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WO2022105922A1 - 源自于ssx2抗原的短肽 - Google Patents

源自于ssx2抗原的短肽 Download PDF

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WO2022105922A1
WO2022105922A1 PCT/CN2021/132192 CN2021132192W WO2022105922A1 WO 2022105922 A1 WO2022105922 A1 WO 2022105922A1 CN 2021132192 W CN2021132192 W CN 2021132192W WO 2022105922 A1 WO2022105922 A1 WO 2022105922A1
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peptide
cell
molecule
pmhc complex
present
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PCT/CN2021/132192
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English (en)
French (fr)
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李懿
余晓红
刘秋萍
杨东雪
吴万里
钟时
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香雪生命科学技术（广东）有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001184—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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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
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/7051—T-cell receptor (TcR)-CD3 complex

Definitions

  • the present invention relates to short peptides derived from SSX2 antigen, in particular, to newly discovered short peptides derived from tumor antigen SSX2, complexes formed by the short peptides with MHC molecules, and uses of the short peptides and complexes.
  • the present invention also relates to molecules that bind to the above-mentioned short peptides or complexes, and uses of these molecules.
  • markers of a pathological or abnormal state can be used not only as markers for disease diagnosis, but also for the production of diagnostic and/or therapeutic agents.
  • markers of cancer are used to generate specific antibodies.
  • these molecules can also effectively stimulate the specific immune response of cytotoxic T lymphocytes (CTL) and exert anti-tumor efficacy.
  • CTL cytotoxic T lymphocytes
  • TCR T cell receptors that can bind to the "marker” through activated CTL. (TCR) as a therapeutic agent. Therefore, these molecules play a very important role in the diagnosis and treatment of related diseases.
  • tumor antigens are proteolytically processed into polypeptide fragments of 8-16 amino acids in length, that is, CTL epitopes, which in turn interact with the major histocompatibility complex (MHC, CTL) in the lumen of the endoplasmic reticulum.
  • MHC major histocompatibility complex
  • Human MHC is usually referred to as HLA gene or HLA complex
  • HLA gene or HLA complex Human MHC molecules combine to form peptide-MHC complex (peptide-MHC complex, pMHC), and finally present pMHC to the cell surface for recognition by TCR on the surface of CD8 + T cells.
  • peptide-MHC complex peptide-MHC complex, pMHC
  • pMHC peptide-MHC complex
  • the discovery and determination of the presented polypeptide fragments is a complex process, because the presentation of polypeptides by HLA is the result of the enzymatic hydrolysis of the antigenic protein and the interaction of the polypeptide fragments with HLA. This shows that the complete tumor antigen molecule cannot provide any information for the discovery and identification of polypeptide fragments.
  • Many literatures have published methods using computer simulation, such as the public databases SYFPEITHI (Rammensee, et al., Immunogenetics. 1999(50): 213-219) and BIMAS (Parker, et al., J. Immunol. 1994. 152: 163), Predictive algorithms are provided to identify which polypeptide fragments are likely to be presented.
  • SSX2 is a synovial sarcoma X breakpoint, also known as HOM-MEL-40.
  • SSX2 is one of ten highly homologous nucleic acid proteins of the SSX family.
  • SSX protein is a tumor testis antigen and is only expressed in tumor cells and testicular blasts without MHC expression.
  • SSX2 is expressed in a variety of human cancer cells including, but not limited to, melanoma, head and neck cancer, lymphoma, various myelomas, pancreatic cancer, prostate cancer, sarcoma, hepatocellular carcinoma, and colon cancer.
  • peptides derived from SSX2 as targets for the above-mentioned cancers, can not only be used as markers for the diagnosis of the above-mentioned diseases, but also can be used to generate prophylactic and/or therapeutic agents for the above-mentioned diseases, such as antibodies or T cell receptors.
  • the present invention utilizes mass spectrometer analysis and identification to discover for the first time a polypeptide fragment derived from tumor antigen SSX2 presented on the surface of tumor cells.
  • the purpose of the present invention is to provide a newly discovered short peptide derived from tumor antigen SSX2, the complex formed by the short peptide and MHC molecules, and the use of the short peptide and the complex.
  • a first aspect of the present invention provides a short peptide derived from the SSX2 antigen, the peptide comprising the amino acid sequence: AQIPEKIQK (SEQ ID NO: 1);
  • the peptide can form a complex with MHC molecules.
  • the peptide consists of 9 or 10 amino acids.
  • the peptide consists of 9 amino acids.
  • amino acid sequence of the peptide is SEQ ID NO: 1.
  • the second aspect of the present invention provides a pMHC complex comprising the peptide of the first aspect of the present invention.
  • amino acid sequence of the peptide in the pMHC complex is SEQ ID NO: 1.
  • the type of MHC molecule is HLA-A*11.
  • the type of MHC molecule is HLA-A*1101.
  • the pMHC complex is a multimer.
  • the pMHC complex is soluble.
  • the pMHC complex is biotinylated.
  • the third aspect of the present invention provides an isolated cell, the cell surface presents the pMHC complex of the second aspect of the present invention.
  • the fourth aspect of the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the peptide of the first aspect of the present invention or a complementary sequence thereof.
  • the fifth aspect of the present invention provides a vector, which contains the nucleic acid molecule described in the fourth aspect of the present invention.
  • the sixth aspect of the present invention provides a host cell containing the vector of the fifth aspect of the present invention.
  • a seventh aspect of the present invention provides a molecule capable of binding the peptide of the first aspect of the present invention and/or the pMHC complex of the second aspect of the present invention.
  • the molecule can specifically bind to the peptide described in the first aspect of the present invention and/or the pMHC complex described in the second aspect of the present invention.
  • the molecule is a T cell receptor.
  • the T cell receptor is soluble.
  • the molecule is an antibody or a binding fragment thereof.
  • the antibody is a monoclonal antibody.
  • an isolated monoclonal T cell obtained by utilizing the peptide of the first aspect of the present invention and/or the pMHC complex of the second aspect of the present invention and/or the present invention
  • the cells are obtained by separation.
  • the monoclonal T cells specifically bind to the pMHC complex of the second aspect of the present invention.
  • the ninth aspect of the present invention provides the use of the peptide of the first aspect of the present invention, the pMHC complex of the second aspect of the present invention or the cell of the third aspect of the present invention for activating and/or isolating T cells.
  • the tenth aspect of the present invention provides the use of the peptide of the first aspect of the present invention and the pMHC complex of the second aspect of the present invention for screening T cell receptors or antibody libraries.
  • the eleventh aspect of the present invention provides the peptide of the first aspect of the present invention, the pMHC complex of the second aspect of the present invention, the cell of the third aspect of the present invention, the nucleic acid molecule of the fourth aspect of the present invention,
  • the use of the molecule described in the seventh aspect of the present invention or the T cell described in the eighth aspect of the present invention is for preparing a medicament for preventing or treating cancer.
  • the twelfth aspect of the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, the peptide of the first aspect of the present invention, the pMHC complex of the second aspect of the present invention, the The cell of the third aspect, the molecule of the seventh aspect of the present invention, or the T cell of the eighth aspect of the present invention.
  • the pharmaceutical composition is a vaccine.
  • the thirteenth aspect of the present invention provides a method for preventing or treating diseases, comprising administering an appropriate amount of the peptide of the first aspect of the present invention, the pMHC complex of the second aspect of the present invention, the first aspect of the present invention to a subject in need.
  • the fourteenth aspect of the present invention provides a method for obtaining a molecule that binds to the pMHC complex described in the second aspect of the present invention, comprising:
  • Figure 1 is a representative mass spectrum for the identification of the short peptides of the present invention.
  • Figure 2 is a native gel map of the soluble pMHC complex of the present invention. Left band is BSA control, right band is pMHC complex.
  • Figure 3 shows the double positive staining results of CD8+ and tetramer-PE of T cell clones.
  • Fig. 4 is a graph showing the results of the Elispot function experiment of the monoclonal cells obtained by using the short peptide of the present invention on tumor cell lines and T2 cells.
  • Figure 5 is a kinetic map of the binding of soluble TCR molecules obtained by using the short peptides of the present invention to the pMHC complexes of the present invention.
  • the present invention obtains a peptide derived from the antigen SSX2, which is presented on the surface of tumor cells by MHC molecules as a tumor marker. Accordingly, the present invention provides peptides derived from the antigen SSX2, complexes formed by such peptides with MHC molecules and uses of said peptides and complexes. At the same time, the present invention also relates to molecules that bind to the above-mentioned peptides or complexes.
  • the peptide of the present invention and the polypeptide of the present invention or the short peptide of the present invention can be used interchangeably, and both refer to the peptide derived from the antigen SSX2 provided by the present invention.
  • the first aspect of the present invention provides a peptide, the amino acid sequence of the peptide is: AQIPEKIQK (SEQ ID NO: 1).
  • the peptides of the present invention may be post-translationally modified at one or more positions between the amino acid sequences.
  • post-translational modifications can be found in Engelhard et al. Curr Opin Immunol. 2006 Feb;18(1):92-7, and include phosphorylation, acetylation, and deamidation.
  • the peptide of the present invention binds to MHC at the peptide binding site of the MHC molecule.
  • the modified amino acids described above do not disrupt the ability of the peptide to bind to MHC.
