antigens are degraded within cells and then carried to the cell surface by MHC molecules. T cell receptors are capable of recognizing peptide-MHC complexes on the surface of antigen presenting cells. Accordingly, in a first aspect the invention provides a TCR molecule capable of specifically binding to the SLLMWITQC-HLA a0201 complex. Preferably, the TCR molecule is isolated or purified. The α and β chains of the TCR each have 3 Complementarity Determining Regions (CDRs).
The alpha chain comprises CDRs having the following amino acid sequences:
α CDR1-ATGYPS(SEQ ID NO:10)
α CDR2-ATKADDK(SEQ ID NO:11)
α CDR3-ALTLNNAGNMLT(SEQ ID NO:12)
the beta chain comprises CDRs having the following amino acid sequences:
β CDR1-SNHLY(SEQ ID NO:13)
β CDR2-FYNNEI(SEQ ID NO:14)
β CDR3-ASLDPRAGTDTQY(SEQ ID NO:15)
chimeric TCRs can be prepared by embedding the above-described amino acid sequences of the CDR regions of the invention into any suitable framework. One skilled in the art can design or synthesize a TCR molecule with the corresponding function based on the CDR regions disclosed herein, so long as the framework structure is compatible with the CDR regions of the TCR of the invention. Thus, the TCR molecules of the invention are those which comprise the above-described α and/or β chain CDR region sequences and any suitable framework structure. The TCR α chain variable domain of the invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity to SEQ ID No. 1; and/or the TCR β chain variable domain of the invention is a variant of SEQ ID NO:5, having at least 90%, preferably 95%, more preferably 98% sequence identity.
In a preferred embodiment of the invention, the TCR molecules of the invention are heterodimers consisting of α and β chains. In particular, in one aspect the α chain of the heterodimeric TCR molecules comprises a variable domain and a constant domain, the α chain variable domain amino acid sequence comprising CDR1(SEQ ID NO: 10), CDR2(SEQ ID NO:11) and CDR3(SEQ ID NO:12) of the above-described α chain. Preferably, the TCR molecule comprises the alpha chain variable domain amino acid sequence SEQ ID NO 1. More preferably, the amino acid sequence of the α chain variable domain of the TCR molecule is SEQ ID NO 1. In another aspect, the β chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the β chain variable domain amino acid sequence comprises CDR1(SEQ ID NO:13), CDR2(SEQ ID NO:14), and CDR3(SEQ ID NO:15) of the above-described β chain. Preferably, the TCR molecule comprises the beta chain variable domain amino acid sequence SEQ ID NO 5. More preferably, the amino acid sequence of the β chain variable domain of the TCR molecule is SEQ ID NO 5.
In a preferred embodiment of the invention, the TCR molecules of the invention are single chain TCR molecules consisting of part or all of the α chain and/or part or all of the β chain. Single chain TCR molecules are described in Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-. From the literature, those skilled in the art are readily able to construct single chain TCR molecules comprising the CDRs regions of the invention. In particular, the single chain TCR molecule comprises V α, V β and C β, preferably linked in order from N-terminus to C-terminus.
The alpha chain variable domain amino acid sequence of the single chain TCR molecule comprises the CDR1(SEQ ID NO: 10), CDR2(SEQ ID NO:11) and CDR3(SEQ ID NO:12) of the alpha chain described above. Preferably, the single chain TCR molecule comprises the alpha chain variable domain amino acid sequence SEQ ID NO 1. More preferably, the α chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO 1. The amino acid sequence of the beta chain variable domain of the single chain TCR molecule comprises the CDR1(SEQ ID NO:13), CDR2(SEQ ID NO:14) and CDR3(SEQ ID NO:15) of the above-described beta chain. Preferably, the single chain TCR molecule comprises the beta chain variable domain amino acid sequence SEQ ID NO 5. More preferably, the amino acid sequence of the β chain variable domain of the single chain TCR molecule is SEQ ID NO 5.
In a preferred embodiment of the invention, the constant domain of the TCR molecules of the invention is a human constant domain. The human constant domain amino acid sequences are known to those skilled in the art or can be obtained by consulting published databases of relevant books or IMGT (international immunogenetic information system). For example, the constant domain sequence of the α chain of the TCR molecules of the invention can be "TRAC 01", and the constant domain sequence of the β chain of the TCR molecules can be "TRBC 1 01" or "TRBC 2 01". Arg at position 53 of the amino acid sequence given in TRAC 01 of IMGT, here denoted: TRAC × 01 Arg53 of exon 1, and so on. Preferably, the amino acid sequence of the α chain of the TCR molecule of the invention is SEQ ID NO 3 and/or the amino acid sequence of the β chain is SEQ ID NO 7.
Naturally occurring TCRs are membrane proteins that are stabilized by their transmembrane regions. Like immunoglobulins (antibodies) as antigen recognition molecules, TCRs can also be developed for diagnostic and therapeutic applications, where soluble TCR molecules are required. Soluble TCR molecules do not include their transmembrane regions. Soluble TCRs have a wide range of uses, not only for studying the interaction of TCRs with pmhcs, but also as diagnostic tools for detecting infections or as markers for autoimmune diseases. Similarly, soluble TCRs can be used to deliver therapeutic agents (e.g., cytotoxic or immunostimulatory compounds) to cells presenting a specific antigen, and in addition, soluble TCRs can be conjugated to other molecules (e.g., anti-CD 3 antibodies) to redirect T cells to target them to cells presenting a particular antigen. The invention also obtains soluble TCR with specificity to NY-ESO-1 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 × 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 TRBC 2x 01; thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1; ser15 and TRBC1 × 01 of TRAC × 01 exon 1 or Glu15 of TRBC2 × 01 exon 1; arg53 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser54 of TRBC2 × 01 exon 1; pro89 and TRBC1 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01; or Tyr10 and TRBC1 and 01 of TRAC 01 exon 1 or Glu20 of TRBC2 and 01 exon 1. I.e., a cysteine residue, in place of any of the above-described alpha and beta chain constant domains. The TCR constant domains of the invention may be truncated at one or more of their C-termini by up to 50, or up to 30, or up to 15, or up to 10, or up to 8 or fewer amino acids, so as not to include a cysteine residue for the purpose of deleting the native disulphide bond, or by mutating the cysteine residue forming the native disulphide bond to another amino acid.
As described above, the TCRs of the invention may comprise artificial disulfide bonds introduced between residues of the constant domains of their alpha and beta chains. It should be noted that the TCRs of the invention may each contain both TRAC constant domain sequences and TRBC1 or TRBC2 constant domain sequences, with or without the artificial disulfide bonds introduced as described above between the constant domains. The TRAC constant domain sequence and TRBC1 or TRBC2 constant domain sequences of the TCR may be linked by the native disulfide bond present in the TCR.
To obtain a soluble TCR, on the other hand, the inventive TCR also comprises a TCR having a mutation in its hydrophobic core region, preferably a mutation that enables an improved stability of the inventive soluble TCR, as described in the patent publication WO 2014/206304. Such TCRs may be mutated at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or positions 3,5,7 of the reciprocal amino acid position of the short peptide of the alpha chain J gene (TRAJ), and/or positions 2,4,6 of the reciprocal amino acid position of the short peptide of the beta chain J gene (TRBJ), wherein the position numbering of the amino acid sequence is according to the position numbering listed in the International immunogenetic information System (IMGT). The above-mentioned international system of immunogenetics information is known to the skilled person and the position numbering of the amino acid residues of the different TCRs in IMGT can be derived from this database.
The TCR with the mutated hydrophobic core region of the invention can be a stable soluble single chain TCR formed by connecting the variable domains of the alpha and beta chains of the TCR by a flexible peptide chain. It should be noted that the flexible peptide chain of the present invention can be any peptide chain suitable for linking the TCR α and β chain variable domains. The single-chain soluble TCR constructed as in example 4 of the invention has an alpha chain variable domain amino acid sequence of SEQ ID NO. 32 and an encoded nucleotide sequence of SEQ ID NO. 33; the amino acid sequence of the beta chain variable domain is SEQ ID NO. 34, and the coded nucleotide sequence is SEQ ID NO. 35.
The TCRs of the invention may also be provided in the form of multivalent complexes. Multivalent TCR complexes of the invention comprise polymers formed by association of two, three, four or more TCRs of the invention, such as might be produced as a tetramer using the tetrameric domain of p53, or a complex formed by association of a plurality of TCRs of the invention with another molecule. The TCR complexes of the invention can be used to track or target cells presenting a particular antigen in vitro or in vivo, and can also be used to generate intermediates for other multivalent TCR complexes having such applications.
The TCRs of the invention may be used alone or in covalent or other association, preferably covalently, with a conjugate. The conjugates include a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the SLLMWITQC-HLA a0201 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.
