CN111138521A

CN111138521A - Short peptides derived from AFP antigens

Info

Publication number: CN111138521A
Application number: CN201911071511.1A
Authority: CN
Inventors: 李懿; 余晓红; 龚海平; 刘秋萍
Original assignee: GUANGZHOU XIANGXUE PHARMACEUTICAL CO Ltd
Current assignee: Xiangxue Life Science Technology Guangdong Co ltd
Priority date: 2018-11-06
Filing date: 2019-11-05
Publication date: 2020-05-12
Anticipated expiration: 2039-11-05
Also published as: CN111138521B

Abstract

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

Description

Short peptides derived from AFP antigens

Technical Field

The present invention relates to short peptides derived from the AFP antigen, in particular, to newly discovered short peptides derived from the tumor antigen AFP, complexes formed between the short peptides and MHC molecules, and uses of the short peptides and complexes. The invention also relates to molecules which bind to the above-mentioned short peptides or complexes, and to the use of these molecules.

Background

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

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

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

AFP (α Fetoprotein), also called α Fetoprotein, is a protein expressed during embryonic development and is the main component of embryonic serum, during development, AFP has relatively high expression levels in yolk sac and liver, and is subsequently inhibited, in liver cancer, AFP expression is activated (Butterfield et al.J. Immunol.,2001, Apr 15; 166(8): 5300-8.) therefore, peptides derived from AFP, as targets of the hepatocellular carcinoma, can be used not only as markers for the diagnosis of the disease, but also as preventive and/or therapeutic agents for the disease, such as antibodies or T cell receptors.

Disclosure of Invention

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

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

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

In another preferred embodiment, the peptide consists of 10 or 11 amino acids.

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

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

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

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

In another preferred embodiment, the MHC molecule is of the type HLA-a 24.

In another preferred embodiment, the MHC molecule is of the type HLA-a 2402.

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

In another preferred embodiment, the pMHC complex is soluble.

In another preferred embodiment, the pMHC complex is biotinylated.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

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

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

FIG. 3 is a graph showing the results of an Elispot functional experiment of monoclonal cells obtained using the short peptide of the present invention on tumor cell lines and T2 cells.

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

FIG. 5 is a kinetic profile of the binding of soluble TCR molecules of the invention to pMHC complexes of the invention.

Detailed Description

The present invention, through extensive and intensive studies, has resulted in the obtaining of peptides derived from the antigen AFP, which are presented by MHC molecules on the surface of tumor cells as tumor markers. Thus, the present invention provides peptides derived from the antigen AFP, complexes of such peptides with MHC molecules and uses of such peptides and complexes. Also, the present invention relates to a molecule bound to the above peptide or complex. It is to be understood that in the present invention, the peptide of the invention is used interchangeably with the polypeptide of the invention or the short peptide of the invention, both referring to the peptide derived from the antigen AFP provided by the invention.

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

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

Preferably, the peptides of the invention bind to MHC a peptide binding site of an MHC molecule. Generally, the modified amino acids described above do not disrupt the ability of the peptide to bind to MHC. In a preferred embodiment, the amino acid modification increases the ability of the peptide to bind to MHC. For example, mutations may occur at the binding site of a peptide to MHC. These binding sites and preferred residues on the binding sites are known in the art, especially for those peptides that bind HLA-A24 (see, e.g., Parkhurst et al, J.Immunol.157: 2539-.

The peptide of the present invention may consist of KWVESIFLIF (SEQ ID NO:1), or consist essentially of KWVESIFLIF (SEQ ID NO:1), which corresponds to the position of residues 2-11 of the full length of the AFP protein.