  • the amino acid modification increases the ability of the peptide to bind to MHC.
  • mutations may occur at the binding site of the peptide to the MHC.
  • the peptides of the present invention may be composed of AQIPEKIQK (SEQ ID NO: 1), or mainly composed of AQIPEKIQK (SEQ ID NO: 1), which correspond to the positions of amino acid residues 15-23 of the full length of the SSX2 protein.
  • the invention also provides analogs of the protein or peptide shown in SEQ ID NO: 1. Differences between these analogs and natural peptides may be differences in amino acid sequence, differences in modified forms that do not affect the sequence, or both. These peptides include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other known molecular biology techniques. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids (eg, beta, gamma-amino acids). It should be understood that the peptides of the present invention are not limited to the representative peptides exemplified above.
  • Modified (usually without altering the primary structure) forms include chemically derivatized forms of the peptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in peptide synthesis and processing or in further processing steps. This modification can be accomplished by exposing the peptide to enzymes that perform glycosylation, such as mammalian glycosylases or deglycosylases. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are peptides modified to increase their resistance to proteolysis or to optimize solubility.
  • the peptides of the present invention can be synthesized simply by the Merrifield synthesis method (also known as polypeptide solid-phase synthesis). GMP grade peptides can be synthesized using solid phase synthesis techniques at Multiple Peptide Systems (San Diego, CA). Alternatively, the peptides can be synthesized recombinantly, if desired, by methods known in the art. Typical of such methods involve the use of vectors comprising nucleic acid sequences encoding the polypeptides to express the polypeptides in vivo; eg, in bacterial, yeast, insect or mammalian cells. Alternatively, in vitro cell-free systems can also be used for expression. Such systems are known in the art and are commercially available.
  • the peptides may be isolated and/or provided in substantially pure form. For example, they may be provided in a form substantially free of other peptides or proteins.
  • a second aspect of the present invention provides a pMHC complex comprising the peptide of the first aspect of the present invention.
  • the polypeptide is bound to the peptide-binding groove of the MHC molecule.
  • the MHC molecule may be an MHC class I molecule or an MHC class II molecule, preferably, the MHC molecule is an MHC class I molecule.
  • the MHC molecule is HLA-A*11, more preferably, the MHC molecule is HLA-A*1101.
  • the pMHC complexes of the present invention may exist in multimeric form, eg, dimers, or tetramers, or pentamers, or hexamers, or octamers, or larger. Appropriate methods for generating pMHC multimers can be found in the relevant literature, eg (Greten et al., Clin. Diagnostic Lab. Immunol. 2002:216-220).
  • pMHC multimers can be produced by complexing pMHC complexes with biotin residues in combination with fluorescently labeled streptavidin.
  • the pMHC multimers can also be formed by immunoglobulins as molecular scaffolds. In this system, the extracellular region of the MHC molecule is joined to the constant region of the immunoglobulin heavy chain by a short linker.
  • the formation of pMHC multimers may utilize carrier molecules such as dextran (WO02072631). pMHC multimers help improve detection of moieties bound to them, such as T cell receptors. Alternatively, enhance the effect of pMHC complexes in related applications, such as activation of T cells.
  • the pMHC complexes of the present invention may be provided in soluble form.
  • the MHC molecules in the pMHC complexes do not contain a transmembrane region.
  • an MHC class I molecule may consist of the extracellular domain of its light chain and all or part of its heavy chain.
  • the MHC molecule is a fragment comprising only its functional domain.
  • MHC molecules in the soluble pMHC complexes of the invention can also be produced synthetically and then refolded with the peptides of the invention. By determining whether the peptides and MHC molecules are capable of refolding, it is possible to determine which class of MHC molecules the peptides of the invention are capable of forming complexes with.
  • the soluble pMHC complexes of the present invention can be used to screen or detect molecules, such as TCRs or antibodies, to which they bind.
  • the method includes contacting the pMHC complex with a binding moiety to be tested, and determining whether the binding moiety to be tested is bound to the complex.
  • Methods for assaying binding of pMHC complexes are well known in the art. Preferred methods include, but are not limited to, surface plasmon resonance, or any other biosensing technique, ELISA, flow cytometry, chromatography, microscopy.
  • the binding can be detected by functional assays of the biological response to binding, such as cytokine release or apoptosis.
  • the soluble pMHC complexes of the invention can also be used to screen TCR or antibody libraries.
  • the construction of antibody libraries using phage display technology is well known in the art, as described in reference Aitken, Antibody phage display: Methods and Protocols (2009, Humana, New York).
  • the pMHC complexes of the invention are used to screen diverse TCR libraries displayed on the surface of phage particles.
  • the TCRs displayed by the library may contain non-native mutations.
  • the soluble pMHC complexes of the present invention can be immobilized on a suitable solid support via a linker.
  • suitable solid supports include, but are not limited to, beads, membranes, agarose gels, magnetic beads, substrates, tubes, columns.
  • the pMHC complexes can be immobilized on ELISA reaction plates, magnetic beads, or surface plasmon resonance biosensor chips.
  • Methods of immobilizing pMHC complexes to solid supports are known to those skilled in the art and include, for example, the use of affinity binding pairs such as biotin and streptavidin, or antibodies and antigens.
  • the pMHC complex is labeled with biotin and immobilized on a streptavidin-coated surface.
  • the peptides of the present invention can be presented to the cell surface together with MHC complexes. Accordingly, the present invention also provides a cell capable of presenting the pMHC complexes of the present invention to its surface.
  • Such cells may be mammalian cells, preferably immune system cells, and preferably specialized antigen presenting cells, such as dendritic cells or B cells.
  • Other preferred cells include T2 cells (Hosken, et al., Science. 1990. 248:367-70).
  • Cells presenting the peptides or pMHC complexes of the present invention may be isolated, preferably, in the form of a population of cells, or provided in substantially pure form.
  • the cells may not naturally present the complexes of the invention, or the cells may present higher levels of the complexes than in the native state.
  • Such cells can be obtained by pulsing with the peptides of the present invention. Pulse treatment involves incubating cells with the peptide for several hours, preferably at a concentration of 10-5-10-12M .
  • the cells can also be transduced with HLA-A*11 molecules to further induce peptide presentation.
  • Cells presenting the pMHC complexes of the present invention can be used to isolate T cells and T cell receptors, which are activated by said cells and further sorted, and can also obtain expression in said T cells. surface T cell receptors.
  • the method of obtaining the above-mentioned T cells comprises stimulating fresh blood obtained from healthy volunteers with the above-mentioned cells presenting the pMHC complexes of the present invention. You can go through several rounds of stimulation, such as 3-4 rounds. Identification of activated T cells can be determined by measuring cytokine release in the presence of peptide-pulsed T2 cells of the invention (eg, IFN- ⁇ ELISpot assay). Using labeled antibodies, activated cells can be sorted by flow cytometry (FACS), and sorted cells can be expanded in culture and further validated, for example, by ELISpot detection and/or cytotoxicity against target cells and/or pMHC multimerization Body staining was verified. TCR chains from validated T cell clones can be amplified by rapid amplification of cDNA ends (RACE) and sequenced.
  • RACE rapid amplification of cDNA ends
  • the present invention also provides a nucleic acid molecule comprising a nucleic acid sequence encoding the peptide of the present invention.
  • the nucleic acid may be cDNA.
  • the nucleic acid molecule may consist essentially of nucleic acid sequences encoding the peptides of the invention, or may only encode the peptides of the invention.
  • Such nucleic acid molecules can be synthesized using methods known in the art. Due to the degeneracy of the genetic code, those skilled in the art will understand that nucleic acid molecules of different nucleic acid sequences may encode the same amino acid sequence.
  • the present invention also provides a vector, which includes the nucleic acid sequence of the present invention.
  • Suitable vectors are known in the art of vector construction, including promoter selection and other regulatory elements, such as enhancer elements.
  • the vectors of the present invention include sequences suitable for introduction into cells.
  • the vector can be an expression vector, in which the coding sequence of the polypeptide is controlled by its own cis-acting regulatory elements, and the design of the vector is convenient for gene integration or gene replacement in host cells.
  • vector includes DNA molecules, such as plasmids, phages, viruses or other vectors, which contain one or more heterologous or recombinant nucleic acid sequences.
  • Suitable phage and viral vectors include, but are not limited to: lambda-phage, EMBL phage, simian virus, bovine wart virus, Epstein-Barr virus, adenovirus, herpes virus, mouse sarcoma virus, murine breast cancer virus, lentivirus, etc. .
  • the present invention also provides a binding molecule that can be used as an immunotherapeutic or diagnostic agent.
  • the binding molecule can bind only to the peptide, or to a complex formed by the peptide and the MHC molecule. In the latter case, the binding molecule may be partially bound to the MHC molecule, while at the same time it is also bound to the peptide of the invention.
  • the binding moieties of the present invention may be isolated and/or soluble, and/or non-naturally occurring, ie, with no equivalents found in nature, and/or pure, and/or artificially synthesized.
  • the binding molecule is a T cell receptor (TCR).
  • TCRs can be described using the International Information System for Immunogenetics (IMGT).
  • IMGT International Information System for Immunogenetics
  • Native ⁇ heterodimeric TCRs have ⁇ and ⁇ chains. Broadly speaking, each chain contains a variable region, a linker region and a constant region, and the beta chain typically also contains a short variable region between the variable region and the linker region, but the variable region is often considered part of the linker region.
