CN110862449B - SSX-derived tumor antigen short peptides - Google Patents

Abstract

The invention relates to a tumor antigen short peptide derived from SSX1, a complex formed by the short peptide and MHC molecules and application of the short peptide and the complex. The invention also relates to molecules which bind to the above-mentioned short peptides or complexes, and to the use of these molecules.

Description

SSX-derived tumor antigen short peptides

Technical Field

The present invention relates to a short peptide derived from tumor antigen SSX, in particular to a newly discovered short peptide derived from tumor antigen SSX1, a complex formed by the short peptide and MHC molecules and the application of the short peptide and the complex. The invention also relates to molecules which bind to the above-mentioned short peptides or complexes, and to the use of these molecules.

Background

It is well known that in many pathological conditions, such as infections, cancer, autoimmune diseases, etc., there is inappropriate expression of certain molecules. Thus, these molecules become "markers" for pathological or abnormal conditions. These molecules can be used not only as markers for disease diagnosis, but also for the production of diagnostic and/or therapeutic agents. For example, markers for cancer are used to generate specific antibodies. In addition, these molecules can also efficiently elicit a specific immune response from Cytotoxic T Lymphocytes (CTLs) to exert anti-tumor efficacy, and at the same time, through activated CTLs, T Cell Receptors (TCRs) capable of binding to this "marker" can be obtained as therapeutic agents. Therefore, these molecules play a very important role in the diagnosis and treatment of related diseases.

For tumors, many different endogenous tumor antigen molecules have been published in the literature. However, this is not a true target for the relevant disease, since the tumor antigen molecules responsible for the CTL immune response are not intact, but rather are derived from specific CTL epitopes (epitopes) of the antigen. In general, tumor antigens are processed intracellularly by proteolysis into 8-16 amino groupsThe acid-long polypeptide fragment, i.e., the CTL epitope, in turn binds to major histocompatibility complex (MHC, human MHC commonly referred to as HLA gene or HLA complex) molecules in the lumen of the endoplasmic reticulum to form a polypeptide-MHC complex (pMHC), and finally presents pMHC to the cell surface for CD8 ⁺ TCR recognition on the T cell surface. Although related endogenous tumor antigen molecules have been published, we are not aware of the specific polypeptide fragments that are presented. Therefore, whether as a vaccine or a diagnostic or therapeutic agent for the production of the relevant disease, it is crucial to identify these 8-16 amino acid long polypeptide fragments presented on the cell surface, i.e. CTL epitopes. Those skilled in the art are working to find and identify such polypeptide fragments that are presented on the surface of a target cell.

The discovery and identification of such presented polypeptide fragments is a complex process, since the presentation of polypeptides by HLA is a combination of proteolytic degradation of the antigen and the interaction of the polypeptide fragments with HLA. This indicates that the complete tumor antigen molecule does not provide any information for the discovery and identification of polypeptide fragments. A number of documents have published methods using computer simulations, such as the public databases SYFPEITHI (Rammensee, et al, immunogenetics.1999 (50): 213-219) and BIMAS (Parker, et al, J.Immunol.1994.152: 163), which provide predictive algorithms to identify which polypeptide fragments are likely to be presented. However, this is only a prediction and has great uncertainty, since it is not a true intracellular process and post-translational modification. After the tumor tissue is treated, the polypeptide fragments presented on the surface of the tumor cells can be directly identified by using a mass spectrometer, and although the process is a complex process, the obtained result is very reliable. At the same time, the sensitivity of the mass spectrometer is sufficient to identify low concentrations of polypeptide fragments and post-translational modifications, and thus it is an ideal tool for the discovery and determination of polypeptide fragments on tumor surfaces.

SSX is the synovial sarcoma X breakpoint and SSX1 is one of ten highly homologous nucleic acid proteins of the SSX family. SSX1 protein is a tumor testis antigen and is expressed only in tumor cells and in testicular germ cells that do not express MHC. SSX1 is expressed in a variety of human cancer cells including, but not limited to, liver cancer, lung cancer, fibrosarcoma, breast cancer, colon cancer, prostate cancer, and chronic myeloma cell leukemia. Therefore, the peptide derived from SSX1, which is a target of the above-mentioned various cancers, can be used not only as a marker for diagnosis of the above-mentioned diseases but also for production of a preventive and/or therapeutic agent for the above-mentioned diseases, such as an antibody or a T cell receptor. The invention utilizes mass spectrometer analysis to identify and discover the polypeptide fragment derived from the tumor antigen SSX1 presented on the surface of the tumor cell for the first time.

