CN116948002A

CN116948002A - Peptide and complex thereof

Info

Publication number: CN116948002A
Application number: CN202210419051.2A
Authority: CN
Inventors: 龚海平; 余晓红; 刘秋萍; 李柳萍; 吴万里; 程健婷; 钟时; 杨东雪; 高咪
Original assignee: Xiangxue Life Science Technology Guangdong Co ltd
Current assignee: Xiangxue Life Science Technology Guangdong Co ltd
Priority date: 2022-04-20
Filing date: 2022-04-20
Publication date: 2023-10-27

Abstract

The present application relates to short peptides derived from AFP antigens, in particular to tumor antigen short peptides derived from AFP, complexes of the short peptides with MHC molecules and uses of the short peptides and complexes. The application also relates to molecules which bind to the above-mentioned short peptides or complexes, and to the use of these molecules.

Description

Peptide and complex thereof

Technical Field

The present application relates to short peptides derived from AFP antigens, in particular to newly discovered short peptides derived from tumor antigen AFP, complexes of the short peptides with MHC molecules and uses of the short peptides and complexes. The application 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 under many pathological conditions, such as infections, cancers, autoimmune diseases, etc., there is inappropriate expression of certain molecules. Thus, these molecules become "markers" of 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, a marker for cancer is used to produce a specific antibody. In addition, these molecules are also effective in eliciting specific immune responses from Cytotoxic T Lymphocytes (CTLs) and exerting anti-tumor efficacy, while also allowing T Cell Receptors (TCRs) capable of binding to the "tag" to be obtained by the activated CTLs as therapeutic agents. Therefore, these molecules play a very important role in the diagnosis and treatment of related diseases.

For tumors, there are many documents that disclose different endogenous tumor antigen molecules. However, this is not a true target for the relevant disease, since not the complete tumor antigen molecule, which elicits the CTL immune response, is derived from a specific CTL Epitope (Epitope) of the antigen. In general, tumor antigens are proteolytically processed in cells into polypeptide fragments of 8-16 amino acids in length, i.e., CTL epitopes, which in turn interact with the main group in the lumen of the endoplasmic reticulumThe binding of a histocompatibility complex (MHC, human MHC is commonly referred to as HLA gene or HLA complex) molecule to form a polypeptide-MHC complex (pMHC), and finally the presentation of pMHC to the cell surface for CD8 ⁺ TCR recognition on T cell surfaces. Although related endogenous tumor antigen molecules have been published, we are unaware of the specific polypeptide fragments that are presented. Thus, it is critical to identify these 8-16 amino acid length polypeptide fragments, i.e., CTL epitopes, that are presented on the cell surface, either as vaccines or as diagnostic or therapeutic agents for the generation of related diseases. Those skilled in the art are working to find and determine these polypeptide fragments that are presented on the surface of target cells.

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

AFP (alpha Fetoprotein), also known as alpha Fetoprotein, is a protein expressed during embryo development and is the main component of embryo serum. During development, AFP has relatively high expression levels in the yolk sac and liver, which are subsequently inhibited. In liver cancer, AFP expression is activated (Butterfield et al J immunol.,2001, apr 15;166 (8): 5300-8). Thus, peptides derived from AFP, which serve as targets for the above hepatocellular carcinoma, are useful not only as markers for the diagnosis of the above-mentioned diseases, but also for the production of preventive and/or therapeutic agents for the above-mentioned diseases, such as antibodies or T cell receptors. The application utilizes mass spectrometer analysis and identification, and discovers the polypeptide fragment which is presented on the surface of tumor cells and is derived from tumor antigen AFP for the first time.

Disclosure of Invention

The object of the present application is to provide a newly discovered short peptide derived from the tumor antigen AFP, a complex of the short peptide with MHC molecules and the use of said short peptide and complex.

In a first aspect of the application, there is provided a peptide comprising the amino acid sequence: VIADFSGLLEK (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 is composed of 11 or 12 amino acids.

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

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

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

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 x 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.

In a third aspect of the application there is provided an isolated cell, which cell surface carries or presents a peptide according to the first aspect of the application.

In another preferred embodiment, the cell surface carries or presents a pMHC complex according to the second aspect of the application.

In another preferred embodiment, the cell is a DC.