  • the TCR of the present invention may be in any form known in the art.
  • the TCR may be a heterodimer, or exist as a single chain.
  • the TCR may be in a soluble form (ie no transmembrane or cytoplasmic domain), in particular, the TCR may comprise all or part of the TCR extracellular domain.
  • the TCR may also be a full-length chain comprising its transmembrane region.
  • the TCR can be presented to the surface of cells, such as T cells.
  • Soluble TCRs can be obtained by combining existing techniques in the art, for example, by introducing artificial disulfide bonds between the constant domains of the ⁇ and ⁇ chains of ⁇ TCR, or between the ⁇ chain variable region and the ⁇ chain constant region of ⁇ TCR. artificial disulfide bonds were introduced.
  • the TCRs of the invention can be used to deliver cytotoxic or immunostimulatory agents to target cells, or be transformed into T cells, enabling T cells expressing the TCR to destroy tumor cells for use in a treatment process known as adoptive immunotherapy given to the patient.
  • the TCR of the present invention may also contain mutations, and preferably, the affinity of the mutated TCR to the pMHC complex of the present invention is improved.
  • the TCR of the present invention can be used alone, or can be combined with the conjugate in a covalent or other manner, preferably in a covalent manner.
  • the conjugate includes a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the pMHC complexes of the invention), a therapeutic agent, a PK (protein kinase) modification moiety, or a combination of any of the above. Combination binding or conjugation.
  • the TCRs of the present invention can also bind to anti-CD3 antibodies, preferably covalently, to redirect T cells to kill target cells.
  • the binding molecule of the present invention is an antibody.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules containing specific binding sites, which may be all-natural, partially synthetic, or fully synthetic.
  • antibody includes antibody fragments, derivatives thereof, functional equivalents thereof, and homologous antibodies, humanized antibodies, which antibody fragments include an immunoglobulin binding region that is or binds to an antibody Region homology. It can be all natural, or partially synthetic, or totally synthetic.
  • a humanized antibody can be a modified antibody that contains the variable regions of a non-human antibody (eg, mouse) and the constant regions of a human antibody.
  • antibodies may be isotype immunoglobulins (eg, IgG, IgE, IgM, IgD, and IgA) and subclasses of their isotypes; fragments include antigen binding regions, such as Fab, scFv, Fv, dAb, Fd; and diabodies.
  • Antibodies can be polyclonal or monoclonal, preferably monoclonal.
  • TCR and antibody preparation methods of the above-mentioned TCR and antibody are known to those skilled in the art, including but not limited to, expression from E. coli cells or insect cells, and purification.
  • the present invention further provides the use of the peptides, pMHC complexes, nucleic acid molecules, vectors, cells and binding molecules of the present invention in pharmaceuticals.
  • the peptides, pMHC complexes, nucleic acids, vectors, cells or binding molecules can be used to treat or prevent cancer, preferably melanoma, bladder cancer, liver cancer, epidermoid cancer, non-small cell lung cancer, squamous cell cancer, and the like.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the peptide of the present invention, the pMHC complex, the nucleic acid molecule of the present invention, the cell of the present invention, or the binding molecule of the present invention, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be in any suitable form, (depending on the method of administration desired by the patient). It can be presented in unit dosage form, usually in a sealed container, and can be presented as part of a kit. Such kits usually, but not necessarily, contain instructions for use. It may contain a plurality of said unit dosage forms.
  • compositions are suitable for any suitable route of administration, such as injection (including subcutaneous, intramuscular, intraperitoneal or intravenous), inhalation or oral, or nasal, or anal routes.
  • routes of administration such as injection (including subcutaneous, intramuscular, intraperitoneal or intravenous), inhalation or oral, or nasal, or anal routes.
  • the compositions may be prepared by any method known in the art of pharmacy, eg, by admixing the active ingredient with a carrier or excipient under sterile conditions.
  • the dosage administered of the formulations of the present invention may vary widely.
  • the appropriate dose to be administered will be ultimately determined by the physician.
  • peptides, pMHC complexes, or cells presenting pMHC complexes, which are presented to the cell surface along with MHC molecules can activate T cells or B cells to function.
  • the peptides, pMHC complexes or cells presenting the pMHC complexes of the invention may be provided in the form of vaccine compositions.
  • the vaccine composition can be used to treat or prevent cancer. All such compositions are included in the present invention. It will be appreciated that the vaccine may be in a variety of forms (Schlom J.J Natl Cancer Inst. 2012 104(8):599-613).
  • the peptides of the invention can be used directly to immunize patients (Salgaller ML. Cancer Res. 1996.56(20):4749-57 and Marchand M.Int J Cancer. 1999.80(2):219-230).
  • the vaccine composition may contain additional peptides such that the peptide of the invention is one of a mixture of peptides.
  • the vaccine composition may be adjuvanted to enhance the immune response.
  • the vaccine composition may be in the form of an antigen presenting cell presenting the peptide of the invention and the MHC complex.
  • the antigen presenting cells are immune cells, more preferably dendritic cells.
  • the peptides can also be pulsed onto the surface of cells (Thurner BI. et al., J. Exp. Med. 1999. 190:1669), or nucleic acids encoding the peptides of the invention can be introduced into dendritic cells, eg, by electroporation Law (Van Tendeloo, VF. et al., Blood 2001.98:49).
  • the present invention uses a digital single-molecule multiplex gene expression profiling system for detection (nanostring), which further verifies the large expression of SSX2 antigen in liver cancer cells.
  • the HLA-short peptide complexes were purified using the commercial antibody A11.1M. Specifically, tumor cells were lysed with a buffer containing a non-ionic surfactant Triton X-100 (1% v/v), 1 ml of lysis buffer was added to 2*10 ⁇ 7 cells, and the cells were incubated at 4°C with rolling for 1 h. Cell debris was removed by centrifugation, the supernatant was incubated with the antibody, and then rProtein A-Sepharose was added to capture the "antibody-HLA-short peptide complex". Pass the column to collect "rProtein A-Sepharose-antibody-HLA-short peptide complex".
  • the column was washed with low-salt and high-salt buffers, and finally the HLA-short peptide complexes on the immunoaffinity column were eluted with 10% acetic acid, heated at 95°C, and subjected to 10KDa (AmiconR Ultr Centrifugal Filters, MILLIPORE) supernatant.
  • 10KDa AmiconR Ultr Centrifugal Filters, MILLIPORE
  • the peptide mixture was fractionated by Agilent 1260 high performance liquid chromatography: ZORBAX 300SB-C18; 1.0*150mm, 3.5um; mobile phase A was 98% water, 2% acetonitrile, 0.1% trifluoroacetic acid, mobile phase B was 98% acetonitrile, 2 % water, 0.1% trifluoroacetic acid, mobile phase gradient from 5% to 70% mobile phase B over 10 minutes. One fraction was collected every minute. The total run time is 30 minutes.
  • HPLC fractions of the peptides were concentrated and injected into the nanoLC-MSMS system for analysis:
  • Eksigent nanoLC-AB Sciex Triple TOF 5600 system Mass spectrometry using IDA analysis method. Liquid chromatography adopts: pre-column: (Eksigent) NanoLC Trap column.5 ⁇ m C18.100 ⁇ m*2.5cm, 910-00050, analytical column: (Eksigent) C18-CL-120, 3 ⁇ m, 0.075 ⁇ 150mm, 805-00120.
  • Dionex Ultimate3000-Thermo QE Plus system Mass spectrometry adopts ddms2 analysis method.
  • Liquid chromatography adopts: Pre-column: (Thermo) Acclaim 100um ⁇ 2cm, nanoViper, C18, 5um, 100A, 164564, analytical column: (Thermo) Acclaim 75um ⁇ 15cm, nanoViper, C18, 3um, 100A, 164568.
  • the mobile phase A of the nanoflow chromatography of the above two systems was 98% water, 2% acetonitrile, 0.1% formic acid, and the mobile phase B was 98% acetonitrile, 2% water, 0.1% formic acid, and the mobile phase gradient was mobile phase within 74 minutes. B increased from 5% to 50%. The total run time is 90 minutes.
  • the heavy chain and light chain ( ⁇ 2m) of type I HLA-A*1101 molecules were expressed in E. coli in the form of inclusion bodies, respectively. It should be noted that in order to obtain soluble pMHC complexes, the heavy chain of the HLA-A*1101 molecule used in this example does not contain its transmembrane and cytoplasmic regions. In addition, to facilitate subsequent biotinylation of the soluble pMHC complex, a biotinylation tag can be added to the C-terminus of the heavy chain.
  • the specific process of preparing the soluble pMHC complex of the present invention is as follows:
  • Collect 100ml of E.coli bacteria that induces the expression of heavy or light chains centrifuge at 8000g at 4°C for 10min, wash the cells once with 10ml PBS, and then use 5ml BugBuster Master Mix Extraction Reagents (Merck) to vigorously shake the cells to resuspend the cells. Incubate with rotation at room temperature for 20 min, then centrifuge at 6000g for 15 min at 4°C, discard the supernatant, and collect the inclusion bodies.
  • the peptide AQIPEKIQK of the present invention was dissolved in DMSO to a concentration of 20 mg/ml.