Therapeutic agents that may be associated or conjugated with the TCRs of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, Cancer metastasis reviews (Cancer metastasis) 24, 539); 2. biotoxicity (Chaudhary et al, 1989, Nature 339, 394; Epel et al, 2002, Cancer Immunology and Immunotherapy 51, 565); 3. cytokines such as IL-2 and the like (Gillies et al, 1992, Proc. Natl. Acad. Sci. USA (PNAS)89, 1428; Card et al, 2004, Cancer Immunology and Immunotherapy)53, 345; Halin et al, 2003, Cancer Research 63, 3202); 4. antibody Fc fragment (Mosquera et al, 2005, Journal Of Immunology 174, 4381); 5. antibody scFv fragments (Zhu et al, 1995, International Journal of Cancer 62,319); 6. gold nanoparticles/nanorods (Lapotko et al, 2005, Cancer letters 239, 36; Huang et al, 2006, Journal of the American Chemical Society 128, 2115); 7. viral particles (Peng et al, 2004, Gene therapy 11, 1234); 8. liposomes (Mamot et al, 2005, Cancer research 65, 11631); 9. nano magnetic particles; 10. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, and the like.
In addition, the TCRs of the invention may also be hybrid TCRs comprising sequences derived from more than one species. For example, studies have shown that murine TCRs are more efficiently expressed in human T cells than human TCRs. Thus, the inventive TCR may comprise a human variable domain and a murine constant domain. The drawback of this approach is the possibility of eliciting an immune response. Thus, there should be a regulatory regimen to immunosuppresse when it is used for adoptive T cell therapy to allow for the engraftment of murine expressing T cells.
It should be understood that the amino acid names herein are expressed in terms of international single-letter or three-letter english letters, and the single-letter english letter and three-letter english letters of the amino acid names correspond to the following relationships: ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y) and Val (V).
Nucleic acid molecules
A second aspect of the invention provides a nucleic acid molecule encoding a TCR molecule of the first aspect of the invention or a portion thereof, which may be one or more CDRs, variable domains of the alpha and/or beta chains, and the alpha and/or beta chains.
The nucleotide sequence encoding the α chain CDR regions of the TCR molecules of the first aspect of the invention is as follows:
α CDR1-gccacaggatacccttcc(SEQ ID NO:16)
α CDR2-gccacgaaggctgatgacaag(SEQ ID NO:17)
α CDR3-gctctgacccttaataatgcaggcaacatgctcacc(SEQ ID NO:18)
the nucleotide sequence encoding the β chain CDR regions of the TCR molecules of the first aspect of the invention is as follows:
β CDR1-tctaatcacttatac(SEQ ID NO:19)
β CDR2-ttttataataatgaaatc(SEQ ID NO:20)
β CDR3-gccagcctggacccacgagcgggcacagatacgcagtat(SEQ ID NO:21)
thus, the nucleotide sequence of the nucleic acid molecule of the invention encoding the TCR alpha chain of the invention comprises SEQ ID NO 16, 17 and 18 and/or the nucleotide sequence of the nucleic acid molecule of the invention encoding the TCR beta chain of the invention comprises SEQ ID NO 19, 20 and 21.
The nucleotide sequence of the nucleic acid molecule of the invention may be single-stranded or double-stranded, the nucleic acid molecule may be RNA or DNA, and may or may not comprise an intron. Preferably, the nucleotide sequence of the nucleic acid molecule of the invention does not comprise an intron but is capable of encoding a polypeptide of the invention, e.g. the nucleotide sequence of the nucleic acid molecule of the invention encoding a TCR alpha chain variable domain of the invention comprises SEQ ID NO 2 and/or the nucleotide sequence of the nucleic acid molecule of the invention encoding a TCR beta chain variable domain of the invention comprises SEQ ID NO 6. Alternatively, the nucleotide sequence of a nucleic acid molecule of the invention encoding a TCR α chain variable domain of the invention comprises SEQ ID NO 33 and/or the nucleotide sequence of a nucleic acid molecule of the invention encoding a TCR β chain variable domain of the invention comprises SEQ ID NO 35. More preferably, the nucleotide sequence of the nucleic acid molecule of the invention comprises SEQ ID NO. 4 and/or SEQ ID NO. 8. Alternatively, the nucleotide sequence of the nucleic acid molecule of the invention is SEQ ID NO. 31.
It will be appreciated that, due to the degeneracy of the genetic code, different nucleotide sequences may encode the same polypeptide. Thus, the nucleic acid sequence encoding the TCR of the present invention may be identical to or a degenerate variant of the nucleic acid sequences shown in the figures of the present invention. As illustrated by one of the examples herein, a "degenerate variant" refers to a nucleic acid sequence that encodes a protein sequence having SEQ ID NO. 1, but differs from the sequence of SEQ ID NO. 2.
The nucleotide sequence may be codon optimized. Different cells differ in the utilization of specific codons, and the expression level can be increased by changing the codons in the sequence according to the type of the cell. Codon usage tables for mammalian cells as well as for various other organisms are well known to those skilled in the art.
The full-length sequence of the nucleic acid molecule of the present invention or a fragment thereof can be obtained by, but not limited to, PCR amplification, recombination, or artificial synthesis. At present, DNA sequences encoding the TCRs of the invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. The DNA may be the coding strand or the non-coding strand.
Carrier
The invention also relates to vectors comprising the nucleic acid molecules of the invention, including expression vectors, i.e. constructs capable of expression in vivo or in vitro. Commonly used vectors include bacterial plasmids, bacteriophages and animal and plant viruses.
Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, baculoviral vectors.
Preferably, the vector can transfer the nucleotide of the invention into a cell, for example a T cell, such that the cell expresses a TCR specific for the NY-ESO-1 antigen. Ideally, the vector should be capable of sustained high level expression in T cells.
Cells
The invention also relates to genetically engineered host cells that have been engineered with the vectors or coding sequences of the invention. The host cell comprises a vector of the invention or has integrated into its chromosome a nucleic acid molecule of the invention. The host cell is selected from: prokaryotic and eukaryotic cells, such as E.coli, yeast cells, CHO cells, and the like.
In addition, the invention also includes isolated cells, particularly T cells, that express the TCRs of the invention. The T cell may be derived from a T cell isolated from a subject, or may be part of a mixed population of cells isolated from a subject, such as a population of Peripheral Blood Lymphocytes (PBLs). For example, the cells may be isolated from Peripheral Blood Mononuclear Cells (PBMC), which may be CD4+Helper T cell or CD8+Cytotoxic T cells. The cell may be in CD4+Helper T cell/CD 8+A mixed population of cytotoxic T cells. Generally, the cells can be activated with an antibody (e.g., an anti-CD 3 or anti-CD 28 antibody) to render them more amenable to transfection, e.g., transfection with a vector comprising a nucleotide sequence encoding a TCR molecule of the invention.
Alternatively, the cell of the invention may also be or be derived from a stem cell, such as a Hematopoietic Stem Cell (HSC). Gene transfer to HSCs does not result in TCR expression on the cell surface, since the CD3 molecule is not expressed on the stem cell surface. However, when stem cells differentiate into lymphoid precursors (lymphoid precursors) that migrate to the thymus, expression of the CD3 molecule will initiate expression of the introduced TCR molecule on the surface of the thymocytes.
There are many methods suitable for T cell transfection using DNA or RNA encoding the TCR of the invention (e.g., Robbins et al, (2008) J.Immunol.180: 6116-. T cells expressing the TCRs of the invention may be used for adoptive immunotherapy. Those skilled in the art will be able to recognize many suitable methods for adoptive therapy (e.g., Rosenberg et al, (2008) Nat Rev Cancer8 (4): 299-308).
NY-ESO-1 antigen related diseases
The invention also relates to a method of treating and/or preventing a NY-ESO-1 related disease in a subject comprising the step of adoptively transferring NY-ESO-1 specific T cells to the subject. The NY-ESO-1 specific T cells recognize SLLMWITQC-HLA A0201 complex.
The NY-ESO-1 specific T cell can be used for treating any NY-ESO-1 related diseases presenting the NY-ESO-1 antigen short peptide SLLMWITQC-HLA A0201 compound. Including but not limited to, neuroblastoma, sarcoma, malignant melanoma, prostate cancer, bladder cancer, breast cancer, multiple myeloma, hepatocellular carcinoma, oral squamous carcinoma, and esophageal cancer.
Method of treatment
Treatment may be effected by isolating T cells from patients or volunteers suffering from a disease associated with the NY-ESO-1 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 NY-ESO-1 related disorder 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 invention are:
(1) the TCR can be specifically combined with the NY-ESO-1 antigen short peptide complex SLLMWITQC-HLA A0201, and cells transduced with the TCR can be specifically activated and have strong killing effect on target cells.
The following specific examples further illustrate the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example as described in Sambrook and Russell et al, Molecular Cloning: A Laboratory Manual (third edition) (2001) CSHL Press, or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.