Disclosure of Invention

The invention aims at providing a newly discovered short peptide derived from a tumor antigen SSX1, a complex formed by the short peptide and an MHC molecule and application of the short peptide and the complex.

In a first aspect of the invention, there is provided a peptide comprising the amino acid sequence: KYSEKISYVYM (SEQ ID NO: 1).

In another preferred embodiment, the peptide is capable of forming a complex with an MHC molecule.

In another preferred embodiment, the peptide consists of 11, 12 or 13 amino acids.

In another preferred embodiment, the peptide consists of 11 or 12 amino acids.

In another preferred embodiment, the amino acid sequence of the peptide is SEQ ID NO 1.

In a second aspect of the invention, there is provided a pMHC complex comprising a peptide according to the first aspect of the invention.

In another preferred embodiment, the amino acid sequence of the peptide in the pMHC complex is SEQ ID NO 1.

In another preferred embodiment, the MHC molecule is of the type HLA-base:Sub>A 24.

In another preferred embodiment, the MHC molecule is of the type HLA-base:Sub>A 2402.

In another preferred embodiment, the pMHC complex is a multimer.

In another preferred embodiment, the pMHC complex is soluble.

In another preferred embodiment, the pMHC complex is biotinylated.

In a third aspect of the invention, there is provided an isolated cell which presents on its surface a pMHC complex according to the second aspect of the invention.

In a fourth aspect of the invention, there is provided a nucleic acid molecule comprising a nucleic acid sequence encoding a peptide according to the first aspect of the invention or a complement thereof.

In a fifth aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the fourth aspect of the invention.

In a sixth aspect of the invention, there is provided a host cell comprising a vector according to the fifth aspect of the invention.

In a seventh aspect of the invention, there is provided a molecule capable of binding a peptide according to the first aspect of the invention and/or a pMHC complex according to the second aspect of the invention.

In another preferred embodiment, the molecule is capable of specifically binding to a peptide according to the first aspect of the invention and/or a pMHC complex according to the second aspect of the invention.

In another preferred embodiment, the molecule is a T cell receptor.

In another preferred embodiment, the T cell receptor is soluble.

In another preferred embodiment, the molecule is an antibody or binding fragment thereof.

In another preferred embodiment, the antibody is a monoclonal antibody.

In an eighth aspect of the present invention there is provided an isolated monoclonal T cell isolated using a peptide according to the first aspect of the present invention and/or a pMHC complex according to the second aspect of the present invention.

In another preferred embodiment, the T cells specifically bind to the pMHC complex of the second aspect of the invention.

In a ninth aspect, the invention provides the use of a peptide according to the first aspect of the invention, a pMHC complex according to the second aspect of the invention or a cell according to the third aspect of the invention for activating and/or isolating T cells.

In a tenth aspect of the invention, there is provided the use of a peptide according to the first aspect of the invention, a pMHC complex according to the second aspect of the invention, for screening a library of T cell receptors or antibodies.

In an eleventh aspect, the present invention provides the use of a peptide according to the first aspect of the invention, a pMHC complex according to the second aspect of the invention, a cell according to the third aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, a molecule according to the seventh aspect of the invention or a T cell according to the eighth aspect of the invention, in the manufacture of a medicament for the prophylaxis or treatment of cancer.

In a twelfth aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a peptide according to the first aspect of the invention, a pMHC complex according to the second aspect of the invention, a cell according to the third aspect of the invention, a molecule according to the seventh aspect of the invention or a T cell according to the eighth aspect of the invention.

In another preferred embodiment, the pharmaceutical composition is a vaccine.

In a thirteenth aspect of the invention, there is provided a method of preventing or treating a disease, comprising administering to a subject in need thereof an amount of a peptide according to the first aspect of the invention, a pMHC complex according to the second aspect of the invention, a cell according to the third aspect of the invention, a molecule according to the seventh aspect of the invention or a T cell according to the eighth aspect of the invention.

In a fourteenth aspect of the present invention, there is provided a method of obtaining a molecule which binds to a pMHC complex according to the second aspect of the present invention, comprising:

contacting a candidate molecule with a pMHC complex according to the second aspect of the invention;

(ii) selecting the molecule which binds to the pMHC complex of (i).

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. 1 is a typical mass spectrum for identifying the short peptide of the present invention.

FIG. 2 is a Native gel map of a soluble pMHC complex of the invention. The left band is BSA control and the right band is pMHC complex.