  • the inclusion bodies of light chain and heavy chain were dissolved with 8M urea, 20mM Tris pH 8.0, 10mM DTT, and further denatured by adding 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA before renaturation.
  • AQIPEKIQK peptide was added to renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4°C), then added 20mg/L light chain and 90mg/L heavy chain in sequence (final concentration, heavy chain was added in three times, 8h/time), and renatured at 4°C for at least 3 days to completion.
  • renaturation buffer 0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4°C
  • renaturation buffer by dialyzing against 10 volumes of 20 mM Tris pH 8.0, at least twice, to sufficiently reduce the ionic strength of the solution.
  • the protein solution was filtered through a 0.45 ⁇ m cellulose acetate filter and loaded onto a HiTrap Q HP (GE) anion exchange column (5 ml bed volume).
  • the protein was eluted using an Akta purifier (GE), a linear gradient of 0-400 mM NaCl prepared in 20 mM Tris pH 8.0, and pMHC was eluted at approximately 250 mM NaCl, and peak fractions were collected.
  • the native gel map of the obtained soluble pMHC complex of the present invention is shown in Figure 2, and the bands are very uniform.
  • Purified pMHC molecules were concentrated with Millipore ultrafiltration tubes while buffer exchanged to 20mM Tris pH 8.0, followed by addition of biotinylation reagents 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50 ⁇ M D-Biotin, 100 ⁇ g/ml BirA Enzyme (GST-BirA), the mixture was incubated overnight at room temperature, and the complete biotinylation was checked by SDS-PAGE.
  • the biotinylated pMHC molecules were concentrated to 1 ml with a Millipore ultrafiltration tube, and the biotinylated pMHC was purified by gel filtration chromatography. HiPrepTM was pre-equilibrated with filtered PBS using an Akta purifier (GE). A 16/60S200HR column (GE), loaded with 1 ml of concentrated biotinylated pMHC molecules, was then eluted with PBS at a flow rate of 1 ml/min.
  • Akta purifier Akta purifier
  • This example provides an illustration of the use of the pMHC complexes of the invention to obtain monoclonal T cells.
  • TCR sequences can be cloned into a suitable vector and then expressed in E. coli, such as E. coli, or on the surface of phage.
  • peripheral blood lymphocytes of healthy volunteers are stimulated with the short peptide of the present invention, sorted, and then monoclonally cultured by the limiting dilution method to obtain T cell clones.
  • the CD8+ and tetramer-PE double positive staining results are shown in Figure 3 Show.
  • the function and specificity of the T cell clone were further tested by ELISPOT assay.
  • the specific response of the T cell clones obtained by using the short peptide of the present invention to the tumor cell line can also indicate that the short peptide of the present invention is indeed presented to the surface of tumor cells by MHC.
  • the effector cells used in the IFN- ⁇ ELISPOT experiment in this example are the T cell clones obtained in the present invention
  • the target cells are T2 cells loaded with the short peptides of the present invention (transformed into HLA A1101 into T2), and T2 cells loaded with other short peptides Cells (transfected with HLA A1101 into T2), negative tumor cell line K562 and positive tumor cell line K562 (A11, SSX2-P2A-GFP) (transfected with HLA A1101 and SSX2 antigen and GFP), which are loaded with other short peptides T2 cells and tumor cell line K562 served as controls.
  • the ELISPOT experiment steps are as follows: Add the components of the test to the ELISPOT plate in the following order: 20,000 target cells/well, 2000 effector cells/well, the amount of short peptide added in the T2 experimental group is 20 ⁇ l, tumor cells 20 ⁇ l culture medium (test medium) was added to the line group, and 2 duplicate wells were set up. It was then incubated overnight (37°C, 5% CO2 ). The plates were then washed and subjected to secondary detection and color development, the plates were dried for 1 hour, and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID Corporation).
  • ELISPOT READER system AID Corporation
  • the experimental results are shown in Fig. 4.
  • the obtained specific antigen-specific T cell clones have specific responses to T2 cells loaded with the short peptide of the present invention, but basically no response to T2 cells loaded with other irrelevant peptides.
  • T cell clones obtained using the short peptides of the present invention have specific responses to positive tumor cell lines, but no response to negative tumor cell lines. It shows that functional T cell clones are obtained by the short peptides of the present invention.