Example 1 cloning of NY-ESO-1 antigen short peptide-specific T cells
Peripheral Blood Lymphocytes (PBLs) from healthy volunteers of genotype HLA-A0201 were stimulated using the synthetic short peptide SLLMWITQC (Baisheng Gene technologies, Beijing). And (3) renaturing the SLLMWITQC short peptide and HLA-A0201 with biotin labels to prepare a 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-CD8-APC cells. The sorted cells were expanded and subjected to secondary sorting as described above, followed by single cloning by limiting dilution. Monoclonal cells were stained with tetramer and double positive clones were selected as shown in FIG. 3.
Example 2 construction of TCR Gene and vector for obtaining NY-ESO-1 antigen short peptide specific T cell clone
Using Quick-RNATMMiniPrep (ZYMO research) extracts total RNA from the T cell clone specific to the antigen short peptide SLLMWITQC and restricted by HLA-A0201 selected in example 1. cDNA was synthesized using the SMART RACE cDNA amplification kit from clontech, using primers designed to preserve the C-terminal region of the human TCR gene. The sequences were cloned into the T vector (TAKARA) and sequenced. Should be takenNote that this sequence is a complementary sequence and does not include an intron. The alpha chain and beta chain sequence structures of the TCR expressed by the double positive clone are respectively shown in figure 1 and figure 2 by sequencing, and figure 1a, figure 1b, figure 1c, figure 1d, figure 1e and figure 1f are respectively a TCR alpha chain variable domain amino acid sequence, a TCR alpha chain variable domain nucleotide sequence, a TCR alpha chain amino acid sequence, a TCR alpha chain nucleotide sequence, a TCR alpha chain amino acid sequence with a leader sequence and a TCR alpha chain nucleotide sequence with the leader sequence; fig. 2a, fig. 2b, fig. 2c, fig. 2d, fig. 2e and fig. 2f are a TCR β 0 chain variable domain amino acid sequence, a TCR β 1 chain variable domain nucleotide sequence, a TCR β 2 chain amino acid sequence, a TCR β 3 chain nucleotide sequence, a TCR β chain amino acid sequence with a leader sequence and a TCR β chain nucleotide sequence with a leader sequence, respectively.
The alpha chain was identified to comprise CDRs with the following amino acid sequences:
α CDR1-ATGYPS(SEQ ID NO:10)
α CDR2-ATKADDK(SEQ ID NO:11)
α CDR3-ALTLNNAGNMLT(SEQ ID NO:12)
the beta chain comprises CDRs having the following amino acid sequences:
β CDR1-SNHLY(SEQ ID NO:13)
β CDR2-FYNNEI(SEQ ID NO:14)
β CDR3-ASLDPRAGTDTQY(SEQ ID NO:15)
full-length genes for TCR α and β chains were cloned into lentiviral expression vectors by overlap (overlap) PCR, respectively. As a control, an eGFP-expressing lentiviral vector was also constructed. The pseudovirus was then packaged again at 293T/17.
Example 3 expression, refolding and purification of NY-ESO-1 antigen short peptide-specific soluble TCR
To obtain soluble TCR molecules, the α and β chains of the TCR molecules of the invention may comprise only the variable and part of the constant domains thereof, respectively, and a cysteine residue has been introduced into the constant domains of the α and β chains, respectively, to form artificial interchain disulfide bonds, at the positions Thr48 of exon 1 TRAC × 01 and Ser57 of exon 1 TRBC2 × 01, respectively; the amino acid sequence and nucleotide sequence of the alpha chain are shown in FIGS. 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 letters. The above-mentioned gene sequences of interest for the TCR alpha and beta chains were synthesized and inserted into the expression vector pET28a + (Novagene) by standard methods described in Molecular Cloning A Laboratory Manual (third edition, Sambrook and Russell), and the upstream and downstream Cloning sites were NcoI and NotI, respectively. The insert was confirmed by sequencing without error.
The expression vectors of TCR alpha and beta chains are transformed into expression bacteria BL21(DE3) by chemical transformation method, and the bacteria are grown in LB culture solution and OD600Inclusion bodies formed after expression of the α and β chains of the TCR were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution several times at 0.6 final induction with final concentration of 0.5mM IPTG, and finally dissolved in 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT),10mM ethylenediaminetetraacetic acid (EDTA),20mM Tris (pH 8.1).
The TCR α and β chains after lysis were separated by 1: 1 was rapidly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1),3.7mM cystamine,6.6mM β -mercaptamine (4 ℃) to a final concentration of 60 mg/mL. After mixing, the solution was dialyzed against 10 volumes of deionized water (4 ℃ C.) and after 12 hours, the deionized water was changed to a buffer (20mM Tris, pH 8.0) and dialysis was continued at 4 ℃ for 12 hours. The solution after completion of dialysis was filtered through a 0.45. mu.M filter and then purified by an anion exchange column (HiTrap Q HP,5ml, GE Healthcare). The TCR eluted with peaks containing successfully renatured α and β dimers was confirmed by SDS-PAGE gel. The TCR was subsequently further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was greater than 90% as determined by SDS-PAGE and the concentration was determined by BCA. The SDS-PAGE gel of the soluble TCR of the invention is shown in FIG. 6.
Example 4 Generation of soluble Single chain TCR specific for NY-ESO-1 antigen short peptides
According to the disclosure of WO2014/206304, the variable domains of TCR α and β chains in example 2 were constructed as a stable soluble single-chain TCR molecule linked by a short flexible peptide (linker) using site-directed mutagenesis. The amino acid sequence and the nucleotide sequence of the single-chain TCR molecule are shown in FIG. 7a and FIG. 7b, respectively. The amino acid sequence and nucleotide sequence of the alpha chain variable domain are shown in FIG. 8a and FIG. 8b, respectively; the amino acid sequence and nucleotide sequence of its beta-chain variable domain are shown in FIG. 9a and FIG. 9b, respectively; the amino acid sequence and the nucleotide sequence of the linker sequence are shown in FIG. 10a and FIG. 10b, respectively.
The target gene was digested simultaneously with Nco I and Not I, and ligated with pET28a vector digested simultaneously with Nco I and Not I. The ligation product was transformed into e.coli DH5 α, spread on LB plates containing kanamycin, cultured at 37 ℃ for overnight inversion, positive clones were selected for PCR screening, positive recombinants were sequenced, and after the sequence was determined to be correct, recombinant plasmids were extracted and transformed into e.coli BL21(DE3) for expression.
Example 5 expression, renaturation and purification of soluble Single-chain TCR specific for NY-ESO-1 antigen short peptides
The BL21(DE3) colony containing the recombinant plasmid pET28 a-template strand prepared in example 4 was inoculated in its entirety into LB medium containing kanamycin, cultured at 37 ℃ to OD600 of 0.6 to 0.8, IPTG was added to a final concentration of 0.5mM, and the culture was continued at 37 ℃ for 4 hours. The cell pellet was harvested by centrifugation at 5000rpm for 15min, the cell pellet was lysed by Bugbuster Master Mix (Merck), inclusion bodies were recovered by centrifugation at 6000rpm for 15min, washed with Bugbuster (Merck) to remove cell debris and membrane components, and centrifuged at 6000rpm for 15min to collect the inclusion bodies. The inclusion bodies were dissolved in buffer (20mM Tris-HCl pH 8.0,8M urea), the insoluble material was removed by high speed centrifugation, the supernatant was quantified by BCA method and split charged, and stored at-80 ℃ for further use.
To 5mg of solubilized single-chain TCR inclusion body protein, 2.5mL of buffer (6M Gua-HCl, 50mM Tris-HCl pH 8.1, 100mM NaCl, 10mM EDTA) was added, DTT was added to a final concentration of 10mM, and treatment was carried out at 37 ℃ for 30 min. The treated single-chain TCR was added dropwise to 125mL of renaturation buffer (100mM Tris-HCl pH 8.1,0.4M L-arginine, 5M urea, 2mM EDTA,6.5mM beta-mercaptoethylamine, 1.87mM Cystamine) with a syringe, stirred at 4 ℃ for 10min, and then the renaturation solution was filled into a cellulose membrane dialysis bag with a cut-off of 4kDa, and the bag was placed in 1L of precooled water and stirred slowly at 4 ℃ overnight. After 17 hours, the dialysate was changed to 1L of pre-chilled buffer (20mM Tris-HCl pH 8.0), dialysis was continued at 4 ℃ for 8h, and then dialysis was continued overnight with the same fresh buffer. After 17 hours, the sample was filtered through a 0.45 μ M filter, vacuum degassed and then passed through an anion exchange column (HiTrap Q HP, GE Healthcare), the protein was purified using a 0-1M NaCl linear gradient eluent formulated in 20mM Tris-HCl pH 8.0, the collected fractions were subjected to SDS-PAGE analysis, the fractions containing single-stranded TCR were concentrated and then further purified using a gel filtration column (Superdex 7510/300, GE Healthcare), and the target fraction was also subjected to SDS-PAGE analysis.