FIG. 3 shows the double positive staining results of monoclonal cells for CD8+ and tetramer-PE.

FIG. 4 is a diagram showing the results of Elispot functional experiments on tumor cell lines and T2 cells using monoclonal cells obtained from the short peptides of the present invention.

Detailed Description

The present invention, through extensive and intensive studies, has obtained a peptide derived from the antigen SSX1, 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 SSX1, complexes of such peptides with MHC molecules and uses of such peptides and complexes. Also, the present invention relates to a molecule bound to the above peptide or complex. It is to be understood that, in the present invention, the peptide of the present invention is used interchangeably with the polypeptide of the present invention or the short peptide of the present invention, and both refer to the peptide derived from the antigen SSX1 provided by the present invention.

In addition, it should be noted that, since ten kinds of nucleic acid proteins of SSX family are highly homologous, the present invention found a short peptide KYSEKISYVYM (SEQ ID NO: 1) in both SSX1 antigen and SSX8 antigen

In particular, a first aspect of the invention provides a peptide comprising the amino acid sequence: KYSEKISYVYM (SEQ ID NO: 1); wherein the peptide consists of 11 or 12 amino acids.

It is known to those skilled in the art that the peptides of the invention may be post-translationally modified at one or more positions between the amino acid sequences. Examples of post-translational modifications can be found in Engelhard et al Curr Opin Immunol.2006, month 2; 18 (1): 92-7 and include phosphorylation, acetylation, and deamidation.

Preferably, the peptides of the invention bind to MHC a peptide binding site of an MHC molecule. Generally, the modified amino acids described above do not disrupt the ability of the peptide to bind to MHC. In a preferred embodiment, the amino acid modification increases the ability of the peptide to bind to MHC. For example, mutations may occur at the binding site of a peptide to MHC. These binding sites and preferred residues on the binding sites are known in the art, especially for those peptides that bind HLA-A.sub.24 (see, e.g., parkhurst et al, J.Immunol.157:2539-2548 (1996)).

More specifically, the amino acids of the peptides of the invention may be 7-25, preferably 8-16, preferably 9, 10, 11 or 12 in length.

The peptides of the invention may consist of KYSEKISYVYM (SEQ ID NO: 1), or consist essentially of KYSEKISYVYM (SEQ ID NO: 1), which corresponds to the positions of residues 41-51 of the full length of the SSX1 protein.

The invention also provides SEQ ID NO:1, or an analog of the protein or peptide of formula 1. These analogs may differ from the native peptide by amino acid sequence differences, by modifications that do not affect the sequence, or by both. These peptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biological techniques. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the peptides of the present invention are not limited to the representative peptides exemplified above.

Modified (generally without altering 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 peptides that result from glycosylation modifications during synthesis and processing of the peptide or during further processing steps. Such modification may be accomplished by exposing the peptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are peptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, "SEQ ID NO:1, and the conservative variant peptide of the protein shown in SEQ ID NO:1, or a peptide formed by substituting at most 3, preferably at most 2, amino acids with amino acids having similar or analogous properties. These conservative variant peptides are preferably generated by amino acid substitutions according to Table 1.

TABLE 1

The peptides of the invention can be simply synthesized by the Merrifield synthesis method (also known as polypeptide solid phase synthesis method). GMP-grade peptides can be synthesized using solid phase synthesis techniques of the polypeptide system (Multiple Peptide Systems, san Diego, calif.). Alternatively, the peptides may be recombined and, if desired, synthesized using methods known in the art. Typical such methods involve the use of a vector comprising a nucleic acid sequence encoding a polypeptide, which is expressed in vivo; for example, in bacterial, yeast, insect or mammalian cells. Alternatively, expression can also be performed using an in vitro cell-free system. 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 that is substantially free of other peptides or proteins.

Tumor antigens are processed intracellularly by proteolysis into polypeptide fragments of 8-16 amino acids in length, i.e., CTL epitopes, which are then bound to MHC molecules in the lumen of the endoplasmic reticulum to form polypeptide-MHC complexes (pMHC) which are presented together on the cell surface. Accordingly, a second aspect of the present invention provides a pMHC complex comprising a peptide according to the first aspect of the invention. Preferably, the polypeptide binds to the peptide binding groove of an 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. inbase:Sub>A preferred embodiment, the MHC molecule is HLA-base:Sub>A 24, more preferably the MHC molecule is HLA-base:Sub>A 2402.