  • RNA of the above T cell clones was extracted with Quick-RNA TM MiniPrep (ZYMO research), and the TCR sequence was obtained.
  • the soluble TCR protein was expressed in E. coli in this example, and its binding to the pMHC complex was detected by BIAcore. It should be noted that soluble TCRs can be obtained according to the prior art, including but not limited to, those described in patent document PCT/CN2015/093806.
  • the BIAcore T200 real-time analysis system was used to detect the binding activity of soluble TCR protein to pMHC complexes.
  • Anti-streptavidin antibody (GenScript) was added to coupling buffer (10 mM sodium acetate buffer, pH 4.77), and the antibody was then flowed through a CM5 chip preactivated with EDC and NHS to immobilize the antibody on the chip. surface, and finally blocked the unreacted activated surface with ethanolamine hydrochloric acid solution to complete the coupling process with a coupling level of about 15,000 RU.
  • a low concentration of streptavidin was flowed over the antibody-coated chip surface, then pMHC prepared in the manner described in Example 2 was flowed through the detection channel, the other channel was used as a reference channel, and 0.05 mM The biotin was flowed through the chip at a flow rate of 10 ⁇ L/min for 2 min to block the remaining binding sites of streptavidin.
  • Figure 5 shows the kinetic map of the combination of soluble TCR molecules and pMHC complexes obtained by using the short peptide of the present invention, and the map shows the combination of the two.
  • the above method was used to detect the binding activity of the soluble TCR molecule to several other irrelevant antigen short peptides and HLA complexes, and the results showed that the TCR molecule of the present invention did not bind to other irrelevant antigens.

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Abstract

本发明涉及源自于SSX2抗原的短肽，具体地，涉及衍生自SSX2的肿瘤抗原短肽，该短肽与MHC分子形成的复合物以及所述短肽与复合物的用途。同时，本发明还涉及与上述短肽或复合物结合的分子，以及这些分子的用途。

Description

源自于SSX2抗原的短肽 技术领域
本发明涉及源自于SSX2抗原的短肽，具体地，涉及新发现的衍生自肿瘤抗原SSX2的短肽，该短肽与MHC分子形成的复合物以及所述短肽与复合物的用途。同时，本发明还涉及与上述短肽或复合物结合的分子，以及这些分子的用途。
背景技术
众所周知，在许多病理条件下，如感染、癌症、自身免疫疾病等，都会有一些特定分子的不适宜表达。因此，这些分子就成了病理或异常状态的“标记”。这些分子不但可以作为疾病诊断的标记物，还可用于产生诊断试剂和/或治疗剂。例如，用癌症的标记物来产生特定的抗体。另外，这些分子还可以有效地激发细胞毒性T淋巴细胞(CTL)的特异性免疫应答，发挥抗肿瘤效能，同时，还可以通过激活的CTL来获得能够与该“标记”结合的T细胞受体(TCR)作为治疗剂。因此，这些分子，在相关疾病的诊断和治疗中发挥着非常重要的作用。
对于肿瘤而言，已有很多文献发表过不同的内源性的肿瘤抗原分子。但这并不是相关疾病真正的靶点，因为引起CTL免疫应答反应的并非完整的肿瘤抗原分子，而是来自于抗原的特异性CTL表位(Epitope)。一般情况下，肿瘤抗原在细胞内通过蛋白水解作用将其加工成为8-16个氨基酸长度的多肽片段，即CTL表位，进而与内质网腔中的主要组织相容性复合体(MHC，人类的MHC通常称为HLA基因或HLA复合体)分子结合形成多肽-MHC复合物(peptide-MHC complex,pMHC),并最终将pMHC递呈到细胞表面供CD8 +T细胞表面的TCR识别。虽然相关的内源性肿瘤抗原分子早已有发表，但我们并不知晓被呈递出的具体多肽片段。因此，无论是作为疫苗还是用于产生相关疾病的诊断试剂或是治疗试剂，鉴定出这些被呈递到细胞表面的8-16个氨基酸长度的多肽片段，即CTL表位，是至关重要的。本领域技术人员致力于发现并确定这些被呈递到靶细胞表面的多肽片段。
这种被递呈的多肽片段的发现与确定是一个复杂的过程，因为多肽被HLA递呈是抗原蛋白的酶解以及多肽片段与HLA的相互作用的共同结果。这说明，完整的肿瘤抗原分子并不能为多肽片段的发现与鉴定提供任何信息。很多文献发表了利用电脑模拟的方法，如公开数据库SYFPEITHI(Rammensee,et al.,Immunogenetics.1999(50):213-219)和BIMAS(Parker,et al.,J.Immunol.1994.152:163)，提供预测算法来鉴定哪个多肽片段可能会被递呈。但这只是一种预测，具有很大的不确定性，因为它并不是真正的胞内处理以及翻译后的修饰过程。肿瘤组织经过处理后，可以利用质谱仪直接鉴定出肿瘤细胞表面被递呈出的多肽片段，虽然这是一个复杂的过程，但这样得到的结果是非常可靠的。同时，质谱仪的灵敏度也足以能够鉴别低浓度的多肽片段以及翻译后的修饰，因此它是发现及确定肿瘤表面多肽片段的理想工具。
SSX2是滑膜肉瘤X断点，也被称为HOM-MEL-40。SSX2是SSX家族十种高度同源的核酸蛋白之一。SSX蛋白是肿瘤睾丸抗原，只在肿瘤细胞以及没有MHC表达的睾丸胚细胞中表达。SSX2在多种人类癌细胞中表达，包括但不限于，黑色素瘤、头颈癌、淋巴瘤、多种骨髓瘤、胰腺癌、前列腺癌、肉瘤、肝细胞癌以及结肠癌。因此，衍生自SSX2的肽，作为上述癌症的靶点，不但可以作为上述疾病诊断的标记物，还可用于产生上述疾病的预防试剂和/或治疗剂，如抗体或T细胞受体。本发明利用质谱仪分析鉴定,首次发现了肿瘤细胞表面被呈递的衍生自肿瘤抗原SSX2的多肽片段。
发明内容
本发明的目的在于提供了一种新发现的衍生自肿瘤抗原SSX2的短肽，该短肽与MHC分子形成的复合物以及所述短肽与复合物的用途。
本发明的第一方面，提供了一种源自于SSX2抗原的短肽，所述肽包含氨基酸序列：AQIPEKIQK(SEQ ID NO:1)；
在另一优选例中，所述肽能够与MHC分子形成复合物。
在另一优选例中，所述肽由9或10个氨基酸组成。
在另一优选例中，所述肽由9个氨基酸组成。
在另一优选例中，所述肽的氨基酸序列为SEQ ID NO:1。
本发明的第二方面，提供了一种pMHC复合物，所述复合物包含本发明第一方面所述的肽。
在另一优选例中，所述pMHC复合物中的肽的氨基酸序列为SEQ ID NO:1。
在另一优选例中，MHC分子的类型是HLA-A*11。
在另一优选例中，MHC分子的类型是HLA-A*1101。
在另一优选例中，所述pMHC复合物为多聚体。
在另一优选例中，所述pMHC复合物是可溶的。
在另一优选例中，所述pMHC复合物是生物素化的。
本发明的第三方面，提供了一种分离的细胞，所述细胞表面呈递本发明第二方面所述pMHC复合物。
本发明的第四方面，提供了一种核酸分子，所述核酸分子包含编码本发明第一方面所述肽的核酸序列或其互补序列。
本发明的第五方面，提供了一种载体，所述载体含有本发明第四方面所述的核酸分子。
本发明的第六方面，提供了一种宿主细胞，所述细胞中含有本发明第五方面所述的载体。
本发明的第七方面，提供了一种分子，所述分子能够结合本发明第一方面所述的肽和/或本发明第二方面所述的pMHC复合物。
在另一优选例中，所述分子能够特异性结合本发明第一方面所述的肽和/或本发明第二方面所述的pMHC复合物。
在另一优选例中，所述分子为T细胞受体。
在另一优选例中，所述T细胞受体是可溶的。
在另一优选例中，所述分子为抗体或其结合片段。
在另一优选例中，所述抗体为单克隆抗体。
本发明的第八方面，提供了一种分离的单克隆T细胞，所述细胞是通过利用本发明第一方面所述肽和/或本发明第二方面所述pMHC复合物和/或本发明第三方面所述细胞分离获得。
在另一优选例中，所述单克隆T细胞特异性结合本发明第二方面所述pMHC复合物。
本发明的第九方面，提供了本发明第一方面所述肽、本发明第二方面所述pMHC复合物或本发明第三方面所述细胞的用途，用于激活和/或分离T细胞。
本发明的第十方面，提供了本发明第一方面所述肽、本发明第二方面所述pMHC复合物的用途，用于筛选T细胞受体或抗体文库。
本发明的第十一方面，提供了本发明第一方面所述肽、本发明第二方面所述pMHC复合物、本发明第三方面所述细胞、本发明第四方面所述的核酸分子、本发明第七方面所述分子或本发明第八方面所述T细胞的用途，用于制备预防或治疗癌症的药物。