The eluted fractions for BIAcore analysis were further tested for purity using gel filtration. The conditions are as follows: the chromatographic column Agilent Bio SEC-3(300A, phi 7.8X 300mM) and the mobile phase are 150mM phosphate buffer solution, the flow rate is 0.5mL/min, the column temperature is 25 ℃, and the ultraviolet detection wavelength is 214 nm.
Example 6 binding characterization
BIAcore analysis
This example demonstrates that soluble TCR molecules of the invention are capable of specifically binding the SLLMWITQC-HLA A0201 complex.
The binding activity of the TCR molecules obtained in example 3 to the SLLMWITQC-HLA A0201 complex was tested 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 through the coated chip surface, then SLLMWITQC-HLA A0201 complex flow through the detection channel, another channel as a reference channel, and then 0.05mM biotin at 10 u L/min flow rate through the chip for 2min, closed streptavidin residual binding sites.
The SLLMWITQC-HLA A0201 complex is prepared as follows:
a. purification of
Collecting 100ml E.coli liquid for inducing expression of heavy chain or light chain, centrifuging at 4 ℃ for 10min at 8000g, washing the thalli once with 10ml PBS, then resuspending the thalli with 5ml BugBuster Master Mix Extraction Reagents (Merck) by vigorous shaking, rotatably incubating at room temperature for 20min, centrifuging at 4 ℃ for 15min at 6000g, discarding supernatant, and collecting inclusion body.
Resuspending the inclusion bodies in 5ml of BugBuster Master Mix, and rotary incubating at room temperature for 5 min; adding 30ml of 10-fold diluted BugBuster, uniformly mixing, and centrifuging at 4 ℃ at 6000g for 15 min; discarding the supernatant, adding 30ml of 10-fold diluted BugBuster to resuspend the inclusion bodies, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, repeating twice, adding 30ml of 20mM Tris-HCl pH 8.0 to resuspend the inclusion bodies, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, finally dissolving the inclusion bodies by using 20mM Tris-HCl 8M urea, detecting the purity of the inclusion bodies by SDS-PAGE, and detecting the concentration by using a BCA kit.
b. Renaturation
The synthesized short peptide SLLMWITQC (Beijing Saibance Gene technology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg/ml. Inclusion of light and heavy chains was solubilized with 8M Urea, 20mM Tris pH 8.0, 10mM DTT and further denatured by addition of 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA prior to renaturation. SLLMWITQC 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 light chain and 90mg/L heavy chain in sequence (final concentration, heavy chain was added in three portions, 8 h/time), and renaturation was performed at 4 ℃ for at least 3 days until completion, and SDS-PAGE checked for success or failure of the renaturation.
c. Purification after renaturation
The renaturation buffer was replaced by dialysis against 10 volumes of 20mM Tris pH 8.0, at least twice to reduce the ionic strength of the solution sufficiently. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and then loaded onto a HiTrap Q HP (GE general electric) anion exchange column (5ml bed volume). The protein was eluted using an Akta purifier (GE general electric) with a 0-400mM NaCl linear gradient prepared in 20mM Tris pH 8.0, pMHC was eluted at about 250mM NaCl, the peak fractions were collected, and the purity was checked by SDS-PAGE.
d. Biotinylation of the compound
The purified pMHC molecules were concentrated using Millipore ultrafiltration tubes while displacing the buffer to 20mM Tris pH 8.0, followed by addition of biotinylation reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50. mu. M D-Biotin, 100. mu.g/ml BirA enzyme (GST-BirA), incubation of the mixture overnight at room temperature, and SDS-PAGE to determine the completion of biotinylation.
e. Purification of biotinylated complexes
The biotinylated pMHC molecules were concentrated to 1ml using Millipore ultrafiltration tubes, the biotinylated pMHC was purified by gel filtration chromatography, and HiPrep was pre-equilibrated with filtered PBS using Akta purifier (GE general electric Co., Ltd.)TM16/60S200HR column (GE general electric) was loaded with 1ml of concentrated biotinylated pMHC molecules and then eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules appeared as a unimodal elution at approximately 55 ml. The fractions containing the protein were pooled, concentrated using Millipore ultrafiltration tubes, protein concentration was determined by BCA (Thermo), and biotinylated pMHC molecules were stored in aliquots at-80 ℃ by addition of the protease inhibitor cocktail (Roche).
Kinetic parameters were calculated using BIAcore Evaluation software, and the kinetic profiles of binding of soluble TCR molecules of the invention to SLLMWITQC-HLA A0201 complex are shown in FIG. 11. Meanwhile, the binding activity of the soluble TCR molecule and other antigen short peptides including KLVALGINAV-HLA A0201 complex and the like is detected by the method, and the result shows that the TCR molecule is not bound with other irrelevant antigen short peptides.
Example 7 function and specificity of TCR
This example demonstrates an illustration that PBL transduced with the TCR obtained by the invention is capable of specifically recognizing NY-ESO-1 antigen positive cancer cells.
(a) Production of lentiviruses by Rapid-mediated transient transfection of 293T cells
A third generation lentiviral packaging system was used to package lentiviruses containing the gene encoding the desired TCR. The 4 plasmids were transiently transfected with PEI into 293T cells (containing pGZ178-HI WT TRA-2A-TRB lentiviral vector, and 3 plasmids containing other components necessary for the construction of infectious but non-replicating lentiviral particles).
For transfection, cells were seeded at day 0 on a 15 cm petri dish at 1.7X 107293T cells, the cells were evenly distributed on the culture dish, and the degree of confluence was slightly higher than 50%. The plasmid was transfected on day 1, packaged pGZ 178-HIWTRA-2A-TRB and pLenti-eGFP pseudovirus, and the above expression plasmid was mixed with packaging plasmids pMDLg/pRRE, pRSV-REV and pMD.2G in a 15 cm diameter plate in the following amounts: 22.5 microgram: 15 microgram: 7.5 micrograms. The ratio of the transfection reagent PEI-MAX to the plasmid was 2:1 and the amount used was 114.75. mu.g per dish. The specific operation is as follows: the expression plasmid and the packaging plasmid were added to 1800. mu.l of OPTI-MEM (Gibbo, catalog No. 31985-; mixing a corresponding amount of PEI with 1800 microliters of OPTI-MEM culture medium uniformly, standing for 5 minutes at room temperature to obtain a PEI mixed solution, mixing the DNA mixed solution with the PEI mixed solution, standing for 30 minutes at room temperature, adding 3150 microliters of OPTI-MEM culture medium, uniformly mixing, adding into 293T cells converted into 11.25 milliliters of OPTI-MEM, gently shaking the culture dish to mix the culture medium uniformly, culturing at 37 deg.C/5% CO2, transfecting for 5-7 hr, the transfection medium was removed and replaced with DMEM (Gibbo, Cat. No. C11995500bt) complete medium containing 10% fetal bovine serum at 37 deg.C/5% CO.2And (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) kit instructions. As a control, a pseudovirus transformed with pLenti-eGFP was also included.
(b) Transduction of PBLs with lentiviruses containing HI-WT specific T cell receptor genes
PBL cells were isolated from the blood of healthy volunteers and transduced with packaged lentiviruses. These cells were counted in 48-well plates in a 1X 10 format in 1640 (Gibbo, Cat. No. C11875500bt) medium containing 10% FBS (Gibbo, Cat. No. C10010500BT) containing 100IU/ml IL-26Cells/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 3: 1.
after overnight stimulation, the concentrated lentivirus of the HI-WT specific T cell receptor gene was added at an MOI of 10 according to the virus titer measured with the p24ELISA kit and centrifuged at 32 ℃ and 900g for 1 hour. After infection, the lentivirus infection solution was removed and the cells were resuspended in 1640 medium containing 10% FBS containing 100IU/ml IL-2 at 37 ℃/5% CO2The cells were cultured for 3 days. Cells were counted 3 days after transduction and diluted to 0.5X 106Individual cells/ml. The cells were counted every two days, replaced or added with fresh medium containing 50IU/ml IL-2, maintaining the cells at 0.5X 106-1×106Individual cells/ml. Lentivirus-transduced PBLs were quantitatively analyzed by flow cytometry starting on day 3 by tetramer staining containing SLLMWITQC short peptides with HLA-A0201. When the tetramer detection positive rate was greater than 10%, it was started on day 5 for functional assays (e.g., ELISPOT for IFN- γ release and non-radioactive cytotoxicity assays).
(c) Tetramer staining of TCR transduced PBLs
NY-ESO-1157-165 SLLMWITQC short peptide (p1A) and HLA-A0201 with biotin label are renatured to prepare pHLA haploid. These haplotypes were combined with PE-labeled streptavidin (BD) into a PE-labeled tetramer, designated p 1A-tetramer-PE. The tetramer was able to label T cells expressing the NY-ESO-1157-165 SLLMWITQC specific T cell receptor gene as positive cells. The transfected T cell sample of (b) was incubated with p1A-tetramer-PE on ice for 30min, followed by the addition of anti-CD8-APC (BioLegent) antibody and further incubation on ice for 15 min. Samples were washed 2 times with PBS containing 2% FBS and then p1A-tetramer-PE and CD8 double positive T cells expressing NY-ESO-1157-165 SLLMWITQC specific T cell receptor genes were detected with Guava 16HT and data analysis was analyzed using FlowJo software (Tree Star Inc, Ashland, OR).
