WO2022105922A1 - Ssx2 antigen derived short peptides - Google Patents

Abstract

The present invention relates to SSX2 antigen derived short peptides, and in particular to SSX2 derived tumor antigen short peptides, complexes formed by the short peptides and MHC molecules, and uses of the short peptides and complexes. In addition, the present invention also relates to molecules bound to the short peptides or complexes described above, and uses of the molecules.

Description

Short peptides derived from SSX2 antigen technical field

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. At the same time, the present invention also relates to molecules that bind to the above-mentioned short peptides or complexes, and uses of these molecules.

Background technique

It is well known that in many pathological conditions, such as infection, cancer, autoimmune diseases, etc., there will be inappropriate expression of some specific molecules. Thus, these molecules become "markers" of a pathological or abnormal state. 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 of cancer are used to generate specific antibodies. In addition, these molecules can also effectively stimulate the specific immune response of cytotoxic T lymphocytes (CTL) and exert anti-tumor efficacy. At the same time, they can also obtain 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.

For tumors, various endogenous tumor antigen molecules have been published in many literatures. But this is not the real target of related diseases, because it is not the complete tumor antigen molecule that causes the CTL immune response, but the specific CTL epitope (Epitope) from the antigen. Under normal circumstances, 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. Human MHC is usually referred to as HLA gene or HLA complex) 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. Although related endogenous tumor antigen molecules have been published, we do not know the specific polypeptide fragments that are presented. Therefore, it is crucial to identify these 8-16 amino acid long polypeptide fragments, ie CTL epitopes, which are presented to the cell surface, whether as vaccines or as diagnostic or therapeutic agents for related diseases. Those skilled in the art are dedicated to discovering and determining these polypeptide fragments that are presented on the surface of target cells.

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. But this is only a prediction, with great uncertainty, because it is not the real intracellular processing and post-translational modification process. After the tumor tissue is processed, the peptide fragments presented on the surface of the tumor cells can be directly identified by mass spectrometer. Although this is a complicated process, the results obtained in this way are very reliable. At the same time, the mass spectrometer is sensitive enough to identify low concentrations of peptide fragments and post-translational modifications, so it is an ideal tool for the discovery and identification of tumor surface peptide fragments.

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. Therefore, 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.

SUMMARY OF THE INVENTION

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);

In another preferred embodiment, the peptide can form a complex with MHC molecules.

In another preferred embodiment, the peptide consists of 9 or 10 amino acids.

In another preferred example, the peptide consists of 9 amino acids.

In another preferred embodiment, the 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.

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

In another preferred example, the type of MHC molecule is HLA-A*11.

In another preferred example, the type of MHC molecule is HLA-A*1101.

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.

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.

In another preferred embodiment, 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.

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 a 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 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 In the third aspect, the cells are obtained by separation.

In another preferred embodiment, 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.

In another preferred embodiment, 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 cells described in the third aspect, the molecules described in the seventh aspect of the present invention, or the T cells described in the eighth aspect of the present invention.

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:

(i) contacting the candidate molecule with the pMHC complex of the second aspect of the invention;

(ii) Screening for molecules that bind to the pMHC complex in (i).

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

Description of drawings

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.

Detailed ways

Through extensive and in-depth research, 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. It should be understood that, in the present invention, 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.

Specifically, the first aspect of the present invention provides a peptide, the amino acid sequence of the peptide is: AQIPEKIQK (SEQ ID NO: 1).

It is known to those skilled in the art that the peptides of the present 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 Feb;18(1):92-7, and include phosphorylation, acetylation, and deamidation.

Preferably, the peptide of the present invention binds to MHC at the peptide binding site of the MHC molecule. In general, 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 the peptide to the MHC. These binding sites and preferred residues at binding sites are known in the art, especially for those peptides that bind HLA-A*11 (see, eg, Parkhurst et al., J. Immunol. 157:2539-2548 (1996)).

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.

Tumor antigens are processed into polypeptide fragments of 8-16 amino acids in length by proteolysis in cells, namely CTL epitopes, and then combined with MHC molecules in the endoplasmic reticulum cavity to form peptide-MHC complexes. , pMHC), and presented together on the cell surface. Accordingly, a second aspect of the present invention provides a pMHC complex comprising the peptide of the first aspect of the present invention. Preferably, 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. In a preferred embodiment, 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).