本发明的第十二方面，提供了一种药物组合物，所述组合物含有药学上可接受的载体以及本发明第一方面所述肽、本发明第二方面所述pMHC复合物、本发明第三方面所述细胞、本发明第七方面所述分子或本发明第八方面所述T细胞。
在另一优选例中，所述药物组合物为疫苗。
本发明的第十三方面，提供了一种预防或治疗疾病的方法，包括给需要的对象施用适量的本发明第一方面所述肽、本发明第二方面所述pMHC复合物、本发明第三方面所述细胞、本发明第七方面所述分子或本发明第八方面所述T细胞。
本发明的第十四方面，提供了一种获得与本发明第二方面所述pMHC复合物结合的分子的方法，包括：
(ⅰ)将备选分子与本发明第二方面所述pMHC复合物接触；
(ⅱ)筛选出与(ⅰ)中pMHC复合物结合的分子。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为鉴定本发明短肽的具有代表性的质谱图。
图2为本发明可溶性pMHC复合物的Native胶图。左侧条带为BSA对照，右侧条带为pMHC复合物。
图3为T细胞克隆的CD8+及四聚体-PE双阳性染色结果。
图4为利用本发明短肽得到的单克隆细胞对肿瘤细胞系及T2细胞的Elispot功能实验结果图。
图5为利用本发明短肽得到的可溶性TCR分子与本发明pMHC复合物结合的动力学图谱。
具体实施方式
本发明通过广泛而深入的研究，获得了衍生自抗原SSX2的肽，该肽由MHC分子呈递到肿瘤细胞表面，作为肿瘤标记物。因此，本发明提供了衍生自抗原SSX2的肽，该肽与MHC分子形成的复合物以及所述肽与复合物的用途。同时，本发明还涉及与上述肽或复合物结合的分子。应理解，在本发明中，本发明的肽与本发明多肽或本发明短肽可互换使用，都指本发明提供的衍生自抗原SSX2的肽。
具体地，本发明的第一方面提供了一种肽，所述肽的氨基酸序列为：AQIPEKIQK(SEQ ID NO:1)。
本领域技术人员已知，本发明所述的肽可以在氨基酸序列之间的一个或多个位置进行翻译后修饰。翻译后修饰的例子可以在Engelhard等Curr Opin Immunol.2006年2月；18(1):92-7中找到，并且包括磷酸化作用、乙酰化作用和脱酰氨基作用。
较佳地，本发明所述的肽与MHC结合于MHC分子的肽结合位点。通常，上述描述的修饰的氨基酸不会破坏所述肽与MHC的结合能力。在一个优选的实施方式中，所述的氨基酸修饰提高了肽与MHC结合的能力。例如，突变可能发生在肽与MHC的结合位点。这些结合位点和结合位点上优选的残基为本领域已知的，尤其是对那些结合HLA-A*11的肽来说(参见，比如Parkhurst等，J.Immunol.157:2539-2548(1996))。
本发明所述的肽可以由AQIPEKIQK(SEQ ID NO:1)组成，或主要由AQIPEKIQK(SEQ ID NO:1)组成，其对应于SSX2蛋白全长15-23氨基酸残基的位置。
发明还提供SEQ ID NO：1所示的蛋白或肽的类似物。这些类似物与天然的肽差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些肽包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分子生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的肽并不限于上述例举的代表性的肽。
修饰(通常不改变一级结构)形式包括：体内或体外的肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在肽的合成和加工中或进一步加工步骤中进行 糖基化修饰而产生的肽。这种修饰可以通过将肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的肽。
本发明所述的肽可以用Merrifield合成方法(又被称为多肽固相合成法)简单合成。GMP级别的肽可以用多肽系统(Multiple Peptide Systems，San Diego，CA)的固相合成技术予以合成。或者，所述肽可以重组合成，如果需要，可以用本领域已知的方法予以合成。典型的此类方法涉及载体的使用，所述载体包括编码多肽的核酸序列，在体内表达多肽；例如，在细菌、酵母、昆虫或哺乳动物细胞中表达。或者，还可以使用体外无细胞体系进行表达。此类系统为本领域已知的，并且可以从商业途径获得。所述肽可以是分离的和/或以基本上纯的形式提供。例如，它们可以以一种基本上没有其他肽或蛋白的形式提供。
肿瘤抗原在细胞内通过蛋白水解作用将其加工成为8-16个氨基酸长度的多肽片段，即CTL表位，进而与内质网腔中的MHC分子结合形成多肽-MHC复合物(peptide-MHC complex,pMHC),一起递呈到细胞表面。因此，本发明的第二方面提供了一种pMHC复合物，所述复合物中包含本发明第一方面所述的肽。较佳地，所述多肽结合于MHC分子的肽结合槽上。所述MHC分子可以是MHC I类分子或MHCⅡ类分子，优选地，所述MHC分子是MHC I类分子。在一个优选的实施方式中，所述MHC分子是HLA-A*11，更优选地，所述MHC分子是HLA-A*1101。
本发明所述的pMHC复合物可以以多聚体形式存在，例如，二聚体、或四聚体、或五聚体、或六聚体、或八聚体、或更大。产生pMHC多聚体的适当方法可以参考相关文献，如(Greten et al.，Clin.Diagnostic Lab.Immunol.2002:216-220)。
通常，可以用带有生物素残基的pMHC复合物与通过荧光标记链霉亲和素复合产生pMHC多聚体。或者，所述pMHC多聚体也可以通过免疫球蛋白作为分子支架来形成。在这个系统中，MHC分子的胞外区与免疫球蛋白重链的恒定区通过一个短的连接序列(linker)结合在一起。另外，形成pMHC多聚体也可以利用载体分子，如右旋糖酐(WO02072631)。pMHC多聚体有助于提高与其结合部分的检测，如T细胞受体。或者，提高pMHC复合物在相关应用中的效应，如激活T细胞。
本发明所述的pMHC复合物可以以可溶形式提供。为获得可溶形式的pMHC复合物，优选地，所述pMHC复合物中MHC分子不含跨膜区。具体地，在pMHC复合物中，MHCⅠ类分子可以由其轻链及全部或部分重链的胞外结构域组成。或者，MHC分子是仅包含其功能结构域的片段。
产生本发明可溶性pMHC复合物的方法是本领域技术人员已知的，包括，但不限于，本发明实施例2中所述的方法。本发明可溶性pMHC复合物中的MHC分子也可以利用合成方法产生，然后与本发明的肽重折叠。通过确定肽与MHC分子是否能够重折叠，可以确定本发明肽能够与哪类MHC分子形成复合物。
本发明的可溶性pMHC复合物可以用于筛选或检测与其结合的分子，如TCR或抗体。所述方法包括将所述pMHC复合物与待测结合部分接触，和测定待测结合部分是否与复合物结合。pMHC复合物的结合的测定方法是本领域熟知的。优选的方法包括但不限于，表面等离子体共振，或任何其他的生物传感技术，ELISA、流式细胞术、色谱法、显微镜检查。或者，此外，所述结合可以通过对结合产生的生物响应进行功能测定来检测，如细胞因子释放或细胞凋亡。
同样地，本发明的可溶性pMHC复合物还可以用于筛选TCR或抗体文库。利用噬菌体展示技术来构建抗体文库是本领域熟知的，如参考文献Aitken,Antibody phage display:Methods and Protocols(2009,Humana,New York)中所述。在一个优选的实施方式中，本发明的pMHC复合物被用于筛选展示于噬菌体颗粒表面的多样性TCR文库。所述文库展示的TCR可能含有非天然的突变。
因此，本发明的可溶性pMHC复合物可以通过连接物固定到适当的固相载体上。固相载体的例子包括，但不限于，珠子、膜、琼脂糖凝胶、磁珠、基板、管子、柱。pMHC复合物可以固定在ELISA反应板、磁珠、或表面等离子体共振生物传感器芯片上。将pMHC复合物固定到固相载体的方法为本领域技术人员已知的，并且包括，例如，使用亲和结合对，比如生物素和链霉亲和素，或抗体和抗原。在一个优选的实施方式中，pMHC复合物用生物素标记，并且固定在链霉亲和素包被的表面。
本发明所述的肽可以与MHC复合物一起递呈到细胞表面。因此，本发明还提供了一种细胞，所述细胞能够递呈本发明的pMHC复合物到其表面。此类细胞可以是哺乳动物细胞，较佳地，免疫系统细胞，并且优选为专门的抗原呈递细胞，比如树突细胞或B细胞。其他优选的细胞包括T2细胞(Hosken,et al.,Science.1990.248:367-70)。呈递本发明所述的肽或pMHC复合物的细胞可以是分离的，较佳地，以细胞群体的形式，或以基本上纯的形式提供。所述细胞可以不是天然递呈本发明所述的复合物的，或所述细胞递呈复合物的水平比天然状态下的高。此类细胞可以用本发明所述的肽进行脉冲处理而获得。脉冲处理涉及用所述肽孵育细胞几小时，优选地，所用肽的浓度为10 -5-10 -12M。此外，所述细胞还可以用HLA-A*11分子进行转导，进一步诱导肽的递呈。递呈本发明所述pMHC复合物的细胞可以被用于分离T细胞和T细胞受体，所述T细胞由所述细胞激活并进一步被分选出来，进而也能够获得表达在所述T细胞表面的T细胞受体。
在一个优选的实施方式中，获得上述T细胞的方法包括利用上述递呈本发明pMHC复合物的细胞刺激从健康志愿者处获得的新鲜血液。可以经过几轮的刺激，如3-4轮。激活的T细胞的鉴定可以通过在本发明的肽脉冲的T2细胞的存在下，来测定细胞因子的释放(比如，IFN-γELISpot实验)。利用标记抗体，激活细胞可以通过流式细胞仪(FACS)进行分选，分选的细胞可以扩大培养和进一步验证，例如，通过ELISpot检测和/或针对靶细胞的细胞毒性和/或pMHC多聚体染色进行验证。来自经验证的T细胞克隆的TCR链可以通过cDNA末端快速扩增(RACE)被放大，并进行测 序。
本发明还提供了一种核酸分子，所述核酸分子包括编码本发明的肽的核酸序列。所述核酸可以是cDNA。所述核酸分子可以主要由编码本发明所述肽的核酸序列组成，或可以仅编码本发明所述的肽。此类核酸分子可以用本领域已知的方法合成。由于遗传密码的简并性，本领域技术人员应理解，不同核酸序列的核酸分子可以编码相同的氨基酸序列。
本发明还提供了一种载体，所述载体中包括本发明所述的核酸序列。合适的载体是载体构建领域已知的，包括启动子的选择和其他调控元件，比如增强子元件。本发明所述的载体包括适合引入细胞的序列。比如，所述载体可以是表达载体，在该载体中，所述多肽的编码序列受到它自身顺式作用调控元件的控制，载体的设计便于宿主细胞的基因整合或基因替换等。
本领域普通技术人员应理解，在本发明中，术语“载体”包括DNA分子，比如质粒、噬菌体、病毒或其他载体，它含有一个或多个异源的或重组的核酸序列。合适的噬菌体和病毒载体包括，但不限于：λ-噬菌体，EMBL噬菌体、猿猴病毒、牛疣病毒、Epstein-Barr病毒、腺病毒、疱疹病毒、小鼠肉瘤病毒、鼠类乳癌病毒、慢病毒等。