The function and specificity of transduction of the PBLs of the invention were further tested by ELISPOT assay. Methods for detecting cell function using the ELISPOT assay are well known to those skilled in the art. The effector cells used in the IFN-gamma ELISPOT experiment in this example were PBL of virus-transduced HI-WT gene obtained in the present invention, the target cells were IM9(ATCC) and U266B1(ATCC) cells positive to antigen (NY-ESO-1), and the control group was 293T cells negative to antigen (NY-ESO-1).
Firstly, preparing an ELISPOT plate, wherein the ELISPOT experiment steps are as follows: each 15 fractions of the assay were added to ELISPOT plates in the following order: groups were added to ELISPOT plates separately: adjusting the culture medium for T2 cells to 2X 105Adding specific short peptide and nonspecific short peptide into corresponding T2 cell diluent according to final volume and short peptide concentration, adding corresponding amount of culture medium into blank group, mixing well, and taking 100 μ L T2 cells and target cell line 2X 105One cell/ml (i.e., 20,000 cells/well), 100 μ L effector cells 2X 104One cell/ml (i.e., 20, 00 virus-transduced cells/well) and three duplicate wells were set. Then incubated overnight (37 ℃, 5% CO)2). The plates were then washed and subjected to secondary detection and color development, dried for 1 hour, and the spots formed on the membrane were counted using an immuno-spot plate READER (ELISPOT READER system; AID20 Co.). As shown in FIG. 12(a and B), the PBL of the virus-transduced HI-WT gene obtained by the present invention has specific reaction to T2 cell loaded with specific polypeptide and IM9 and U266B1 cell lines positive to antigen (NY-ESO-1) compared with 293T cell of control group.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Guangzhou Xiangxue pharmaceutical products Co., Ltd
<120> T cell receptor recognizing NY-ESO-1 antigen short peptide
<130> P2016-1668
<150> 201510228932.6
<151> 2015-05-06
<160> 37
<170> PatentIn version 3.5
<210> 1
<211> 114
<212> PRT
<213> Artificial sequence
<400> 1
Gly Asn Ser Val Thr Gln Met Glu Gly Pro Val Thr Leu Ser Glu Glu
1 5 10 15
Ala Phe Leu Thr Ile Asn Cys Thr Tyr Thr Ala Thr Gly Tyr Pro Ser
20 25 30
Leu Phe Trp Tyr Val Gln Tyr Pro Gly Glu Gly Leu Gln Leu Leu Leu
35 40 45
Lys Ala Thr Lys Ala Asp Asp Lys Gly Ser Asn Lys Gly Phe Glu Ala
50 55 60
Thr Tyr Arg Lys Glu Thr Thr Ser Phe His Leu Glu Lys Gly Ser Val
65 70 75 80
Gln Val Ser Asp Ser Ala Val Tyr Phe Cys Ala Leu Thr Leu Asn Asn
85 90 95
Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys
100 105 110
Pro His
<210> 2
<211> 342
<212> DNA
<213> Artificial sequence
<400> 2
ggaaattcag tgacccagat ggaagggcca gtgactctct cagaagaggc cttcctgact 60
ataaactgca cgtacacagc cacaggatac ccttcccttt tctggtatgt ccaatatcct 120
ggagaaggtc tacagctcct cctgaaagcc acgaaggctg atgacaaggg aagcaacaaa 180
ggttttgaag ccacataccg taaagaaacc acttctttcc acttggagaa aggctcagtt 240
caagtgtcag actcagcggt gtacttctgt gctctgaccc ttaataatgc aggcaacatg 300
ctcacctttg gagggggaac aaggttaatg gtcaaacccc at 342
<210> 3
<211> 254
<212> PRT
<213> Artificial sequence
<400> 3
Gly Asn Ser Val Thr Gln Met Glu Gly Pro Val Thr Leu Ser Glu Glu
1 5 10 15
Ala Phe Leu Thr Ile Asn Cys Thr Tyr Thr Ala Thr Gly Tyr Pro Ser
20 25 30
Leu Phe Trp Tyr Val Gln Tyr Pro Gly Glu Gly Leu Gln Leu Leu Leu
35 40 45
Lys Ala Thr Lys Ala Asp Asp Lys Gly Ser Asn Lys Gly Phe Glu Ala
50 55 60
Thr Tyr Arg Lys Glu Thr Thr Ser Phe His Leu Glu Lys Gly Ser Val
65 70 75 80
Gln Val Ser Asp Ser Ala Val Tyr Phe Cys Ala Leu Thr Leu Asn Asn
85 90 95
Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys
100 105 110
Pro His 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> 762
<212> DNA
<213> Artificial sequence
<400> 4
ggaaattcag tgacccagat ggaagggcca gtgactctct cagaagaggc cttcctgact 60
ataaactgca cgtacacagc cacaggatac ccttcccttt tctggtatgt ccaatatcct 120
ggagaaggtc tacagctcct cctgaaagcc acgaaggctg atgacaaggg aagcaacaaa 180
ggttttgaag ccacataccg taaagaaacc acttctttcc acttggagaa aggctcagtt 240
caagtgtcag actcagcggt gtacttctgt gctctgaccc ttaataatgc aggcaacatg 300
ctcacctttg gagggggaac aaggttaatg gtcaaacccc 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> 5
<211> 115
<212> PRT
<213> Artificial sequence
<400> 5
Glu Pro Glu Val Thr Gln Thr Pro Ser His Gln Val Thr Gln Met Gly
1 5 10 15
Gln Glu Val Ile Leu Arg Cys Val Pro Ile Ser Asn His Leu Tyr Phe
20 25 30
Tyr Trp Tyr Arg Gln Ile Leu Gly Gln Lys Val Glu Phe Leu Val Ser
35 40 45
Phe Tyr Asn Asn Glu Ile Ser Glu Lys Ser Glu Ile Phe Asp Asp Gln
50 55 60
Phe Ser Val Glu Arg Pro Asp Gly Ser Asn Phe Thr Leu Lys Ile Arg
65 70 75 80
Ser Thr Lys Leu Glu Asp Ser Ala Met Tyr Phe Cys Ala Ser Leu Asp
85 90 95
Pro Arg Ala Gly Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu
100 105 110
Thr Val Leu
115
<210> 6
<211> 345
<212> DNA
<213> Artificial sequence
<400> 6
gaacctgaag tcacccagac tcccagccat caggtcacac agatgggaca ggaagtgatc 60
ttgcgctgtg tccccatctc taatcactta tacttctatt ggtacagaca aatcttgggg 120
cagaaagtcg agtttctggt ttccttttat aataatgaaa tctcagagaa gtctgaaata 180
ttcgatgatc aattctcagt tgaaaggcct gatggatcaa atttcactct gaagatccgg 240
tccacaaagc tggaggactc agccatgtac ttctgtgcca gcctggaccc acgagcgggc 300
acagatacgc agtattttgg cccaggcacc cggctgacag tgctc 345
<210> 7
<211> 294
<212> PRT
<213> Artificial sequence
<400> 7
Glu Pro Glu Val Thr Gln Thr Pro Ser His Gln Val Thr Gln Met Gly
1 5 10 15
Gln Glu Val Ile Leu Arg Cys Val Pro Ile Ser Asn His Leu Tyr Phe
20 25 30
Tyr Trp Tyr Arg Gln Ile Leu Gly Gln Lys Val Glu Phe Leu Val Ser
35 40 45
Phe Tyr Asn Asn Glu Ile Ser Glu Lys Ser Glu Ile Phe Asp Asp Gln
50 55 60
Phe Ser Val Glu Arg Pro Asp Gly Ser Asn Phe Thr Leu Lys Ile Arg
65 70 75 80
Ser Thr Lys Leu Glu Asp Ser Ala Met Tyr Phe Cys Ala Ser Leu Asp
85 90 95
Pro Arg Ala Gly Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu
100 105 110
Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val
115 120 125
Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu
130 135 140
Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp
145 150 155 160
Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln
165 170 175
Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser
180 185 190
Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His
195 200 205
Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp
210 215 220
Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala
225 230 235 240
Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly
245 250 255
Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr
260 265 270
Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys
275 280 285
Arg Lys Asp Ser Arg Gly
290
<210> 8
<211> 882
<212> DNA
<213> Artificial sequence
<400> 8
gaacctgaag tcacccagac tcccagccat caggtcacac agatgggaca ggaagtgatc 60
ttgcgctgtg tccccatctc taatcactta tacttctatt ggtacagaca aatcttgggg 120
cagaaagtcg agtttctggt ttccttttat aataatgaaa tctcagagaa gtctgaaata 180
ttcgatgatc aattctcagt tgaaaggcct gatggatcaa atttcactct gaagatccgg 240
tccacaaagc tggaggactc agccatgtac ttctgtgcca gcctggaccc acgagcgggc 300
acagatacgc agtattttgg cccaggcacc cggctgacag tgctcgagga cctgaaaaac 360