In another preferred embodiment, the cell is a T2 cell.

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

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

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

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

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

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 application there is provided an isolated monoclonal T cell obtained by isolation using a peptide according to the first aspect of the application and/or a pMHC complex according to the second aspect of the application and/or a cell according to the third aspect of the application.

In another preferred embodiment, the isolated monoclonal T cells are obtained by in vitro stimulation screening of the peptides of the first aspect of the application and/or the pMHC complexes of the second aspect of the application and/or the cells of the third aspect of the application.

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

In a ninth aspect of the application there is provided the use of a peptide according to the first aspect of the application, a pMHC complex according to the second aspect of the application or a cell according to the third aspect of the application for in vitro activation and/or isolation of T cells.

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

According to an eleventh aspect of the present application there is provided the use of a peptide according to the first aspect of the present application, a pMHC complex according to the second aspect of the present application, a cell according to the third aspect of the present application, a nucleic acid molecule according to the fourth aspect of the present application, a molecule according to the seventh aspect of the present application or a T cell according to the eighth aspect of the present application for the manufacture of a medicament for the prevention or treatment of cancer.

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

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

In another preferred embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

In a thirteenth aspect of the application 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 application, a pMHC complex according to the second aspect of the application, a cell according to the third aspect of the application, a molecule according to the seventh aspect of the application or a T cell according to the eighth aspect of the application.

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

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

(ii) screening for molecules which bind to the pMHC complex of (i).

It is understood that within the scope of the present application, the above-described technical features of the present application and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 is a mass spectrum representative of the identification of the short peptides of the application.

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

FIG. 3 is a graph showing the results of Elispot function experiments on tumor cell lines and T2 cells using monoclonal cells obtained from the short peptides of the present application.

FIG. 4 shows the results of double cationic staining of CD8+ and tetramer-PE of T cell clones.

FIG. 5 is a kinetic profile of binding of a soluble TCR molecule of the application to a pMHC complex of the application

Detailed Description

The present application has been extensively studied to obtain peptides derived from the antigen AFP, which are presented on the surface of tumor cells by MHC molecules as tumor markers. Thus, the present application provides peptides derived from the antigen AFP, complexes of the peptides with MHC molecules and uses of the peptides and complexes. The application also relates to molecules that bind to the peptides or complexes. It will be appreciated that in the present application, the peptides of the application are used interchangeably with the polypeptides of the application or the short peptides of the application, and all refer to peptides derived from the antigen AFP provided by the application.

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

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

Preferably, the peptides of the application bind to MHC at the peptide binding site of an MHC molecule. In general, the modified amino acids described above do not disrupt the binding capacity of the peptide 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 binding sites of peptides 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.11 (see, e.g., parkhurst et al, J. Immunol.157:2539-2548 (1996)).

The peptides of the application may consist of VIADFSGLLEK (SEQ ID NO: 1) or consist essentially of VIADFSGLLEK (SEQ ID NO: 1), which corresponds to the position of the full length 571-581 residues of the AFP protein.

The application also provides SEQ ID NO:1 or a peptide analogue of a protein or peptide as shown in 1. These analogs may differ from the native peptide by differences in amino acid sequence, by differences in modified forms 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, by site-directed mutagenesis or other known techniques of molecular biology. Analogs also include analogs having residues other than the natural L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gammA-Amino acids). It should be understood that the peptides of the present application are not limited to the representative peptides exemplified above.

Modified (typically without altering the primary structure) forms include: chemical derivatization forms of peptides in vivo or in vitro such as acetylation or carboxylation. Modifications also include glycosylation, such as those resulting 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 (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Peptides modified to improve their proteolytic resistance or to optimize their solubility properties are also included.

The peptides of the application can be simply synthesized using Merrifield synthesis (also known as solid phase synthesis of polypeptides). 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 synthesized recombinantly, and if desired, by methods known in the art. Typically such methods involve the use of vectors comprising a nucleic acid sequence encoding a polypeptide for expression of the polypeptide in vivo; for example, expression in bacterial, yeast, insect or mammalian cells. Alternatively, expression may 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 into polypeptide fragments of 8-16 amino acids in length, i.e., CTL epitopes, by proteolysis within cells, which in turn bind to MHC molecules in the lumen of the endoplasmic reticulum to form polypeptide-MHC complexes (pMHC) that are presented together to the cell surface. Accordingly, in a second aspect the present application provides a pMHC complex comprising a peptide according to the first aspect of the application. Preferably, the polypeptide is bound to a 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. In a preferred embodiment, the MHC molecule is HLA-A x 11, more preferably the MHC molecule is HLA-A x 1101.