本发明还提供了一种结合分子，所述分子可以用来作为免疫治疗剂或诊断试剂。该结合分子可以仅和肽结合，或与肽和MHC分子形成的复合物结合。在后一种情况，所述结合分子可以部分结合到MHC分子上，同时，它还与本发明的肽结合。本发明的结合部分可以是分离的和/或可溶的，和/或非天然存在的，即大自然中没有等效物，和/或纯的，和/或人工合成的。
在本发明的一个优选例中，所述结合分子是T细胞受体(TCR)。可以采用国际免疫遗传学信息系统(IMGT)来描述TCR。天然αβ异源二聚TCR具有α链和β链。广义上讲，各链包含可变区、连接区和恒定区，β链通常还在可变区和连接区之间含有短的多变区，但该多变区常视作连接区的一部分。
本发明的TCR可以是本领域已知的任何形式。例如，所述TCR可以是异二聚体，或以单链的形式存在。所述TCR可以是可溶形式(即无跨膜或胞浆区)，具体地，所述TCR可以包含全部或部分TCR胞外结构域。所述TCR也可以是包含其跨膜区的全长链。所述TCR可以被提供到细胞表面，比如T细胞。
可以结合本领域中的现有技术来获得可溶性的TCR，例如，在αβTCR的α与β链的恒定域之间引入人工二硫键，或者在αβTCR的α链可变区与β链恒定区之间引入人工二硫键。
本发明的TCR可用于将细胞毒性剂或免疫刺激剂递送到靶细胞，或被转化入T细胞，使表达该TCR的T细胞能够破坏肿瘤细胞，以便在被称为过继免疫治疗的治疗过程中给予患者。另外，本发明的TCR中也可以含有突变，优选地，突变后的TCR对本发明pMHC复合物的亲和力有所提高。本发明的TCR可以单独使用，也可与偶联物以共价或其他方式结合，优选以共价方式结合。所述偶联物包括可检测标记物(为 诊断目的，其中所述TCR用于检测呈递本发明pMHC复合物的细胞的存在)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。本发明的TCR还可以与抗-CD3的抗体结合，优选以共价方式结合，以重定向T细胞，从而杀伤靶细胞。
在另一优选例中，本发明的结合分子是抗体。如本文所用，术语“抗体”指免疫球蛋白分子和免疫球蛋白分子的免疫活性部分，即含有特异性结合位点的分子，其可以全天然、或部分人工合成、或全部人工合成。术语“抗体”包括抗体片段、其衍生物、功能性等效物以及同源抗体、人源化抗体，所述抗体片段包括免疫球蛋白结合区，所述结合区是抗体结合区或与抗体结合区同源。其可以全天然、或部分人工合成、或全部人工合成。人源化抗体可以是修饰的抗体，其含有非人抗体的可变区(例如，小鼠)以及人抗体的恒定区。
抗体的例子可以是同型免疫球蛋白(例如IgG、IgE、IgM、IgD以及IgA)以及它们同型的亚类；片段包括抗原结合区，比如Fab、scFv、Fv、dAb、Fd；以及双链抗体。抗体可以是多抗或单抗，优选为单克隆抗体。
上述TCR与抗体的制备方法是本领域技术人员已知的，包括但不限于，从大肠杆菌细胞或昆虫细胞中表达，并纯化出来。
在另一方面，本发明进一步提供了本发明的肽、pMHC复合物、核酸分子、载体、细胞以及结合分子在制药方面的用途。所述肽、pMHC复合物、核酸、载体、细胞或结合分子可以被用于治疗或预防癌症，优选黑色素瘤、膀胱癌、肝癌、表皮样癌、非小细胞肺癌和鳞状细胞癌等。
本发明还提供了一种药物组合物，其包含本发明的肽、pMHC复合物、本发明的核酸分子、本发明的细胞或本发明的结合分子，以及药学上可接受的载体。所述药物组合物可以是任何合适的形式，(取决于患者需要的给药方法)。其可以以单位剂型的形式提供，通常置于密封容器中，并且可以作为试剂盒的一部分提供。此类试剂盒通常(但不是必须)包含使用说明。其可以包含多个所述的单位剂型。
所述药物组合物适用于任何适当的给药途径，如注射(包括皮下，肌肉，腹腔或静脉注射)、吸入或口服、或经鼻、或经肛门等途径。所述组合物可以通过药学领域已知的任何方法制备，例如在无菌条件下，通过将活性成分与载体或赋形剂混合。
根据用于治疗的疾病或病症(例如癌症、病毒性感染或自身免疫疾病)、患者的个体年龄和状况等，本发明制剂的给药剂量可以在较宽的范围内变化。恰当的给药剂量将由医师最终决定。
根据本领域的现有技术，与MHC分子一起被呈递到细胞表面的肽、pMHC复合物或递呈pMHC复合物的细胞，可以激活T细胞或B细胞，使其发挥作用。
因此，本发明的肽、pMHC复合物或递呈pMHC复合物的细胞可以以疫苗组合物的形式提供。所述疫苗组合物可以用于治疗或预防癌症。所有的这类组合物都包括在本发明中。应理解，所述疫苗可以为多种形式(Schlom J.J Natl Cancer Inst.2012 104(8):599-613)。例如，本发明的肽可以直接用于免疫患者(Salgaller ML.Cancer Res. 1996.56(20):4749-57和Marchand M.Int J Cancer.1999.80(2):219-230)。所述疫苗组合物可以包含额外的肽，使得本发明的肽是肽混合物中的一个。所述疫苗组合物可以加入佐剂，以增强免疫反应。或者，所述疫苗组合物可以是呈递本发明肽和MHC复合物的抗原递呈细胞的形式。优选地，所述抗原呈递细胞为免疫细胞，更优选地为树突细胞。所述肽也可以脉冲到细胞的表面(Thurner BI.et al.,J.Exp.Med.1999.190:1669)，或者可以将本发明肽的编码核酸引入到树突细胞中，例如，利用电穿孔法(Van Tendeloo,VF.et al.,Blood 2001.98:49)。
下面的具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如(Sambrook和Russell等人，分子克隆：实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)(2001)CSHL出版社)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
如无特别说明，本发明实施例中所用材料均为市售产品。
实施例1 通过质谱鉴定衍生自SSX2抗原的多肽
在进行质谱鉴定之前，本发明利用数字式单分子多重基因表达谱分析系统进行检测(nanostring)，进一步验证了SSX2抗原在肝癌细胞中的大量表达。
使用商业化抗体A11.1M来纯化HLA-短肽复合物。具体地，用含有非离子型表面活性剂Triton X-100(1％v/v)的缓冲液裂解肿瘤细胞，按2*10^7细胞加入1ml裂解液，在4℃滚动孵育1h。离心去除细胞碎片，上清先与抗体孵育，再加入rProtein A-Sepharose捕获“抗体-HLA-短肽复合物”。过柱，收集“rProtein A-Sepharose-抗体-HLA-短肽复合物”。用低盐和高盐缓冲液洗涤柱子，最后用10％的乙酸洗脱挂于免疫亲和柱上HLA-短肽复合物，再经过95℃加热，以及10KDa(AmiconR Ultr Centrifugal Filters,MILLIPORE)超滤膜过滤掉大分子，最终得到多肽混合物。
多肽混合物经Agilent 1260高效液相色谱分馏：ZORBAX 300SB-C18；1.0*150mm,3.5um；流动相A为98％水，2％乙腈，0.1％三氟乙酸，流动相B为98％乙腈，2％水，0.1％三氟乙酸，流动相梯度为10分钟内流动相B由5％升到70％。每一分钟收集一馏份。总运行时间为30分钟。
多肽的HPLC馏份经浓缩后，进样nanoLC-MSMS系统分析：
Eksigent nanoLC-AB Sciex Triple TOF 5600系统：质谱采用IDA分析方法。液相色谱采用：预柱：(Eksigent)NanoLC Trap column.5μm C18.100μm*2.5cm，910-00050，分析柱：(Eksigent)C18-CL-120,3μm,
Figure PCTCN2021132192-appb-000001
0.075×150mm，805-00120。
Dionex Ultimate3000-Thermo QE Plus系统：质谱采用ddms2分析方法.液相色谱采用：预柱：(Thermo)Acclaim
Figure PCTCN2021132192-appb-000002
100um×2cm,nanoViper，C18,5um,100A，164564，分析柱：(Thermo)Acclaim
Figure PCTCN2021132192-appb-000003
75um×15cm,nanoViper，C18, 3um,100A，164568。
上述两个系统的纳流色谱的流动相A为98％水，2％乙腈，0.1％甲酸，流动相B为98％乙腈，2％水，0.1％甲酸，流动相梯度为74分钟内流动相B由5％升到50％。总运行时间为90分钟。
质谱分析结果，借助搜库软件ProteinPilot和Peaks，搜索人类蛋白的Uniprot数据库。根据软件的结果，综合分析，最终得出本发明的抗原短肽序列，如图1所示。
实施例2 可溶性pMHC复合物的制备
Ⅰ型HLA-A*1101分子的重链和轻链(β2m)分别以包涵体的形式在大肠杆菌中(E.coli)表达。应注意，为获得可溶性的pMHC复合物，本实施例中所用的HLA-A*1101分子的重链不包含其跨膜区和胞质区。另外，为方便后续对可溶性的pMHC复合物进行生物素化，可以在重链的C末端加入生物素化标签。制备本发明可溶性pMHC复合物的具体过程如下：
a.纯化
收集100ml诱导表达重链或轻链的E.coli菌液，于4℃8000g离心10min后用10ml PBS洗涤菌体一次，之后用5ml BugBuster Master Mix Extraction Reagents(Merck)剧烈震荡重悬菌体，并于室温旋转孵育20min，之后于4℃，6000g离心15min，弃去上清，收集包涵体。
将上述包涵体重悬于5ml BugBuster Master Mix中，室温旋转孵育5min；加30ml稀释10倍的BugBuster，混匀，4℃6000g离心15min；弃去上清，加30ml稀释10倍的BugBuster重悬包涵体，混匀，4℃6000g离心15min，重复两次，加30ml 20mM Tris-HCl pH 8.0重悬包涵体，混匀，4℃6000g离心15min，最后用20mM Tris-HCl8M尿素溶解包涵体，SDS-PAGE检测包涵体纯度，BCA试剂盒测浓度。
b.复性
将本发明的肽AQIPEKIQK(北京赛百盛基因技术有限公司合成)溶解于DMSO至20mg/ml的浓度。轻链和重链的包涵体用8M尿素、20mM Tris pH 8.0、10mM DTT来溶解，复性前加入3M盐酸胍、10mM醋酸钠、10mM EDTA进一步变性。将AQIPEKIQK肽以25mg/L(终浓度)加入复性缓冲液(0.4M L-精氨酸、100mM Tris pH 8.3、2mM EDTA、0.5mM氧化性谷胱甘肽、5mM还原型谷胱甘肽、0.2mM PMSF，冷却至4℃)，然后依次加入20mg/L的轻链和90mg/L的重链(终浓度，重链分三次加入，8h/次)，复性在4℃进行至少3天至完成。
c.复性后纯化