The invention also provides analogs of the proteins or peptides of SEQ ID NO. 1, which analogs may differ from the native peptide by amino acid sequence differences, by modifications that do not affect the sequence, or by both, which peptides include natural or induced genetic variants, induced variants may be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, by site-directed mutagenesis, or other known molecular biological techniques.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the peptide such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those peptides that result from glycosylation modifications during synthesis and processing of the peptide or during further processing steps. Such modification may be accomplished by exposing the peptide to an enzyme that performs glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are peptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

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

Tumor antigens are processed into polypeptide fragments with the length of 8-16 amino acids, namely CTL epitopes, in cells through proteolysis, and then are combined with MHC molecules in endoplasmic reticulum cavities to form polypeptide-MHC complexes (pMHC) to be presented on the surfaces of the cells together. Accordingly, a second aspect of the present invention provides a pMHC complex comprising a peptide according to the first aspect of the invention. Preferably, the polypeptide binds to the peptide binding groove of an MHC molecule. The MHC molecule may be an MHC class I molecule or an MHC class ii molecule, preferably the MHC molecule is an MHC class I molecule. In a preferred embodiment, the MHC molecule is HLA-a 24, more preferably the MHC molecule is HLA-a 2402.

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

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

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

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

The soluble pMHC complexes of the invention can be used to screen for or detect molecules, such as TCRs or antibodies, that bind to them. The method comprises contacting the pMHC complex with a test binding moiety and determining whether the test binding moiety binds to the complex. Assays for binding of pMHC complexes are well known in the art. Preferred methods include, but are not limited to, surface plasmon resonance, or any other biosensing technique, ELISA, flow cytometry, chromatography, microscopy. Alternatively, or in addition, the binding may be detected by functional assays for the biological response produced by the binding, such as cytokine release or apoptosis.

Likewise, the soluble pMHC complexes of the invention may also be used to screen TCR or antibody libraries. The construction of Antibody libraries using phage display technology is well known in the art, as described in the references Aitken, Antibody phase display: Methods and Protocols (2009, Humana, New York). In a preferred embodiment, the pMHC complexes of the invention are used to screen diverse TCR libraries displayed on the surface of phage particles. The TCR displayed by the library may contain non-native mutations.

Thus, the soluble pMHC complexes of the invention may be immobilised to a suitable solid support via a linker. Examples of solid supports include, but are not limited to, beads, membranes, sepharoses, magnetic beads, substrates, tubes, columns. The pMHC complexes can be immobilized on ELISA reaction plates, magnetic beads, or surface plasmon resonance biosensor chips. Methods of immobilising pMHC complexes to solid phase carriers are known to those skilled in the art and include, for example, the use of affinity binding pairs such as biotin and streptavidin, or antibodies and antigens. In a preferred embodiment, the pMHC complexes are labeled with biotin and are immobilized on a streptavidin-coated surface.

The peptides of the invention may be presented to the cell surface together with MHC complexes. Accordingly, the present invention also provides a cell capable of presenting a pMHC complex of the invention to its surface. Such cells may be mammalian cells, preferably cells of the immune system, and preferably specialized antigen presenting cells, such as dendritic cells or B cells. Other preferred cells include T2 cells (Hosken, et al., science.1990.248: 367-70). The cells presenting the peptides or pMHC complexes of the invention may be isolated, preferably in the form of a population of cells, or provided in substantially pure form. The cell may not naturally present the complex of the invention, or the cell may present the complex at a higher level than in its natural state. Such cells can be obtained by pulsing with the peptides of the invention. The pulse treatment involves incubating the cells with the peptide for several hours, preferably at a concentration of 10^-5-10^-12And M. In addition, the cells may be transduced with HLA-A x 24 molecules to further induce peptide presentation. Cells presenting the pMHC complexes of the invention can be used to isolate T cells and T cell receptors, which are activated by the cells and further sorted out, and can also obtain the T cell receptors expressed on the surface of the T cells.

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

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

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

It will be understood by those of ordinary skill in the art that, in the present invention, the term "vector" includes DNA molecules, such as plasmids, phages, viruses or other vectors, which contain one or more heterologous or recombinant nucleic acid sequences. Suitable phage and viral vectors include, but are not limited to: lambda-phage, EMBL phage, simian virus, verruca bovis, Epstein-Barr virus, adenovirus, herpes virus, murine sarcoma virus, murine mammary carcinoma virus, lentivirus, etc.