gtgttcccac ccgaggtcgc tgtgtttgag ccatcagaag cagagatctc ccacacccaa 420
aaggccacac tggtatgcct ggccacaggc ttctaccccg accacgtgga gctgagctgg 480
tgggtgaatg ggaaggaggt gcacagtggg gtcagcacag acccgcagcc cctcaaggag 540
cagcccgccc tcaatgactc cagatactgc ctgagcagcc gcctgagggt ctcggccacc 600
ttctggcaga acccccgcaa ccacttccgc tgtcaagtcc agttctacgg gctctcggag 660
aatgacgagt ggacccagga tagggccaaa cccgtcaccc agatcgtcag cgccgaggcc 720
tggggtagag cagactgtgg cttcacctcc gagtcttacc agcaaggggt cctgtctgcc 780
accatcctct atgagatctt gctagggaag gccaccttgt atgccgtgct ggtcagtgcc 840
ctcgtgctga tggccatggt caagagaaag gattccagag gc 882
<210> 9
<211> 9
<212> PRT
<213> NY-ESO-1
<400> 9
Ser Leu Leu Met Trp Ile Thr Gln Cys
1 5
<210> 10
<211> 6
<212> PRT
<213> Artificial sequence
<400> 10
Ala Thr Gly Tyr Pro Ser
1 5
<210> 11
<211> 7
<212> PRT
<213> Artificial sequence
<400> 11
Ala Thr Lys Ala Asp Asp Lys
1 5
<210> 12
<211> 12
<212> PRT
<213> Artificial sequence
<400> 12
Ala Leu Thr Leu Asn Asn Ala Gly Asn Met Leu Thr
1 5 10
<210> 13
<211> 5
<212> PRT
<213> Artificial sequence
<400> 13
Ser Asn His Leu Tyr
1 5
<210> 14
<211> 6
<212> PRT
<213> Artificial sequence
<400> 14
Phe Tyr Asn Asn Glu Ile
1 5
<210> 15
<211> 13
<212> PRT
<213> Artificial sequence
<400> 15
Ala Ser Leu Asp Pro Arg Ala Gly Thr Asp Thr Gln Tyr
1 5 10
<210> 16
<211> 18
<212> DNA
<213> Artificial sequence
<400> 16
gccacaggat acccttcc 18
<210> 17
<211> 21
<212> DNA
<213> Artificial sequence
<400> 17
gccacgaagg ctgatgacaa g 21
<210> 18
<211> 36
<212> DNA
<213> Artificial sequence
<400> 18
gctctgaccc ttaataatgc aggcaacatg ctcacc 36
<210> 19
<211> 15
<212> DNA
<213> Artificial sequence
<400> 19
tctaatcact tatac 15
<210> 20
<211> 18
<212> DNA
<213> Artificial sequence
<400> 20
ttttataata atgaaatc 18
<210> 21
<211> 39
<212> DNA
<213> Artificial sequence
<400> 21
gccagcctgg acccacgagc gggcacagat acgcagtat 39
<210> 22
<211> 273
<212> PRT
<213> Artificial sequence
<400> 22
Met Asn Tyr Ser Pro Gly Leu Val Ser Leu Ile Leu Leu Leu Leu Gly
1 5 10 15
Arg Thr Arg Gly Asn Ser Val Thr Gln Met Glu Gly Pro Val Thr Leu
20 25 30
Ser Glu Glu Ala Phe Leu Thr Ile Asn Cys Thr Tyr Thr Ala Thr Gly
35 40 45
Tyr Pro Ser Leu Phe Trp Tyr Val Gln Tyr Pro Gly Glu Gly Leu Gln
50 55 60
Leu Leu Leu Lys Ala Thr Lys Ala Asp Asp Lys Gly Ser Asn Lys Gly
65 70 75 80
Phe Glu Ala Thr Tyr Arg Lys Glu Thr Thr Ser Phe His Leu Glu Lys
85 90 95
Gly Ser Val Gln Val Ser Asp Ser Ala Val Tyr Phe Cys Ala Leu Thr
100 105 110
Leu Asn Asn Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu
115 120 125
Met Val Lys Pro His Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu
130 135 140
Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe
145 150 155 160
Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile
165 170 175
Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn
180 185 190
Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala
195 200 205
Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu
210 215 220
Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr
225 230 235 240
Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu
245 250 255
Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser
260 265 270
Ser
<210> 23
<211> 822
<212> DNA
<213> Artificial sequence
<400> 23
atgaactatt ctccaggctt agtatctctg atactcttac tgcttggaag aacccgtgga 60
aattcagtga cccagatgga agggccagtg actctctcag aagaggcctt cctgactata 120
aactgcacgt acacagccac aggataccct tcccttttct ggtatgtcca atatcctgga 180
gaaggtctac agctcctcct gaaagccacg aaggctgatg acaagggaag caacaaaggt 240
tttgaagcca cataccgtaa agaaaccact tctttccact tggagaaagg ctcagttcaa 300
gtgtcagact cagcggtgta cttctgtgct ctgaccctta ataatgcagg caacatgctc 360
acctttggag ggggaacaag gttaatggtc aaaccccata tccagaaccc tgaccctgcc 420
gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 480
gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 540
gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 600
tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 660
cccagcccag aaagttcctg tgatgtcaag ctggtcgaga aaagctttga aacagatacg 720
aacctaaact ttcaaaacct gtcagtgatt gggttccgaa tcctcctcct gaaagtggcc 780
gggtttaatc tgctcatgac gctgcggctg tggtccagct ag 822
<210> 24
<211> 313
<212> PRT
<213> Artificial sequence
<400> 24
Met Asp Thr Trp Leu Val Cys Trp Ala Ile Phe Ser Leu Leu Lys Ala
1 5 10 15
Gly Leu Thr Glu Pro Glu Val Thr Gln Thr Pro Ser His Gln Val Thr
20 25 30
Gln Met Gly Gln Glu Val Ile Leu Arg Cys Val Pro Ile Ser Asn His
35 40 45
Leu Tyr Phe Tyr Trp Tyr Arg Gln Ile Leu Gly Gln Lys Val Glu Phe
50 55 60
Leu Val Ser Phe Tyr Asn Asn Glu Ile Ser Glu Lys Ser Glu Ile Phe
65 70 75 80
Asp Asp Gln Phe Ser Val Glu Arg Pro Asp Gly Ser Asn Phe Thr Leu
85 90 95
Lys Ile Arg Ser Thr Lys Leu Glu Asp Ser Ala Met Tyr Phe Cys Ala
100 105 110
Ser Leu Asp Pro Arg Ala Gly Thr Asp Thr Gln Tyr Phe Gly Pro Gly
115 120 125
Thr Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu
130 135 140
Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys
145 150 155 160
Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu
165 170 175
Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr
180 185 190
Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr
195 200 205
Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro
210 215 220
Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn
225 230 235 240
Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser
245 250 255
Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr
260 265 270
Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly
275 280 285
Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala
290 295 300
Met Val Lys Arg Lys Asp Ser Arg Gly
305 310
<210> 25
<211> 942
<212> DNA
<213> Artificial sequence
<400> 25
atggatacct ggctcgtatg ctgggcaatt tttagtctct tgaaagcagg actcacagaa 60
cctgaagtca cccagactcc cagccatcag gtcacacaga tgggacagga agtgatcttg 120
cgctgtgtcc ccatctctaa tcacttatac ttctattggt acagacaaat cttggggcag 180
aaagtcgagt ttctggtttc cttttataat aatgaaatct cagagaagtc tgaaatattc 240
gatgatcaat tctcagttga aaggcctgat ggatcaaatt tcactctgaa gatccggtcc 300
acaaagctgg aggactcagc catgtacttc tgtgccagcc tggacccacg agcgggcaca 360
gatacgcagt attttggccc aggcacccgg ctgacagtgc tcgaggacct gaaaaacgtg 420
ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 480
gccacactgg tatgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 540
gtgaatggga aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag 600
cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 660
tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 720
gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 780
ggtagagcag actgtggctt cacctccgag tcttaccagc aaggggtcct gtctgccacc 840
atcctctatg agatcttgct agggaaggcc accttgtatg ccgtgctggt cagtgccctc 900
gtgctgatgg ccatggtcaa gagaaaggat tccagaggct ag 942
<210> 26
<211> 207
<212> PRT
<213> Artificial sequence
<400> 26
Gly Asn Ser Val Thr Gln Met Glu Gly Pro Val Thr Leu Ser Glu Glu
1 5 10 15
Ala Phe Leu Thr Ile Asn Cys Thr Tyr Thr Ala Thr Gly Tyr Pro Ser
20 25 30
Leu Phe Trp Tyr Val Gln Tyr Pro Gly Glu Gly Leu Gln Leu Leu Leu
35 40 45
Lys Ala Thr Lys Ala Asp Asp Lys Gly Ser Asn Lys Gly Phe Glu Ala
50 55 60