The pMHC complexes described herein may exist in multimeric forms, for example, dimeric, or tetrameric, or pentameric, or hexameric, or octameric, or larger. Suitable methods for generating pMHC multimers can be found in the relevant literature, e.g. (Green et al, clin. Diagnostic Lab. Immunol.2002: 216-220).

Typically, pMHC multimers can be generated by complexing a pMHC complex with biotin residues to streptavidin via fluorescent labeling. Alternatively, the pMHC multimer may also be formed by an immunoglobulin as a molecular scaffold. In this system, the extracellular domain of the MHC molecule is joined to the constant region of an immunoglobulin heavy chain by a short linker sequence (linker). Alternatively, formation of pMHC multimers may utilize carrier molecules such as dextran (WO 02072631). pMHC multimers help to improve detection of binding moieties, such as T cell receptors, thereto. Alternatively, the effect of the pMHC complex in related applications is enhanced, such as the activation of T cells.

The pMHC complexes described herein may be provided in soluble form. To obtain the soluble form of the pMHC complex, preferably the MHC molecules in the pMHC complex are devoid of a transmembrane region. In particular, in a pMHC complex, MHC class i molecules may consist of the extracellular domain of their light chain and all or part of their heavy chain. Alternatively, an MHC molecule is a fragment comprising only its functional domain.

Methods of producing soluble pMHC complexes of the invention are known to those skilled in the art and include, but are not limited to, the methods described in example 2 of the invention. The MHC molecules in the soluble pMHC complexes of the invention may also be produced synthetically and then refolded with the peptides of the invention. By determining whether a peptide is capable of refolding with an MHC molecule, it can be determined with which MHC molecule the peptide of the invention is capable of forming a complex.

The soluble pMHC complexes of the invention can be used to screen for or detect molecules, such as TCRs or antibodies, that bind to them. The method comprises contacting the pMHC complex with a binding moiety to be tested and determining whether the binding moiety to be tested binds to the complex. Methods for determining 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. Alternatively, or in addition, the binding may be detected by functional assays for the biological response produced by the binding, such as cytokine release or apoptosis.

Likewise, the soluble pMHC complexes of the invention may 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 the references Aitken, antibody phase display: methods and Protocols (2009, humana, new York). In a preferred embodiment, the pMHC complexes of the invention are used to screen diverse TCR libraries displayed on the surface of phage particles. The TCR displayed by the library may contain non-native mutations.

Thus, the soluble pMHC complexes of the invention may be immobilised to a suitable solid support via a linker. Examples of solid phase carriers include, but are not limited to, beads, membranes, sepharose, 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 immobilising pMHC complexes to solid phase carriers 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. In a preferred embodiment, the pMHC complexes are labeled with biotin and are immobilized on a streptavidin-coated surface.

The peptides of the invention may be presented to the cell surface together with MHC complexes. Accordingly, the present invention also provides a cell capable of presenting a pMHC complex of the invention to its surface. Such cells may be mammalian cells, preferably cells of the immune system, 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). The cells presenting the peptides or pMHC complexes of the invention may be isolated, preferablyProvided in the form of a population of cells, or in substantially pure form. The cell may not naturally present the complex of the invention, or the cell may present the complex at a higher level than in its natural state. Such cells can be obtained by pulsing with the peptides of the invention. The pulse treatment involves incubating the cells with the peptide for several hours, preferably at a concentration of 10 ^-5 -10 ^-12 And M. In addition, the cells may be transduced with HLA-A x 24 molecules to further induce peptide presentation. Cells presenting the pMHC complexes of the invention can be used to isolate T cells and T cell receptors, which are activated by the cells and further sorted out, and can also obtain the T cell receptors expressed on the surface of the T cells.

In a preferred embodiment, the method of obtaining the above-described T cells comprises stimulating fresh blood obtained from healthy volunteers with the above-described cells presenting the pMHC complex of the invention. Several rounds of stimulation may be performed, such as 3-4 rounds. Identification of activated T cells cytokine release can be measured by the presence of peptide-pulsed T2 cells of the invention (e.g., IFN- γ ELISpot assay). Using labeled antibodies, activated cells can be sorted by flow cytometry (FACS), and sorted cells can be expanded and further validated, e.g., by ELISpot detection and/or cytotoxicity against target cells and/or pMHC multimer staining. TCR chains from validated T cell clones can be amplified by cDNA end Rapid Amplification (RACE) and sequenced.