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

In a preferred embodiment of the invention, the binding molecule is a T Cell Receptor (TCR). an International immunogenetic information System (IMGT) may be used to describe a TCR. native αβ heterodimeric TCR having α and β chains. broadly, each chain comprises a variable region, a linking region and a constant region, and β chains typically also contain a short diversity region between the variable and linking regions, but the diversity region is often considered part of the linking region.

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

Soluble TCRs can be obtained by combining techniques known in the art, for example, introducing an artificial disulfide bond between the constant domains of the α and β chains of the αβ TCR, or between the α variable region and the β chain constant region of the αβ TCR.

The TCRs of the invention are useful for delivering cytotoxic or immunostimulatory agents to target cells, or are transformed into T cells, enabling T cells expressing the TCR to destroy tumor cells for administration to patients in a course of treatment known as adoptive immunotherapy. In addition, the TCR of the invention may also comprise a mutation, preferably the mutated TCR has an improved affinity for the pMHC complex of the invention. The TCRs of the invention may be used alone or in covalent or other association, preferably covalently, with a conjugate. The conjugates include a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the pMHC complex of the invention), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination or conjugate of any of the above. The TCRs of the invention may also be conjugated, preferably covalently, to an anti-CD 3 antibody to redirect T cells, thereby killing target cells.

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

Examples of antibodies may be isotypic immunoglobulins (e.g., IgG, IgE, IgM, IgD, and IgA) and their isotypic subclasses; fragments include antigen binding regions, such as Fab, scFv, Fv, dAb, Fd; and diabodies. The antibody may be a polyclonal or monoclonal antibody, preferably a monoclonal antibody.

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

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

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

The pharmaceutical composition is suitable for any suitable route of administration, such as injection (including subcutaneous, intramuscular, intraperitoneal or intravenous), inhalation or oral, or nasal, or anal routes. The compositions may be prepared by any method known in the art of pharmacy, for example, by mixing the active ingredient with the carrier or excipient under sterile conditions.

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

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

Thus, the peptides, pMHC complexes or cells presenting pMHC complexes of the invention may be provided in the form of vaccine compositions. The vaccine composition may be used for the treatment or prevention of cancer. All such compositions are included in the present invention. It will be appreciated that the vaccine may be in a variety of forms (Schlom J.J Natl Cancer Inst.2012104 (8): 599-. For example, the peptides of the invention can be used directly to immunize patients (Salgaleller ML. cancer Res.1996.56 (20): 4749-57and Marchand M. int J cancer.1999.80 (2): 219-230). The vaccine composition may comprise additional peptides such that the peptide of the invention is one of a mixture of peptides. The vaccine composition may be adjuvanted to enhance the immune response. Alternatively, the vaccine composition may be in the form of an antigen presenting cell presenting a complex of a peptide of the invention and an MHC. Preferably, the antigen presenting cell is an immune cell, more preferably a dendritic cell. The peptides may also be pulsed onto the surface of cells (Thurner BI.et al, J.Exp.Med.1999.190:1669), or nucleic acids encoding the peptides of the invention may be introduced into dendritic cells, for example, using electroporation (Van Tendeloo, VF.et al, Blood 2001.98: 49).

The following specific examples further illustrate the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example as described in Sambrook and Russell et al, Molecular Cloning: A laboratory Manual (third edition) (2001) CSHL Press, or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.

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

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

Before mass spectrometric identification, the invention utilizes a digital single-molecule multiplex gene expression profiling system to carry out detection (nanostring), and further verifies the mass expression of the AFP antigen in liver cancer cells.

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

The polypeptide mixture was fractionated by Agilent 1260 hplc: ZORBAX 300 SB-C18; 1.0 x 150mm,3.5 um; mobile phase a was 98% water, 2% acetonitrile, 0.1% trifluoroacetic acid, mobile phase B was 98% acetonitrile, 2% water, 0.1% trifluoroacetic acid, mobile phase gradient was mobile phase B rose from 5% to 70% within 10 minutes. One fraction was collected every minute. The total run time was 30 minutes.