Thr Tyr Arg Lys Glu Thr Thr Ser Phe His Leu Glu Lys Gly Ser Val
65 70 75 80
Gln Val Ser Asp Ser Ala Val Tyr Phe Cys Ala Leu Thr Leu Asn Asn
85 90 95
Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys
100 105 110
Pro His 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
Cys 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
195 200 205
<210> 27
<211> 621
<212> DNA
<213> Artificial sequence
<400> 27
ggcaacagcg tgacccagat ggaagggcca gtgactctct cagaagaggc cttcctgact 60
ataaactgca cgtacacagc cacaggatac ccttcccttt tctggtatgt ccaatatcct 120
ggagaaggtc tacagctcct cctgaaagcc acgaaggctg atgacaaggg aagcaacaaa 180
ggttttgaag ccacataccg taaagaaacc acttctttcc acttggagaa aggctcagtt 240
caagtgtcag actcagcggt gtacttctgt gctctgaccc ttaataatgc aggcaacatg 300
ctcacctttg gagggggaac aaggttaatg gtcaaacccc atatccagaa ccctgaccct 360
gccgtgtacc agctgagaga ctctaagtcg agtgacaagt ctgtctgcct attcaccgat 420
tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 480
tgcgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 540
aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 600
ttccccagcc cagaaagttc c 621
<210> 28
<211> 245
<212> PRT
<213> Artificial sequence
<400> 28
Glu Pro Glu Val Thr Gln Thr Pro Ser His Gln Val Thr Gln Met Gly
1 5 10 15
Gln Glu Val Ile Leu Arg Cys Val Pro Ile Ser Asn His Leu Tyr Phe
20 25 30
Tyr Trp Tyr Arg Gln Ile Leu Gly Gln Lys Val Glu Phe Leu Val Ser
35 40 45
Phe Tyr Asn Asn Glu Ile Ser Glu Lys Ser Glu Ile Phe Asp Asp Gln
50 55 60
Phe Ser Val Glu Arg Pro Asp Gly Ser Asn Phe Thr Leu Lys Ile Arg
65 70 75 80
Ser Thr Lys Leu Glu Asp Ser Ala Met Tyr Phe Cys Ala Ser Leu Asp
85 90 95
Pro Arg Ala Gly Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu
100 105 110
Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val
115 120 125
Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu
130 135 140
Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp
145 150 155 160
Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln
165 170 175
Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser
180 185 190
Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His
195 200 205
Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp
210 215 220
Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala
225 230 235 240
Trp Gly Arg Ala Asp
245
<210> 29
<211> 735
<212> DNA
<213> Artificial sequence
<400> 29
gaaccggaag tgacccagac tcccagccat caggtcacac agatgggaca ggaagtgatc 60
ttgcgctgtg tccccatctc taatcactta tacttctatt ggtacagaca aatcttgggg 120
cagaaagtcg agtttctggt ttccttttat aataatgaaa tctcagagaa gtctgaaata 180
ttcgatgatc aattctcagt tgaaaggcct gatggatcaa atttcactct gaagatccgg 240
tccacaaagc tggaggactc agccatgtac ttctgtgcca gcctggaccc acgagcgggc 300
acagatacgc agtattttgg cccaggcacc cggctgacag tgctcgagga cctgaaaaac 360
gtgttcccac ccgaggtcgc tgtgtttgag ccatcagaag cagagatctc ccacacccaa 420
aaggccacac tggtgtgcct ggccaccggt ttctaccccg accacgtgga gctgagctgg 480
tgggtgaatg ggaaggaggt gcacagtggg gtctgcacag acccgcagcc cctcaaggag 540
cagcccgccc tcaatgactc cagatacgct ctgagcagcc gcctgagggt ctcggccacc 600
ttctggcagg acccccgcaa ccacttccgc tgtcaagtcc agttctacgg gctctcggag 660
aatgacgagt ggacccagga tagggccaaa cccgtcaccc agatcgtcag cgccgaggcc 720
tggggtagag cagac 735
<210> 30
<211> 253
<212> PRT
<213> Artificial sequence
<400> 30
Ala Gly Asn Ser Val Thr Gln Ser Glu Gly Pro Leu Thr Val Ser Glu
1 5 10 15
Glu Glu Asn Val Thr Ile Asn Cys Thr Tyr Thr Ala Thr Gly Tyr Pro
20 25 30
Ser Leu Phe Trp Tyr Arg Gln Tyr Pro Gly Glu Gly Leu Gln Leu Leu
35 40 45
Leu Lys Ala Thr Lys Ala Asp Asp Lys Gly Ser Asn Lys Arg Phe Glu
50 55 60
Ala Thr Tyr Arg Lys Glu Thr Thr Ser Phe His Leu Glu Ile Glu Arg
65 70 75 80
Ile Gln Pro Asn Asp Ser Ala Thr Tyr Phe Cys Ala Leu Thr Leu Asn
85 90 95
Asn Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Lys Leu Ser Val
100 105 110
His Asn Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser
115 120 125
Glu Gly Gly Gly Ser Glu Gly Gly Thr Gly Glu Pro Glu Ile Thr Gln
130 135 140
Thr Pro Ser His Leu Ser Val Gln Thr Gly Gln Glu Val Thr Leu Arg
145 150 155 160
Cys Val Pro Ile Ser Asn His Leu Tyr Phe Tyr Trp Tyr Arg Gln Asp
165 170 175
Pro Gly Gln Lys Val Arg Phe Leu Val Ser Phe Tyr Asn Asn Glu Ile
180 185 190
Ser Glu Lys Ser Glu Ile Pro Asp Asp Arg Phe Ser Val Glu Arg Pro
195 200 205
Asp Gly Ser Asn Phe Thr Leu Lys Ile Arg Ser Val Lys Pro Glu Asp
210 215 220
Ser Ala Met Tyr Leu Cys Ala Ser Leu Asp Pro Arg Ala Gly Thr Asp
225 230 235 240
Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Glu Val Asp
245 250
<210> 31
<211> 759
<212> DNA
<213> Artificial sequence
<400> 31
gcaggtaatt ctgttacgca gtcggaaggc ccgctgacgg tctcggaaga agaaaatgtg 60
acgattaatt gtacctatac ggcaacgggt tacccgagcc tgttttggta tcgtcagtat 120
ccgggtgaag gcctgcaact gctgctgaaa gcgaccaaag ccgatgacaa aggtagcaac 180
aaacgttttg aagcaacgta ccgcaaagaa accacgagct tccatctgga aattgaacgc 240
atccagccga atgattctgc aacctatttt tgcgctctga cgctgaacaa tgctggcaac 300
atgctgacct tcggcggtgg cacgaaactg agtgtgcaca atggtggcgg ttcagaaggc 360
ggtggctcgg aaggtggcgg tagcgaaggc ggtggctctg aaggtggcac cggtgaaccg 420
gaaattaccc aaacgccgag ccatctgtct gttcagaccg gccaagaagt cacgctgcgt 480
tgcgtgccga tcagtaacca cctgtatttt tactggtatc gtcaagatcc gggccaaaaa 540
gtgcgctttc tggtttcctt ctacaacaac gaaattagtg aaaaatccga aatcccggat 600
gaccgttttt cagttgaacg cccggatggt tcgaatttca ccctgaaaat tcgcagtgtc 660
aaaccggaag actccgcgat gtacctgtgt gcgagcctgg acccgcgtgc gggcaccgac 720
acgcagtatt tcggtccggg cacccgcctg gaagtggat 759
<210> 32
<211> 114
<212> PRT
<213> Artificial sequence
<400> 32
Ala Gly Asn Ser Val Thr Gln Ser Glu Gly Pro Leu Thr Val Ser Glu
1 5 10 15
Glu Glu Asn Val Thr Ile Asn Cys Thr Tyr Thr Ala Thr Gly Tyr Pro
20 25 30
Ser Leu Phe Trp Tyr Arg Gln Tyr Pro Gly Glu Gly Leu Gln Leu Leu
35 40 45
Leu Lys Ala Thr Lys Ala Asp Asp Lys Gly Ser Asn Lys Arg Phe Glu
50 55 60
Ala Thr Tyr Arg Lys Glu Thr Thr Ser Phe His Leu Glu Ile Glu Arg
65 70 75 80
Ile Gln Pro Asn Asp Ser Ala Thr Tyr Phe Cys Ala Leu Thr Leu Asn
85 90 95
Asn Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Lys Leu Ser Val
100 105 110
His Asn
<210> 33
<211> 342
<212> DNA
<213> Artificial sequence
<400> 33
gcaggtaatt ctgttacgca gtcggaaggc ccgctgacgg tctcggaaga agaaaatgtg 60
acgattaatt gtacctatac ggcaacgggt tacccgagcc tgttttggta tcgtcagtat 120
ccgggtgaag gcctgcaact gctgctgaaa gcgaccaaag ccgatgacaa aggtagcaac 180
aaacgttttg aagcaacgta ccgcaaagaa accacgagct tccatctgga aattgaacgc 240
atccagccga atgattctgc aacctatttt tgcgctctga cgctgaacaa tgctggcaac 300
atgctgacct tcggcggtgg cacgaaactg agtgtgcaca at 342
<210> 34
<211> 115
<212> PRT
<213> Artificial sequence
<400> 34
Glu Pro Glu Ile Thr Gln Thr Pro Ser His Leu Ser Val Gln Thr Gly
1 5 10 15
Gln Glu Val Thr Leu Arg Cys Val Pro Ile Ser Asn His Leu Tyr Phe
20 25 30
Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Lys Val Arg Phe Leu Val Ser
35 40 45
Phe Tyr Asn Asn Glu Ile Ser Glu Lys Ser Glu Ile Pro Asp Asp Arg
50 55 60
Phe Ser Val Glu Arg Pro Asp Gly Ser Asn Phe Thr Leu Lys Ile Arg
65 70 75 80
Ser Val Lys Pro Glu Asp Ser Ala Met Tyr Leu Cys Ala Ser Leu Asp
85 90 95
Pro Arg Ala Gly Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu
100 105 110
Glu Val Asp
115
<210> 35
<211> 345