The pMHC complexes of the application may exist in multimeric form, for example, as dimers, or tetramers, or pentamers, or hexamers, or octamers, or greater. Suitable methods for producing pMHC multimers can be found in the literature, for example (Greten et al, clin.diagnostic Lab.immunol.2002:216-220).

In general, pMHC multimers can be generated by complexing a pMHC complex with biotin residues with streptavidin labeled by fluorescence. Alternatively, the pMHC multimer may also be formed by immunoglobulins as a molecular scaffold. 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 sequence (linker). In addition, the formation of pMHC multimers may also utilize carrier molecules, such as dextran (WO 02072631). pMHC multimers help to enhance detection of binding moieties, such as T cell receptors. Alternatively, the effect of the pMHC complex in related applications, such as activating T cells, is increased.

The pMHC complexes of the application may be provided in soluble form. To obtain a soluble form of the pMHC complex, preferably the MHC molecules in the pMHC complex do not contain a transmembrane region. In particular, in pMHC complexes, MHC class i molecules may consist of the extracellular domain of their light chain and all or part of the heavy chain. Alternatively, an MHC molecule is a fragment comprising only its functional domain.

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

The soluble pMHC complexes of the application can be used to screen or detect molecules, such as TCRs or antibodies, bound thereto. The method comprises contacting the pMHC complex with a test binding moiety, and determining whether the test binding moiety binds to the complex. Methods for determining the 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, and in addition, the binding may be detected by performing a functional assay of the biological response produced by the binding, such as cytokine release or apoptosis.

Likewise, the soluble pMHC complexes of the application 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 reference Aitken, antibody phage display: methods and Protocols (2009,Humana,New York). In a preferred embodiment, the pMHC complexes of the application are used to screen diverse TCR libraries displayed on the surface of phage particles. The library may display TCRs that contain unnatural mutations.

Thus, the soluble pMHC complexes of the application may be immobilized via a linker to a suitable solid support. 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 is immobilized on a streptavidin-coated surface.

The peptides of the application may be presented to the cell surface together with MHC complexes. Thus, the present application also provides a cell capable of presenting the pMHC complex of the application to its surface. Such cells may be mammalian cells, preferably cells of the immune system, and preferably are 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 peptide or pMHC complex of the application 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 application, or the cells may present the complexes at a higher level than in the natural state. Such cells can be obtained by pulsing with the peptides of the application. The pulse treatment involves incubating the cells with the peptide for several hours, preferably at a concentration of 10 ^-5 -10 ^-12 M. In addition, the cells may also be transduced with HLA-A x 11 molecules, further inducing peptide presentation.Cells presenting the pMHC complexes of the application can be used to isolate T cells activated by the cells and further sorted to obtain T cell receptors expressed on the surface of the T cells.

In a preferred embodiment, the method of obtaining the T cells described above comprises stimulating fresh blood obtained from healthy volunteers with the cells presenting pMHC complexes of the application described above. Several rounds of stimulation, such as 3-4 rounds, may be performed. Identification of activated T cells cytokine release can be determined by the presence of peptide-pulsed T2 cells of the application (e.g., IFN- γ ELISpot assay). With labeled antibodies, activated cells can be sorted by flow cytometry (FACS), and the sorted cells can be expanded cultured and further validated, for example, 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 Rapid Amplification of CDNA Ends (RACE) and sequenced.

The application also provides a nucleic acid molecule comprising a nucleic acid sequence encoding a peptide of the application. The nucleic acid may be cDNA. The nucleic acid molecule may consist essentially of a nucleic acid sequence encoding the peptide of the application, or may encode only the peptide of the application. Such nucleic acid molecules can be synthesized using methods known in the art. Because of 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 application also provides a vector comprising the nucleic acid sequence of the application. Suitable vectors are known in the art of vector construction and include selection of promoters and other regulatory elements, such as enhancer elements. The vectors of the present application include sequences suitable for introduction into cells. For example, the vector may be an expression vector in which the coding sequence of the polypeptide is under the control of its own cis-acting regulatory element, the vector being designed to facilitate gene integration or gene replacement in a host cell, etc.