The invention also provides a nucleic acid molecule comprising a nucleic acid sequence encoding a peptide of the invention. The nucleic acid may be cDNA. The nucleic acid molecule may consist essentially of a nucleic acid sequence encoding a peptide according to the invention, or may encode only a peptide according to the invention. Such nucleic acid molecules can be synthesized using methods known in the art. Due to the degeneracy of the genetic code, it will be understood by those skilled in the art that nucleic acid molecules of different nucleic acid sequences may encode the same amino acid sequence.

The invention also provides a vector, wherein the vector comprises the nucleic acid sequence. Suitable vectors are known in the art of vector construction and include promoter selection and other regulatory elements, such as enhancer elements. The vectors of the invention include sequences suitable for introduction into a cell. For example, the vector may be an expression vector in which the coding sequence for the polypeptide is under the control of its own cis-acting regulatory elements, designed to facilitate gene integration or gene replacement in a host cell, and the like.

It will be understood by those of ordinary skill in the art that, in the present invention, the term "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, verruca bovis, epstein-Barr virus, adenovirus, herpes virus, murine sarcoma virus, murine mammary carcinoma virus, lentivirus, etc.

The invention also provides a binding molecule which may be used as an immunotherapeutic or diagnostic agent. The binding molecule may be bound to the peptide alone or to a complex formed between the peptide and an MHC molecule. In the latter case, the binding molecule may be partially bound to an MHC molecule, while it is also bound to a peptide of the invention. Binding moieties of the invention may be isolated and/or soluble, and/or non-naturally occurring, i.e., without an equivalent in nature, and/or pure, and/or synthetically produced.

In a preferred embodiment of the invention, the binding molecule is a T Cell Receptor (TCR). The TCR may be described using the international immunogenetics information system (IMGT). Native α β heterodimeric TCRs have an α chain and a β chain. In a broad sense, each chain comprises a variable region, a linker region and a constant region, and the beta chain also typically contains a short diversity region between the variable region and the linker region, but the diversity region is often considered part of the linker region.

The TCRs of the invention may be in any form known in the art. For example, the TCR may be heterodimeric, or present as a single chain. The TCR may be in soluble form (i.e. without a transmembrane or cytoplasmic region), 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 may be provided to a cell surface, such as a T cell.

Soluble TCRs can be obtained in combination with techniques known in the art, e.g., introducing an artificial disulfide bond between the constant domains of the α and β chains of an α β TCR, or between the α chain variable region and the β chain constant region of an α β TCR.

The TCRs of the invention are useful for delivering cytotoxic or immunostimulatory agents to target cells, or are transformed into T cells, enabling the T cells expressing the TCR to destroy tumor cells for administration to patients in a therapeutic process known as adoptive immunotherapy. In addition, the TCR of the invention may also comprise a mutation, preferably the mutated TCR has an improved affinity for the pMHC complex of the invention. 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 pMHC complex of the invention), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination or conjugate of any of the above. The TCRs of the invention may also be conjugated, preferably covalently, to an anti-CD 3 antibody to redirect T cells, thereby killing target cells.

In another preferred embodiment, the binding molecule of the invention is an antibody. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain specific binding sites, which can be wholly natural, or partially or wholly artificial. The term "antibody" includes antibody fragments, including immunoglobulin binding regions that are or are homologous to antibody binding regions, derivatives, functional equivalents, and homologous, humanized antibodies thereof. It may be all natural, partially synthetic, or fully synthetic. A humanized antibody may be a modified antibody comprising the variable regions of a non-human antibody (e.g., mouse) and the constant regions of a human antibody.

Examples of antibodies may be isotypic immunoglobulins (e.g., igG, igE, igM, igD, and IgA) and their isotypic subclasses; fragments include antigen binding regions, such as Fab, scFv, fv, dAb, fd; and diabodies. The antibody may be a polyclonal or monoclonal antibody, preferably a monoclonal antibody.

Methods for making the above-described TCRs and antibodies are known to those skilled in the art and include, but are not limited to, expression from e.coli cells or insect cells and purification.

In a further aspect, the invention further provides the use of the peptides, pMHC complexes, nucleic acid molecules, vectors, cells and binding molecules of the invention in the manufacture of a medicament. The peptides, pMHC complexes, nucleic acids, vectors, cells or binding molecules may be used to treat or prevent cancer, preferably melanoma, bladder cancer, liver cancer, epidermoid cancer, non-small cell lung cancer, squamous cell carcinoma and the like.