用10体积的20mM Tris pH 8.0作透析来更换复性缓冲液，至少更换缓冲液两次来充分降低溶液的离子强度。透析后用0.45μm醋酸纤维素滤膜过滤蛋白质溶液，然后加载到HiTrap Q HP(GE通用电气公司)阴离子交换柱上(5ml床体积)。利用Akta纯化仪(GE通用电气公司)，20mM Tris pH 8.0配制的0-400mM NaCl线性梯度液洗脱 蛋白，pMHC约在250mM NaCl处洗脱，收集诸峰组分。得到的本发明可溶性pMHC复合物的Native胶图如图2所示，条带非常均一。
d.生物素化
用Millipore超滤管将纯化的pMHC分子浓缩，同时将缓冲液置换为20mM Tris pH 8.0，然后加入生物素化试剂0.05M Bicine pH 8.3、10mM ATP、10mM MgOAc、50μM D-Biotin、100μg/ml BirA酶(GST-BirA)，室温孵育混合物过夜，SDS-PAGE检测生物素化是否完全。
e.纯化生物素化后的复合物
用Millipore超滤管将生物素化标记后的pMHC分子浓缩至1ml，采用凝胶过滤层析纯化生物素化的pMHC，利用Akta纯化仪(GE通用电气公司)，用过滤过的PBS预平衡HiPrepTM 16/60S200HR柱(GE通用电气公司)，加载1ml浓缩过的生物素化pMHC分子，然后用PBS以1ml/min流速洗脱。
实施例3 利用本发明短肽获得的T细胞克隆
本实施例提供了利用本发明pMHC复合物来获得单克隆T细胞的例证。
本领域技术人员熟知获得TCR的多种方法，包括但不限于，从T细胞克隆中将TCRα与β链的序列分离出来，所述T细胞克隆是被递呈本发明pMHC复合物的细胞刺激的。获得的TCR序列可以被克隆到适合的载体上面，然后在大肠杆菌如E.coli中表达，或表达在噬菌体的表面。
利用本发明的短肽刺激健康志愿者的外周血淋巴细胞，分选，随后用有限稀释法进行单克隆培养来获得T细胞克隆，其CD8+及四聚体-PE双阳性染色结果如图3所示。
实施例4 T细胞克隆的功能
通过ELISPOT实验进一步检测该T细胞克隆的功能及特异性。同时，利用本发明短肽获得的T细胞克隆对肿瘤细胞系的特异性反应也能够说明本发明的短肽确实被MHC递呈到肿瘤细胞表面。
本领域技术人员熟知利用ELISPOT实验检测细胞功能的方法。本实施例IFN-γELISPOT实验中所用的效应细胞为本发明中获得的T细胞克隆，靶细胞为负载了本发明短肽的T2细胞(转了HLA A1101到T2中)，负载其他短肽的T2细胞(转了HLA A1101到T2中)，阴性肿瘤细胞系K562和阳性肿瘤细胞系K562(A11，SSX2-P2A-GFP)(转染了HLA A1101及SSX2抗原和GFP)，其中，负载其他短肽的T2细胞和肿瘤细胞系K562作为对照。
首先准备ELISPOT平板，ELISPOT实验步骤如下：按以下顺序将试验的各个组分加入ELISPOT平板：20,000个靶细胞/孔、2000个效应细胞/孔、T2实验组加入短肽的量为20μl，肿瘤细胞系组加入20μl培养基(试验培养基)，并设置2复孔。然后温 育过夜(37℃，5％CO 2)。随后洗涤平板并进行二级检测和显色，干燥平板1小时，再利用免疫斑点平板读数计(ELISPOT READER system；AID公司)计数膜上形成的斑点。
实验结果如图4所示，得到的特定抗原特异性T细胞克隆对负载本发明短肽的T2细胞有特异性反应，而对负载其他无关肽的T2细胞基本没有反应。另外，利用本发明短肽得到的T细胞克隆对阳性肿瘤细胞系有特异性反应，对阴性肿瘤细胞系没有反应。说明通过本发明的短肽得到了有功能的T细胞克隆。
实施例5 结合本发明pMHC复合物的TCR
用Quick-RNA TM MiniPrep(ZYMO research)抽提上述T细胞克隆的总RNA，并获得TCR序列。为进一步验证获得的TCR能够结合本发明的pMHC复合物，本实施例在E.coli中表达出了可溶的TCR蛋白，并通过BIAcore检测其与pMHC复合物的结合。应注意，可以根据现有技术来获得可溶性的TCR，包括但不限于，专利文献PCT/CN2015/093806中所述。
使用BIAcore T200实时分析系统检测可溶性TCR蛋白与pMHC复合物的结合活性。将抗链霉亲和素的抗体(GenScript)加入偶联缓冲液(10mM醋酸钠缓冲液，pH4.77)，然后将抗体流过预先用EDC和NHS活化过的CM5芯片，使抗体固定在芯片表面，最后用乙醇胺的盐酸溶液封闭未反应的活化表面，完成偶联过程，偶联水平约为15,000RU。使低浓度的链霉亲和素流过已包被抗体的芯片表面，然后将按实施例2中所述方式制得的pMHC物流过检测通道，另一通道作为参比通道，再将0.05mM的生物素以10μL/min的流速流过芯片2min，封闭链霉亲和素剩余的结合位点。
利用本发明短肽得到可溶性的TCR分子与pMHC复合物结合的动力学图谱如图5所示，图谱显示出了二者的结合。同时，还利用上述方法检测了可溶性的TCR分子与其他几种无关抗原短肽与HLA复合物的结合活性，结果显示本发明TCR分子与其他无关抗原均无结合。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的。

Claims (24)

  1. 一种源自于AFP抗原的短肽，其特征在于，所述短肽包含氨基酸序列：
    AQIPEKIQK(SEQ ID NO:1)；其中，所述肽由9或10个氨基酸组成。
  2. 如权利要求1所述的肽，其特征在于，所述肽的氨基酸序列为SEQ ID NO:1。
  3. 一种pMHC复合物，其特征在于，所述复合物包含权利要求1中所述的肽。
  4. 如权利要求3所述的pMHC复合物，其特征在于，所述pMHC复合物中的肽的氨基酸序列为SEQ ID NO:1。
  5. 如权利要求3中所述pMHC复合物，其特征在于，MHC分子的类型是HLA-A*11。
  6. 如权利要求3中所述pMHC复合物，其特征在于，MHC分子的类型是HLA-A*1101。
  7. 如权利要求3中所述pMHC复合物，其特征在于，所述pMHC复合物为多聚体。
  8. 如权利要求3中所述pMHC复合物，其特征在于，所述pMHC复合物是可溶的。
  9. 一种分离的细胞，其特征在于，所述细胞表面呈递或负载权利要求3-7中任一所述pMHC复合物，优选地，所述细胞为DC。
  10. 一种核酸分子，其特征在于，所述核酸分子包含编码权利要求1中所述肽的核酸序列或其互补序列。
  11. 一种载体，其特征在于，所述载体含有权利要求10中所述的核酸分子。
  12. 一种宿主细胞，其特征在于，所述细胞中含有权利要求11中所述的载体。
  13. 一种分子，其特征在于，所述分子能够结合权利要求1中所述肽和/或权利要求3-8中任一所述pMHC复合物。
  14. 如权利要求13所述的分子，其特征在于，所述分子为T细胞受体。
  15. 如权利要求14所述的T细胞受体，其特征在于，所述T细胞受体是可溶的。
  16. 如权利要求13所述的分子，其特征在于，所述分子为抗体或其结合片段。
  17. 如权利要求16所述的抗体，其特征在于，所述抗体为单克隆抗体。
  18. 一种分离的单克隆T细胞，其特征在于，所述细胞是通过权利要求1中所述肽或权利要求3-8中任一所述pMHC复合物或权利要求9中所述细胞体外刺激筛选获得。
  19. 权利要求1中所述肽、权利要求3-8中任一所述pMHC复合物或权利要求9中所述细胞的用途，其特征在于，用于体外激活和/或分离T细胞。
  20. 权利要求1中所述肽、权利要求3-8中任一所述pMHC复合物的用途，其特征在于，用于筛选T细胞受体或抗体文库。
  21. 权利要求1中所述肽、权利要求3-8中任一所述pMHC复合物、权利要求9 中所述细胞、权利要求10中所述的核酸分子、权利要求13-17中所述分子或权利要求18中所述T细胞的用途，其特征在于，用于制备预防或治疗癌症的药物。
  22. 一种药物组合物，其特征在于，所述组合物含有药学上可接受的载体以及权利要求1中所述肽、权利要求3-8中任一所述pMHC复合物、权利要求9中所述细胞、权利要求13-17中所述分子或权利要求18中所述T细胞。
  23. 如权利要求22中所述药物组合物，其特征在于，所述药物组合物为疫苗。
  24. 一种获得与权利要求3-8中任一所述pMHC复合物结合的分子的方法，包括：
    (i)将备选分子与权利要求3-8中任一所述pMHC复合物接触；
    (ii)筛选出与(i)中pMHC复合物结合的分子。
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CN117304301A (zh) * 2022-06-17 2023-12-29 香雪生命科学技术（广东）有限公司 一种结合ssx2抗原的tcr分子及其应用

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CN1691964A (zh) * 2002-09-06 2005-11-02 曼康公司 表位序列
CN101287755A (zh) * 2005-09-05 2008-10-15 伊玛提克斯生物技术有限公司 结合于人类白细胞抗原(hla)ⅰ类或ⅱ类分子的肿瘤相关肽及相关的抗癌疫苗
CN110343167A (zh) * 2018-04-03 2019-10-18 广东香雪精准医疗技术有限公司 识别ssx2抗原短肽的t细胞受体
CA3100775A1 (en) * 2018-05-18 2019-11-21 Children's National Medical Center Improved targeted t-cell therapy
CN110950949A (zh) * 2018-09-26 2020-04-03 广东香雪精准医疗技术有限公司 一种识别ssx2抗原的t细胞受体

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CN1691964A (zh) * 2002-09-06 2005-11-02 曼康公司 表位序列
CN101287755A (zh) * 2005-09-05 2008-10-15 伊玛提克斯生物技术有限公司 结合于人类白细胞抗原(hla)ⅰ类或ⅱ类分子的肿瘤相关肽及相关的抗癌疫苗
CN110343167A (zh) * 2018-04-03 2019-10-18 广东香雪精准医疗技术有限公司 识别ssx2抗原短肽的t细胞受体
CA3100775A1 (en) * 2018-05-18 2019-11-21 Children's National Medical Center Improved targeted t-cell therapy
CN110950949A (zh) * 2018-09-26 2020-04-03 广东香雪精准医疗技术有限公司 一种识别ssx2抗原的t细胞受体

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