As understood by those of ordinary skill in the art, in the present application, 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 application also provides a binding molecule that can be used as an immunotherapeutic or diagnostic agent. The binding molecule may bind to the peptide alone or to a complex formed by the peptide and an MHC molecule. In the latter case, the binding molecule may be partially bound to an MHC molecule, while it also binds to the peptide of the application. The binding moieties of the application may be isolated and/or soluble and/or non-naturally occurring, i.e., without equivalents in nature, and/or pure, and/or synthetic.

In a preferred embodiment of the application, the binding molecule is a T Cell Receptor (TCR). TCRs can be described using the international immunogenetic information system (IMGT). The native αβ heterodimeric TCR has an α chain and a β chain. In a broad sense, each chain comprises a variable region, a linking region and a constant region, and the β chain also typically contains a short polytropic region between the variable region and the linking region, but this polytropic region is often considered part of the linking region.

The TCRs of the present application may be in any form known in the art. For example, the TCR may be heterodimeric, or exist in a single chain form. The TCR may be in a soluble form (i.e. without transmembrane or cytoplasmic domains), in particular the TCR may comprise all or part of the TCR extracellular domain. The TCR may also be a full long chain comprising its transmembrane region. The TCR may be provided to a cell surface, such as a T cell.

Soluble TCRs may be obtained in combination with the prior art in the art, for example, by introducing artificial disulfide bonds between the α and β chain constant domains of an αβ TCR, or by introducing artificial disulfide bonds between the α chain variable region and the β chain constant region of an αβ TCR.

The TCRs of the present application may be used to deliver a cytotoxic or immunostimulatory agent to a target cell, or be transformed into T cells, enabling T cells expressing the TCR to destroy tumor cells for administration to a patient during a treatment process known as adoptive immunotherapy. In addition, the TCR of the application may also comprise a mutation, preferably a mutated TCR with increased affinity for the pMHC complex of the application. The TCRs of the present application may be used alone or may be covalently or otherwise bound to the conjugate, preferably covalently. The conjugate includes a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the pMHC complex of the application), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination or coupling of any of the above. The TCRs of the application may also be conjugated, preferably covalently, with an anti-CD 3 antibody to redirect T cells, thereby killing target cells.

In another preferred embodiment, the binding molecule of the application 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, that can be all natural, or partially or fully synthetic. The term "antibody" includes antibody fragments, derivatives, functional equivalents, and homologous, humanized antibodies, which comprise immunoglobulin binding regions, which are or are homologous to antibody binding regions. It may be entirely natural, or partially or entirely synthetic. The humanized antibody may be a modified antibody that contains the variable region of a non-human antibody (e.g., mouse) as well as the constant region of a human antibody.

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

Methods for preparing such 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 application further provides the use of the peptides, pMHC complexes, nucleic acid molecules, vectors, cells and binding molecules of the application in the pharmaceutical field. The peptides, pMHC complexes, nucleic acids, vectors, cells or binding molecules may be used for the treatment or prevention of cancer, preferably melanoma, bladder cancer, liver cancer, epidermoid carcinoma, non-small cell lung cancer, squamous cell carcinoma, and the like.

The application also provides a pharmaceutical composition comprising a peptide of the application, a pMHC complex, a nucleic acid molecule of the application, a cell of the application or a binding molecule of the application, 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) comprise instructions for use. Which 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. The compositions may be prepared by any method known in the pharmaceutical arts, for example, by mixing the active ingredient with a carrier or excipient under sterile conditions.

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

According to the state of the art, a peptide presented to the cell surface together with an MHC molecule, pMHC complex or a cell presenting pMHC complex can activate T cells or B cells to function.