The invention also provides a pharmaceutical composition comprising a peptide of the invention, a pMHC complex, a nucleic acid molecule of the invention, a cell of the invention or a binding molecule of the invention, and a pharmaceutically acceptable carrier. The pharmaceutical composition may be in any suitable form (depending on the method of administration required by the patient). It may be provided in unit dosage form, typically in a sealed container, and may be provided as part of a kit. Such kits typically (but not necessarily) contain instructions for use. It may comprise a plurality of said unit dosage forms.

The pharmaceutical composition is suitable for any suitable route 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, for example, by mixing the active ingredient with the carrier or excipient under sterile conditions.

The dosage of the formulations of the present invention may vary over a wide range depending on the disease or disorder being treated (e.g., cancer, viral infection or autoimmune disease), the age and condition of the individual patient, and the like. The appropriate dosage to be administered will be ultimately determined by the physician.

According to the state of the art, peptides presented to the cell surface together with MHC molecules, pMHC complexes or cells presenting pMHC complexes can activate T cells or B cells to function.

Thus, the peptide, pMHC complex or cells presenting pMHC complexes of the invention may be provided in the form of a vaccine composition. The vaccine composition may be used for the treatment or prevention of 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). For example, the peptides of the invention can be used directly to immunize patients (Salgaleller ML. Cancer Res.1996.56 (20): 4749-57and Marchand M. Int J cancer.1999.80 (2): 219-230). The vaccine composition may comprise 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. Alternatively, the vaccine composition may be in the form of an antigen presenting cell presenting a complex of a peptide of the invention and an MHC. Preferably, the antigen presenting cell is an immune cell, more preferably a dendritic cell. The peptides may 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 may be introduced into dendritic cells, for example, using electroporation (Van endeloo, vf.et al, blood 2001.98.

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 noted 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 publisher), or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.

The materials used in the examples of the present invention are all commercially available products unless otherwise specified.

Example 1 identification of polypeptides derived from SSX antigens by Mass Spectrometry

Prior to mass spectrometric identification, the present invention further demonstrates that SSX1 antigen is expressed in a variety of tumor cells. Specifically, the detection is carried out by using a digital single-molecule multiplex gene expression profiling system (nanostring). The results show that SSX1 antigen is expressed in tumor tissues such as liver cancer, lung cancer, fibrosarcoma, breast cancer, prostatic cancer, chronic myeloma cell leukemia and the like.

Commercial antibody A11.1M was used to purify HLA-short peptide complexes. Specifically, tumor cells were lysed in a buffer containing the nonionic surfactant Triton X-100 (1%v/v), 2 × 10^7 cells were added to 1ml of the lysate, and incubated at 4 ℃ for 1 hour with rolling. Cell debris was removed by centrifugation, and the supernatant was incubated with the antibody, followed by addition of rProtein A-Sepharose to capture the "antibody-HLA-short peptide complex". And (4) passing through a column, and collecting the rProtein A-Sepharose-antibody-HLA-short peptide complex. Washing the column with low-salt and high-salt buffer solutions, eluting the HLA-short peptide complex suspended on the immunoaffinity column with 10% acetic acid, heating at 95 deg.C, and filtering with 10kDa (amicon R Ultr Centrifugal Filters, MILLIPORE) ultrafiltration membrane to remove macromolecules to obtain polypeptide mixture.

The polypeptide mixture was fractionated by Agilent 1260 hplc: ZORBAX 300SB-C18;1.0 x 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 was mobile phase B rose from 5% to 70% within 10 minutes. One fraction was collected every minute. The total run time was 30 minutes.

Concentrating the HPLC fraction of the polypeptide, injecting a sample of a NanoLC-MSMS system for analysis:

eksigent nanolC-AB Sciex Triple TOF 5600 System: the mass spectrum was analyzed by IDA analysis. The liquid chromatography adopts: pre-column: (Eksigent) NanoLC Trap column.5. Mu. M C18.100. Mu. M. By 2.5cm,910-00050, analytical column: (Eksigent) C18-CL-120,3 μm,

0.075×150mm，805-00120。

dionex Ultimate3000-Thermo QE Plus System: mass spectrometry using ddms2 analytical method, liquid phaseThe chromatogram adopts: pre-column: (Thermo) Acclaim

100,100um X2cm, nanoViper, C18,5um,100A,164564, analytical column: (Thermo) Acclaim

100，75um×15cm,nanoViper，C18,3um,100A，164568。

The mobile phase a of the nanofluidic chromatography of the two systems described above was 98% water, 2% acetonitrile, 0.1% formic acid, and the mobile phase B was 98% acetonitrile, 2% water, 0.1% formic acid, with a mobile phase gradient of mobile phase B rising from 5% to 50% in 74 minutes. The total run time was 90 minutes.