<212> DNA
<213> Artificial sequence
<400> 35
gaaccggaaa ttacccaaac gccgagccat ctgtctgttc agaccggcca agaagtcacg 60
ctgcgttgcg tgccgatcag taaccacctg tatttttact ggtatcgtca agatccgggc 120
caaaaagtgc gctttctggt ttccttctac aacaacgaaa ttagtgaaaa atccgaaatc 180
ccggatgacc gtttttcagt tgaacgcccg gatggttcga atttcaccct gaaaattcgc 240
agtgtcaaac cggaagactc cgcgatgtac ctgtgtgcga gcctggaccc gcgtgcgggc 300
accgacacgc agtatttcgg tccgggcacc cgcctggaag tggat 345
<210> 36
<211> 24
<212> PRT
<213> Artificial sequence
<400> 36
Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly
1 5 10 15
Gly Gly Ser Glu Gly Gly Thr Gly
20
<210> 37
<211> 72
<212> DNA
<213> Artificial sequence
<400> 37
ggtggcggtt cagaaggcgg tggctcggaa ggtggcggta gcgaaggcgg tggctctgaa 60
ggtggcaccg gt 72

Claims (33)

1. A T Cell Receptor (TCR) which is capable of specifically binding to a SLLMWITQC-HLA complex, which comprises a TCR a chain variable domain and a TCR β chain variable domain, and wherein the TCR a chain variable domain is an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1;
and, the 3 Complementarity Determining Regions (CDRs) of the TCR α chain variable domain are:
α CDR1- ATGYPS (SEQ ID NO: 10)
α CDR2- ATKADDK (SEQ ID NO: 11)
alpha CDR3-ALTLNNAGNMLT (SEQ ID NO: 12); and
the 3 complementarity determining regions of the TCR β chain variable domain are:
β CDR1- SNHLY (SEQ ID NO: 13)
β CDR2- FYNNEI (SEQ ID NO: 14)
β CDR3- ASLDPRAGTDTQY (SEQ ID NO: 15)。
2. a TCR as claimed in claim 1 wherein the TCR β chain variable domain is substantially identical to SEQ ID NO:5 an amino acid sequence having at least 90% sequence identity.
3. A TCR as claimed in claim 1 which comprises the α chain variable domain amino acid sequence SEQ ID NO 1.
4. A TCR as claimed in claim 1 which comprises the β chain variable domain amino acid sequence SEQ ID NO 5.
5. A TCR as claimed in claim 1 which is an α β heterodimer comprising a TCR α chain constant region TRAC 01 and a TCR β chain constant region TRBC1 01 or TRBC2 01.
6. A TCR as claimed in claim 5 wherein the α chain amino acid sequence of the TCR is SEQ ID NO:3 and/or the beta chain amino acid sequence of the TCR is SEQ ID NO 7.
7. A TCR as claimed in any one of claims 1 to 4 which is soluble.
8. A TCR as claimed in claim 7 which is single chain.
9. A TCR as claimed in claim 8 which is formed by the α chain variable domain linked to the β chain variable domain by a peptide linker sequence.
10. A TCR as claimed in claim 9 which has one or more mutations in the alpha chain variable region amino acid 11, 13, 19, 21, 53, 76, 89, 91 or 94 and/or the alpha chain J gene short peptide amino acid penultimate 3, penultimate 5 or penultimate 7 position; and/or the TCR has one or more mutations in beta chain variable region amino acid 11, 13, 19, 21, 53, 76, 89, 91, or 94 th, and/or beta chain J gene short peptide amino acid penultimate 2,4 or 6 th, wherein the amino acid position numbering is according to the position numbering listed in IMGT (international immunogenetic information system).
11. A TCR as claimed in claim 10 wherein the α chain variable domain amino acid sequence of the TCR comprises SEQ ID No. 32 and/or the β chain variable domain amino acid sequence of the TCR comprises SEQ ID No. 34.
12. A TCR as claimed in claim 11 which has the amino acid sequence SEQ ID No. 30.
13. A TCR as claimed in claim 7 which comprises (a) all or part of the TCR α chain, excluding the transmembrane domain; and (b) all or part of a TCR β chain, excluding the transmembrane domain;
and (a) and (b) each comprise a functional variable domain.
14. A TCR as claimed in claim 13 wherein (a) and (b) each further comprise at least a portion of the constant domain of the TCR chain.
15. A TCR as claimed in claim 14 in which the cysteine residues form an artificial disulphide bond between the α and β chain constant domains of the TCR.
16. A TCR as claimed in claim 15 wherein the cysteine residues which form the artificial disulphide bond in the TCR are substituted at one or more groups selected from:
thr48 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser57 of TRBC2 × 01 exon 1;
thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser77 of TRBC2 × 01 exon 1;
tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC 2x 01;
thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1;
ser15 and TRBC1 × 01 of TRAC × 01 exon 1 or Glu15 of TRBC2 × 01 exon 1;
arg53 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser54 of TRBC2 × 01 exon 1;
pro89 and TRBC1 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01; and
tyr10 and TRBC1 × 01 of exon 1 of TRAC × 01 or Glu20 of exon 1 of TRBC2 × 01.
17. A TCR as claimed in claim 16 wherein the α chain amino acid sequence of the TCR is SEQ ID No. 26 and/or the β chain amino acid sequence of the TCR is SEQ ID No. 28.
18. A TCR as claimed in claim 1 wherein a conjugate is attached to the C-or N-terminus of the α and/or β chains of the TCR.
19. A TCR as claimed in claim 18 wherein the conjugate to which the TCR is bound is a detectable label, a therapeutic agent, a PK modifying moiety or a combination thereof.
20. A TCR as claimed in claim 19 wherein the therapeutic agent is an anti-CD 3 antibody.
21. A multivalent TCR complex comprising at least two TCR molecules, and wherein at least one TCR molecule is a TCR as claimed in any one of claims 1 to 20.
22. A nucleic acid molecule comprising a nucleic acid sequence encoding a TCR according to any one of claims 1 to 20, or the complement thereof.
23. The nucleic acid molecule of claim 22, comprising the nucleotide sequence encoding the TCR α chain variable domain of SEQ ID NO:2 or SEQ ID NO: 33.
24. The nucleic acid molecule of claim 22 or 23, comprising the nucleotide sequence of SEQ ID NO:6 or SEQ ID NO 35.
25. The nucleic acid molecule of claim 22, comprising the nucleotide sequence encoding a TCR α chain of SEQ ID NO:4 and/or comprises the nucleotide sequence encoding the TCR β chain SEQ ID NO: 8.
26. a vector comprising the nucleic acid molecule of any one of claims 22-25.
27. The vector of claim 26, wherein said vector is a viral vector.
28. The vector of claim 27, wherein said vector is a lentiviral vector.
29. An isolated host cell comprising the vector of claim 26 or a nucleic acid molecule of any one of claims 22-25 integrated into the chromosome.
30. A cell transduced with the nucleic acid molecule of any one of claims 22 to 25 or the vector of claim 26.
31. The cell of claim 30, wherein the cell is a T cell or a stem cell.
32. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to any one of claims 1 to 20, a TCR complex according to claim 21, a nucleic acid molecule according to any one of claims 22 to 25, or a cell according to claim 30.
33. Use of a TCR as claimed in any one of claims 1 to 20 or a TCR complex as claimed in claim 21 or a cell as claimed in claim 30 in the manufacture of a medicament for the treatment of a tumour.
CN201680001318.9A 2015-05-06 2016-05-05 T cell receptor for recognizing NY-ESO-1 antigen short peptide Active CN107197625B (en)

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