In general, pMHC multimers can be produced by complexing pMHC complexes with biotin residues in combination with fluorescently labeled streptavidin. Alternatively, 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. Alternatively, 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. To obtain pMHC complexes in soluble form, preferably, the MHC molecules in the pMHC complexes do not contain a transmembrane region. Specifically, in a pMHC complex, an MHC class I molecule may consist of the extracellular domain of its light chain and all or part of its heavy chain. Alternatively, the MHC molecule is a fragment comprising only its functional domain.

Methods for producing soluble pMHC complexes of the present invention are known to those skilled in the art, including, but not limited to, the methods described in Example 2 of the present invention. The 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. Alternatively, in addition, the binding can be detected by functional assays of the biological response to binding, such as cytokine release or apoptosis.

Likewise, 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). 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 TCRs displayed by the library may contain non-native mutations.

Thus, the soluble pMHC complexes of the present invention can be immobilized on a suitable solid support via a linker. Examples of 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. In a preferred embodiment, 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 .} In addition, 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.

In a preferred embodiment, 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-γ 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.

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. For example, 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.

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, 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.

In a preferred embodiment of the present invention, the binding molecule is a T cell receptor (TCR). TCRs can be described using the International Information System for Immunogenetics (IMGT). Native αβ heterodimeric TCRs have α and β 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. For example, 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 α and β chains of αβTCR, or between the α chain variable region and the β chain constant region of αβTCR. 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. In addition, 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.

In another preferred embodiment, the binding molecule of the present invention is an antibody. As used herein, the term "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. The term "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.

Examples of 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.

The 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.

In another aspect, 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 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.

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, eg, by admixing the active ingredient with a carrier or excipient under sterile conditions.

Depending on the disease or condition being treated (eg, cancer, viral infection or autoimmune disease), the individual age and condition of the patient, and the like, 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.

According to the state of the art, 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.

Accordingly, 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). For example, 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. Alternatively, the vaccine composition may be in the form of an antigen presenting cell presenting the peptide of the invention and the MHC complex. Preferably, 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 following specific examples further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted specific conditions in the following examples, usually according to conventional conditions, such as (Sambrook and Russell et al., Molecular Cloning: Laboratory Manual (Molecular Cloning-A Laboratory Manual) (Third Edition) (2001) CSHL Publishing company), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

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

Example 1 Identification of polypeptides derived from SSX2 antigen by mass spectrometry

Before performing mass spectrometry identification, 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. The filter membrane filters out the macromolecules, and finally the peptide mixture is obtained.

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.

The 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.5μm C18.100μm*2.5cm, 910-00050, analytical column: (Eksigent) C18-CL-120, 3μm,

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 results of mass spectrometry analysis were used to search the Uniprot database of human proteins with the help of library search software ProteinPilot and Peaks. According to the results of the software and comprehensive analysis, the antigenic short peptide sequence of the present invention is finally obtained, as shown in FIG. 1 .

Example 2 Preparation of soluble pMHC complexes

The heavy chain and light chain (β2m) 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:

a. Purification

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.

Resuspend the above inclusions in 5ml of BugBuster Master Mix, rotate and incubate at room temperature for 5min; add 30ml of BugBuster diluted 10 times, mix well, and centrifuge at 6000g at 4°C for 15min; discard the supernatant and add 30ml of BugBuster diluted 10 times to resuspend the inclusion bodies , Mix well, centrifuge at 6000g at 4°C for 15min, repeat twice, add 30ml 20mM Tris-HCl pH 8.0 to resuspend the inclusion bodies, mix well, centrifuge at 6000g at 4°C for 15min, and finally dissolve the inclusion bodies with 20mM Tris-HCl8M urea, SDS-PAGE The purity of inclusion bodies was detected, and the concentration was measured by BCA kit.

b. Refolding

The peptide AQIPEKIQK of the present invention (synthesized by Beijing Saibaisheng Gene Technology Co., Ltd.) 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.

c. Purification after renaturation

Replace the 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. After dialysis, the protein solution was filtered through a 0.45 μm 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.

d. Biotinylation

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, 50μM D-Biotin, 100μg/ml BirA Enzyme (GST-BirA), the mixture was incubated overnight at room temperature, and the complete biotinylation was checked by SDS-PAGE.

e. Purification of biotinylated complexes

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.