And (3) searching a Uniprot database of human proteins by virtue of library searching software Proteinpilot and Peaks according to the mass spectrum analysis result. According to the results of the software, the antigen short peptide sequence of the invention is finally obtained by comprehensive analysis, as shown in figure 1.

Example 2 preparation of soluble pMHC complexes

HLA class i-base:Sub>A 2402 molecules heavy and light chains (β 2 m) were expressed in e.coli (e.coli) as inclusion bodies, respectively. It should be noted that the heavy chain of the HLA-base:Sub>A 2402 molecule used in this example does not comprise its transmembrane and cytoplasmic domains in order to obtain soluble pMHC complexes. In addition, in order to facilitate subsequent biotinylation of soluble pMHC complexes, a biotinylation tag may be added at the C-terminus of the heavy chain. The specific process for preparing the soluble pMHC complex of the present invention is 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 5min; adding 30ml of 10-fold diluted BugBuster, uniformly mixing, and centrifuging at 4 ℃ at 6000g for 15min; discarding supernatant, adding 30ml of 10-fold diluted BugBuster to resuspend the inclusion bodies, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, repeating twice, adding 30ml of 20mM Tris-HCl with pH of 8.0 to resuspend the inclusion bodies, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, finally dissolving the inclusion bodies by using 20mM Tris-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 peptide KYSEKISYVYM (synthesized by Beijing Saibance Gene technology Co., ltd.) of the present invention was dissolved in DMSO to a concentration of 20 mg/ml. Inclusion of light and heavy chains was solubilized using 8M urea, 20mM Tris pH 8.0, 10mM DTT, and further denatured by addition of 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA prior to renaturation. KYSEKISYVYM peptide was added at 25mg/L (final concentration) 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 ℃), followed by the addition of 20mg/L of light chain and 90mg/L of heavy chain in sequence (final concentration, heavy chain added in three additions, 8 h/time), renaturation was performed at 4 ℃ for at least 3 days to completion.

c. Purification after renaturation

The renaturation buffer was replaced by dialysis against 10 volumes of 20mM Tris pH 8.0, at least twice to reduce the ionic strength of the solution sufficiently. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and then loaded onto a HiTrap Q HP (GE general electric) anion exchange column (5 ml bed volume). The protein was eluted using a 0-400mM NaCl linear gradient in an Akta purifier (GE general electric), 2mM Tris pH 8.0, pMHC was eluted at about 250mM NaCl, and the peak fractions were collected. The Native gel pattern of the soluble pMHC complex of the present invention obtained is shown in FIG. 2, and the bands are very uniform.

d. Biotinylation of the compound

The purified pMHC molecules were concentrated using Millipore ultrafiltration tubes while replacing the buffer with 20mM Tris pH 8.0, followed by addition of biotinylation reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50. Mu. M D-Biotin, 100. Mu.g/ml BirA enzyme (GST-BirA), incubation of the mixture overnight at room temperature, and SDS-PAGE to determine whether biotinylation was complete.

e. Purification of the biotinylated Complex

The biotinylated-labeled pMHC molecules were concentrated to 1ml using Millipore ultrafiltration tubes, the biotinylated pMHC was purified by gel filtration chromatography, the HiPrepTM 16/60S200HR column (GE general electric) was pre-equilibrated with filtered PBS using an Akta purifier (GE general electric), loaded with 1ml of concentrated biotinylated pMHC molecules and then eluted with PBS at a flow rate of 1 ml/min.

Example 3T cell clone obtained by Using the short peptide of the present invention

This example provides an illustration of the use of the pMHC complexes of the invention to obtain monoclonal T cells.

A variety of methods for obtaining TCRs are well known to those skilled in the art, including, but not limited to, isolating the sequences of TCR α and β chains from T cell clones stimulated by cells presenting the pMHC complex of the invention. The TCR sequences obtained can be cloned into a suitable vector and then expressed in e.coli, e.g. e.

The short peptide of the invention is used for stimulating peripheral blood lymphocytes of healthy volunteers, sorting and then carrying out monoclonal culture by a limiting dilution method to obtain T cell clones, and the double positive staining results of CD8+ and tetramer-PE are shown in figure 3.

Example 4 function of T cell cloning

The function and specificity of the T cell clone were further tested by ELISPOT assay. Meanwhile, the specific reaction of the T cell clone obtained by using the short peptide of the invention on a tumor cell line can also show that the short peptide of the invention is really presented on the surface of the tumor cell by MHC.