Thus, the peptides, pMHC complexes or cells presenting pMHC complexes of the application may be provided in the form of a vaccine composition. The vaccine composition may be used to treat or prevent cancer. All such compositions are included in the present application. It will be appreciated that the vaccine may take a variety of forms (Schlom J.J Natl Cancer Inst.2012104 (8): 599-613). For example, the peptides of the application can be used directly in immunization of patients (Salteller 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 application is one of a mixture of peptides. The vaccine composition may be added with an adjuvant to enhance the immune response. Alternatively, the vaccine composition may be in the form of antigen presenting cells presenting the peptide and MHC complex of the application. Preferably, the antigen presenting cells are immune cells, more preferably dendritic cells. The peptides may also be pulsed onto the surface of cells (Thurer BI.et al, J. Exp. Med. 1999.190:1669), or nucleic acids encoding the peptides of the application may be introduced into dendritic cells, for example, using electroporation (Van Tendelloo, VF. Et al., blood 2001.98:49).

The following specific examples further illustrate the application. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental procedure, which does not address specific conditions in the examples below, is generally followed by conventional conditions, for example those described in the laboratory Manual (Molecular Cloning-A Laboratory Manual) (third edition) (2001) CSHL Press, or by the manufacturer's recommendations (Sambrook and Russell et al, molecular cloning). Percentages and parts are by weight unless otherwise indicated.

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

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

Before mass spectrometry identification, the application utilizes a digital single-molecule multiple gene expression profile analysis system to detect (nanostring), and further verifies the massive expression of AFP antigen in liver cancer cells.

The HLA-short peptide complex was purified using a commercial antibody W6/32 (purchased from ATCC). Specifically, tumor cells were lysed with a buffer containing the non-ionic surfactant Triton X-100 (1% v/v), 1ml of lysate was added to 2X 10≡7 cells and incubated at 4℃for 1h with rolling. Cell debris was removed by centrifugation, and the supernatant was incubated with antibody before the "antibody-HLA-short peptide complex" was captured by the addition of rProtein A-Sepharose. And (3) 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 solution, eluting HLA-short peptide complex hung on the immunoaffinity column with 10% acetic acid, heating at 95deg.C, and filtering out macromolecules with 10kDa (AmiconR Ultr Centrifugal Filters, MILLIPORE) ultrafiltration membrane to obtain polypeptide mixture.

The polypeptide mixture was fractionated by Agilent 1260 high performance liquid chromatography: 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 10 minutes, mobile phase B rising from 5% to 70%. One fraction was collected every minute. The total run time was 30 minutes.

After concentrating the HPLC fraction of the polypeptide, the sample was analyzed by the nanoLC-MSMS system:

eksigent nanoLC-AB Sciex Triple TOF 5600 System: the mass spectrum uses the IDA analysis method. The liquid chromatography is as follows: pre-column: (eksig) NanoLC Trap column.5 μm C18.100 μm 2.5cm,910-00050, analytical column: (eksig) C18-CL-120,3 μm,0.075×150mm，805-00120。

dionex Ultimate3000-Thermo QE Plus System: the mass spectrum was analyzed using ddms2 liquid chromatography using: pre-column: (Thermo) Acclaim100,100um×2cm, 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 is 98% of water, 2% of acetonitrile, 0.1% of formic acid, the mobile phase B is 98% of acetonitrile, 2% of water, 0.1% of formic acid, and the mobile phase gradient is from 5% to 50% in 74 minutes. The total run time was 90 minutes.

Mass spectrometry results, search Uniprot database of human proteins by means of search software proteonpilot and Peaks. According to the result of the software, the antigen short peptide sequence of the application is finally obtained through comprehensive analysis, and the mass spectrum result is shown in figure 1.

EXAMPLE 2 preparation of soluble pMHC complexes

The heavy and light chains (β2m) of type i HLA-A.1101 molecules are expressed in escherichia coli (e.coli) in the form of inclusion bodies, respectively. It should be noted that, in order to obtain a soluble pMHC complex, the heavy chain of the HLA-A x 1101 molecule used in this example does not comprise its transmembrane and cytoplasmic domains. In addition, to facilitate subsequent biotinylation of the soluble pMHC complex, a biotinylation tag may be added at the C-terminus of the heavy chain.

The specific procedure for preparing the soluble pMHC complexes of the application is as follows:

a. purification

Collecting 100ml E.coli bacterial liquid for inducing expression of heavy chain or light chain, centrifuging at 8000g at 4 ℃ for 10min, washing the bacterial body once with 10ml PBS, then severely shaking and re-suspending the bacterial body with 5ml BugBuster Master Mix Extraction Reagents (Merck), rotating at room temperature for 20min, centrifuging at 6000g at 4 ℃ for 15min, discarding the supernatant, and collecting inclusion bodies.