Example 3 T cell clones obtained using the short peptides of the present invention

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

Those skilled in the art are familiar with various methods of obtaining TCR, including, but not limited to, isolating the sequences of the TCR alpha and beta chains from T cell clones stimulated by cells presenting the pMHC complexes of the invention . The obtained 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.

The 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.

Example 4 Function of T cell clones

The function and specificity of the T cell clone were further tested by ELISPOT assay. At the same time, 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.

Those skilled in the art are familiar with methods for detecting cell function using ELISPOT assays. The effector cells used in the IFN-γ 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.

First, prepare the ELISPOT plate. 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 μl, tumor cells 20 μl 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).

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. In addition, 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.

Example 5 TCR binding to the pMHC complex of the present invention

The total RNA of the above T cell clones was extracted with Quick-RNA ^™ MiniPrep (ZYMO research), and the TCR sequence was obtained. In order to further verify that the obtained TCR can bind to the pMHC complex of the present invention, 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 μL/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. At the same time, 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.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms are also defined by the appended claims of the present application.

Claims (24)

1. A kind of short peptide derived from AFP antigen, it is characterized in that, described short peptide comprises amino acid sequence:

   AQIPEKIQK (SEQ ID NO: 1); wherein the peptide consists of 9 or 10 amino acids.
2. The peptide of claim 1, wherein the amino acid sequence of the peptide is SEQ ID NO: 1.
3. A pMHC complex comprising the peptide of claim 1.
4. The pMHC complex of claim 3, wherein the amino acid sequence of the peptide in the pMHC complex is SEQ ID NO: 1.
5. The pMHC complex of claim 3, wherein the type of MHC molecule is HLA-A*11.
6. The pMHC complex of claim 3, wherein the type of MHC molecule is HLA-A*1101.
7. The pMHC complex of claim 3, wherein the pMHC complex is a multimer.
8. The pMHC complex of claim 3, wherein the pMHC complex is soluble.
9. An isolated cell, characterized in that the cell surface presents or carries the pMHC complex according to any one of claims 3-7, preferably, the cell is a DC.
10. A nucleic acid molecule comprising a nucleic acid sequence encoding the peptide of claim 1 or a complementary sequence thereof.
11. A vector, characterized in that the vector contains the nucleic acid molecule described in claim 10.
12. A host cell, wherein the cell contains the vector described in claim 11.
13. A molecule which is capable of binding the peptide of claim 1 and/or the pMHC complex of any one of claims 3-8.
14. 14. The molecule of claim 13, wherein the molecule is a T cell receptor.
15. 15. The T cell receptor of claim 14, wherein the T cell receptor is soluble.
16. The molecule of claim 13, wherein the molecule is an antibody or a binding fragment thereof.
17. The antibody of claim 16, wherein the antibody is a monoclonal antibody.
18. An isolated monoclonal T cell, characterized in that the cell is screened by the peptide described in claim 1 or the pMHC complex described in any one of claims 3-8 or the cell described in claim 9 in vitro stimulation get.
19. Use of the peptide according to claim 1, the pMHC complex according to any one of claims 3 to 8, or the cell according to claim 9, for activating and/or isolating T cells in vitro.
20. The use of the peptide of claim 1 and the pMHC complex of any one of claims 3 to 8, characterized in that it is used to screen T cell receptors or antibody libraries.
21. The peptide of claim 1, the pMHC complex of any one of claims 3-8, the cell of claim 9, the nucleic acid molecule of claim 10, the molecule of claim 13-17, or The use of the T cells in claim 18, characterized in that it is used to prepare a medicament for preventing or treating cancer.
22. A pharmaceutical composition, characterized in that the composition contains a pharmaceutically acceptable carrier, the peptide described in claim 1, the pMHC complex described in any one of claims 3-8, and the pMHC complex described in claim 9. A cell, a molecule as claimed in claims 13-17 or a T cell as claimed in claim 18.
23. The pharmaceutical composition of claim 22, wherein the pharmaceutical composition is a vaccine.
24. A method of obtaining a molecule bound to the pMHC complex of any one of claims 3-8, comprising:

    (i) contacting the candidate molecule with the pMHC complex of any one of claims 3-8;

    (ii) Screening for molecules that bind to the pMHC complex in (i).

Images