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- γ ELISPOT experiment in this example were T cell clones obtained in the present invention, the target cells were T2 (a 2402) cells loaded with the short peptide of the present invention (HLA a 2402-transferred to T2), T2 (a 2402) cells loaded with other short peptides, positive tumor cell lines HLF, K562 (a 2402) (HLA a 2402-transferred to K562), and negative tumor cell lines K562.

Firstly, preparing an ELISPOT plate, wherein the ELISPOT experiment steps are as follows: push buttonThe components of the assay were added to the ELISPOT plate in the following order: the amount of short peptide added to the T2 experimental group was 20. Mu.l, and 20. Mu.l of the culture medium (test medium) was added to the tumor cell line group, and 2 wells were set, with 20,000 target cells/well and 2000 effector cells/well. Then incubated overnight (37 ℃,5% CO) ₂ ). The plates were then washed and subjected to secondary detection and color development, the plates were dried for 1 hour, and spots formed on the membrane were counted using an immuno-spot plate READER (ELISPOT READER system; AID Co.).

The results of the experiment are shown in FIG. 4, and the obtained T cell clone specific to the specific antigen has specific response to the T2 cell loaded with the short peptide of the present invention, but has no substantial response to the T2 cell loaded with other irrelevant peptides. In addition, T cell clones obtained by using the short peptide of the invention have specific reaction on positive tumor cell lines and have no reaction on negative tumor cell lines.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Guangdong Xiangxue accurate medical technology Limited

<120> SSX-derived tumor antigen short peptide

<130> P2018-1591

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 11

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 1

Lys Tyr Ser Glu Lys Ile Ser Tyr Val Tyr Met

1 5 10

Claims (23)

1. A peptide having the amino acid sequence KYSEKISYVYM (SEQ ID NO: 1);

wherein the peptide is capable of forming a complex with an MHC molecule.

2. A pMHC complex comprising a peptide according to claim 1.

3. The pMHC complex of claim 2, wherein the MHC molecule is of the type HLA-base:Sub>A x 24.

4. The pMHC complexes of claim 3, wherein the class of MHC molecules is HLA-A2402.

5. The pMHC complex of any one of claims 2 to 4, wherein the pMHC complex is a multimer.

6. The pMHC complex of any one of claims 2 to 4, wherein the pMHC complex is soluble.

7. An isolated cell which presents on its surface the pMHC complex of any one of claims 2 to 4.

8. A nucleic acid molecule which is a nucleic acid sequence encoding the peptide of claim 1 or the complete complement of said nucleic acid sequence.

9. A vector comprising the nucleic acid molecule of claim 8.

10. A host cell comprising the vector of claim 9.

11. A molecule capable of binding the peptide of claim 1 and/or the pMHC complexes of any of claims 2 to 6.

12. The molecule of claim 11, wherein said molecule is a T cell receptor.

13. The molecule of claim 11, wherein said molecule is an antibody or binding fragment thereof.

14. The molecule of claim 13, wherein said antibody is a monoclonal antibody.

15. A T cell receptor capable of binding the peptide of claim 1 and/or the pMHC complex of claim 2.

16. The T cell receptor of claim 15, wherein said T cell receptor is soluble.

17. An isolated monoclonal T cell, wherein the cell binds the pMHC complex of any one of claims 2 to 6.

18. Use of a peptide according to claim 1, a pMHC complex according to any one of claims 2 to 6, for screening a library of T-cell receptors or antibodies.

19. Use of a peptide according to claim 1, a pMHC complex according to any one of claims 2 to 6 or a cell according to claim 7 for activating and/or isolating T cells.

20. Use of a peptide according to claim 1, a pMHC complex according to any one of claims 2 to 6, a cell according to claim 7, a nucleic acid molecule according to claim 8, a molecule according to claims 11 to 14 or a T-cell according to claim 17, for the preparation of a medicament for the prophylaxis or treatment of liver cancer, chronic myelogenous leukemia, melanoma, multiple myeloma.

21. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the peptide of claim 1, the pMHC complex of any one of claims 2-6, the cell of claim 7 or the monoclonal T cell of claim 17.

22. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition is a vaccine.

23. A method of obtaining a molecule which binds to a pMHC complex described in any one of claims 2 to 6, comprising:

contacting a candidate molecule with a pMHC complex according to any one of claims 2 to 6;

(ii) selecting the molecule which binds to the pMHC complex of (i).

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