The inclusion body is resuspended in 5ml BugBuster Master Mix and incubated for 5min at room temperature; adding 30ml of BugBuster diluted 10 times, mixing, and centrifuging at 4deg.C for 15min at 6000 g; removing the supernatant, adding 30ml of BugBuster diluted 10 times, mixing, centrifuging at 4 ℃ for 15min, repeating twice, adding 30ml of 20mM Tris-HCl pH 8.0, mixing, centrifuging at 4 ℃ for 15min, dissolving the inclusion body with 20mM Tris-HCl 8M urea, detecting purity of the inclusion body by SDS-PAGE, and detecting concentration by BCA kit.

b. Renaturation

The peptide VIADFSGLLEK of the present application (synthesized by Beijing Saint Biotechnology Co., ltd.) was dissolved in DMSO to a concentration of 20 mg/ml. The inclusion bodies of the light and heavy chains were solubilized with 8M urea, 20mM Tris pH 8.0, 10mM DTT, and further denatured by adding 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA prior to renaturation. VIADFSGLLEK 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 ℃) at 25mg/L (final concentration), followed by sequential addition of 20mg/L light chain and 90mg/L heavy chain (final concentration, heavy chain added in three portions, 8 h/times), renaturation was performed at 4℃for at least 3 days to completion.

c. Purification after renaturation

The renaturation buffer was exchanged with 10 volumes of 20mM Tris pH 8.0 for dialysis, 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 then loaded onto a HiTrap Q HP (GE general electric company) anion exchange column (5 ml bed volume). Using an Akta purifier (GE general electric), a linear gradient of 0-400mM NaCl in 20mM Tris pH 8.0 was used to elute the protein, pMHC was eluted at about 250mM NaCl, and the peak fractions were collected. The Native gel pattern of the resulting soluble pMHC complexes of the application is shown in fig. 2, with very uniform bands.

d. Biotinylation

Purified pMHC molecules were concentrated using Millipore ultrafiltration tubes while buffer was replaced with 20mM Tris pH 8.0, and then biotinylated reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50. Mu. M D-Biotin, 100. Mu.g/ml birA enzyme (GST-birA), the mixture incubated overnight at room temperature and SDS-PAGE was performed to determine whether biotinylation was complete.

e. Purification of biotinylated complexes

Biotinylated pMHC molecules were concentrated to 1ml using a Millipore ultrafiltration tube, biotinylated pMHC was purified using gel filtration chromatography, hiPrepTM 16/60 s200 HR column (GE general electric company) was pre-equilibrated with filtered PBS using Akta purifier (GE general electric company), 1ml of concentrated biotinylated pMHC molecules were loaded, and then eluted with PBS at a flow rate of 1 ml/min.

Example 3T cell clones obtained with the short peptides of the application

This example provides an illustration of the use of pMHC complexes of the application 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 alpha and beta chains from T cell clones stimulated by cells presenting the pMHC complexes of the application. The obtained TCR sequences may be cloned onto a suitable vector and then expressed in e.coli, e.g. e.coli, or on the surface of a phage.

The short peptides of the present application were used to stimulate peripheral blood lymphocytes of healthy volunteers, and then were subjected to sorting and then to monoclonal culture by limiting dilution to obtain T cell clones, and the results of CD8+ and tetramer-PE double positive staining thereof are shown in FIG. 4, which illustrates that T cells can be screened by the short peptides of the present application in vitro stimulation.

Example 4 function of T cell cloning

The function and specificity of the T cell clone was further examined by ELISPOT experiments. Meanwhile, the specific reaction of the T cell clone obtained by using the short peptide of the application on a tumor cell line can also prove that the short peptide of the application is truly presented on the surface of tumor cells by MHC.

Methods for detecting cellular function using ELISPOT assays are well known to those skilled in the art. The effector cells used in the IFN-. Gamma.ELISPOT experiments were the T cell clones obtained in the present application, and the target cells were T2 (1101) cells loaded with the short peptides of the present application (HLA A1101 to T2 was transformed), T2 (1101) cells loaded with other short peptides, positive tumor cell line SK-MEL-28 (AFP) (transfected with AFP antigen) and negative tumor cell line SK-MEL-28. Among them, the cell line SK-MEL-28 was purchased from Guangzhou Saku Biotechnology Co., ltd, and T2 was purchased from ATCC.

First, prepare an ELISPOT plate, the ELISPOT experiment procedure is as follows: the individual components tested were added to the ELISPOT plates in the following order: the amount of short peptide added to the experimental group of 20,000 target cells/well, 2000 effector cells/well, T2 was 20 μl, the tumor cell line group was added to 20 μl of medium (test medium), and 2 multiplex wells were set. Then incubated overnight (37 ℃,5% co) ₂ ). The plates were then washed and subjected to secondary detection and development, and the plates were dried for 1 hour, and spots formed on the films were counted using an immunoblotter plate reader (ELISPOT READER system; AID company).

As shown in FIG. 3, the obtained specific antigen-specific T cell clone has a specific reaction to T2 cells loaded with the short peptide of the present application, but has no reaction to T2 cells loaded with other unrelated peptides. In addition, the T cell clone obtained by utilizing the short peptide has specific reaction to a positive tumor cell line and has no reaction to a negative tumor cell line.

EXAMPLE 5 TCR binding to pMHC complexes of the application

With Quick-RNA ^TM MiniPrep (ZYMO research) total RNA from the above T cell clone was extracted and TCR sequences were obtained. To further verify that the obtained TCR was able to bind to the pMHC complex of the application, this example expressed soluble TCR proteins in e.coli and its binding to pMHC complex was detected by BIAcore. It should be noted that soluble TCRs may be obtained according to the prior art, including but not limited to those described in patent document PCT/CN 2015/093806.

Binding activity of soluble TCR proteins to pMHC complexes was detected using a BIAcore T200 real-time assay system. The anti-streptavidin antibody (GenScript) was added to the coupling buffer (10 mM sodium acetate buffer, pH 4.77), then the antibody was passed through a CM5 chip pre-activated with EDC and NHS to immobilize the antibody on the chip surface, and finally the unreacted activated surface was blocked with ethanolamine in hydrochloric acid to complete the coupling process at a level of about 15,000RU. The chip surface coated with antibody was subjected to low concentration of streptavidin, then the pMHC prepared in the manner described in example 2 was passed through the detection channel, the other channel was used as a reference channel, and 0.05mM biotin was passed through the chip at a flow rate of 10. Mu.L/min for 2min, blocking the remaining binding sites for streptavidin.

Kinetic parameters were calculated using BIAcore Evaluation software, and a kinetic profile of the binding of the soluble TCR molecules of the application to the pMHC complexes of the application was obtained as shown in figure 5, which shows the binding of the two. Meanwhile, the binding activity of the soluble TCR molecules of the application and other irrelevant antigens of short peptides and HLA complexes is also detected by using the method, and the result shows that the TCR molecules of the application are not bound with other irrelevant antigens.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it is understood that various changes and modifications of the present application may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Sequence listing

<110> Xiangxue life science and technology (Guangdong) Co., ltd

<120> a peptide and its complex

<130> 2022-04-15

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 11

<212> PRT

<213> artificial sequence

<400> 1

Val Ile Ala Asp Phe Ser Gly Leu Leu Glu Lys

1 5 10

Claims

1. A peptide comprising the amino acid sequence: VIADFSGLLEK (SEQ ID NO: 1);

wherein the peptide consists of 11 or 12 amino acids.

2. A pMHC complex, characterized in that the complex comprises the peptide of claim 1.

3. An isolated cell, wherein the cell surface presents the pMHC complex of claim 2.

4. A nucleic acid molecule, wherein said nucleic acid molecule is a nucleic acid sequence encoding the peptide of claim 1 or a complete complement of said nucleic acid sequence.

5. A vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the vector of claim 5.

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

8. An isolated monoclonal T cell obtained by isolation using the peptide of claim 1 and/or the pMHC complex of claim 2.

9. Use of the peptide of claim 1, the pMHC complex of claim 2, for screening a T cell receptor or an antibody library.

10. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the peptide of claim 1, the pMHC complex of claim 2, the cell of claim 3 or the monoclonal T cell of claim 8.