Concentrating the HPLC fraction of the polypeptide, and analyzing by a sample introduction nanoLC-MSMS system:

Eksigent nanoLC-AB Sciex Triple TOF 5600 system: the mass spectrum was analyzed by IDA analysis. The liquid chromatography adopts the following steps: pre-column: (Eksigent) NanoLC Trap column.5. mu. m C18.100. mu. m.2.5 cm, 910-: (Eksigent) C18-CL-120,3 μm,

0.075×150mm，805-00120。

dionex Ultimate3000-Thermo QE Plus System: mass spectrometry was performed using ddms2 analytical methods liquid chromatography was performed using: pre-column: (Thermo) Acclaim

100,100um × 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 described above was 98% water, 2% acetonitrile, 0.1% formic acid, and the mobile phase B was 98% acetonitrile, 2% water, 0.1% formic acid, with a mobile phase gradient of mobile phase B rising from 5% to 50% in 74 minutes. The total run time was 90 minutes.

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

Example 2 preparation of soluble pMHC complexes

It should be noted that to obtain soluble pMHC complexes, the heavy chain of the HLA-a 2402 molecule used in this example does not contain its transmembrane and cytoplasmic regions, and in addition, to facilitate subsequent biotinylation of the soluble pMHC complexes, a biotinylation tag may be added to the C-terminus of the heavy chain.

a. Purification of

Collecting 100ml E.coli liquid for inducing expression of heavy chain or light chain, centrifuging at 4 ℃ for 10min at 8000g, washing the thalli once with 10ml PBS, then resuspending the thalli with 5ml BugBuster Master Mix Extraction Reagents (Merck) by vigorous shaking, rotatably incubating at room temperature for 20min, centrifuging at 4 ℃ for 15min at 6000g, discarding supernatant, and collecting inclusion body.

Resuspending the inclusion bodies in 5ml of BugBuster Master Mix, and rotary incubating at room temperature for 5 min; adding 30ml of 10-fold diluted BugBuster, uniformly mixing, and centrifuging at 4 ℃ at 6000g for 15 min; discarding the supernatant, adding 30ml of 10-fold diluted BugBuster to resuspend the inclusion bodies, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, repeating twice, adding 30ml of 20mM Tris-HCl pH8.0 to resuspend the inclusion bodies, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, finally dissolving the inclusion bodies by using 20mM Tris-HCl 8M urea, detecting the purity of the inclusion bodies by SDS-PAGE, and detecting the concentration by using a BCA kit.

b. Renaturation

The peptide KWVESIFLIF (synthesized by Beijing Baisheng Gene technology Co., Ltd.) of the present invention was dissolved in DMSO to a concentration of 20 mg/ml. Inclusion of light and heavy chains was solubilized with 8M Urea, 20mM Tris pH8.0, 10mM DTT and further denatured by addition of 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA prior to renaturation. KWVESIFLIF peptide was added at 25mg/L (final concentration) to renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidative glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4 ℃), then 20mg/L of light chain and 90mg/L of heavy chain (final concentration, heavy chain added in three portions, 8 h/time) were added in sequence, and renaturation was carried out at 4 ℃ for at least 3 days to completion.

c. Purification after renaturation

The renaturation buffer was replaced by dialysis against 10 volumes of 20mM Tris pH8.0, at least twice to reduce the ionic strength of the solution sufficiently. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and then loaded onto a HiTrap Q HP (GE general electric) anion exchange column (5ml bed volume). The protein was eluted using an Akta purifier (GE general electric) with a 0-400mM NaCl linear gradient of 20mM Tris pH8.0 and pMHC at about 250mM NaCl and the peak fractions were collected. The Native gel pattern of the soluble pMHC complex of the present invention obtained is shown in FIG. 2, and the bands are very uniform.

d. Biotinylation of the compound

The purified pMHC molecules were concentrated using a Mill ipore ultrafiltration tube while replacing the buffer with 20mM Tris pH8.0, followed by addition of biotinylation reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50. mu. M D-Biotin, 100. mu.g/ml BirA enzyme (GST-BirA), incubation of the mixture overnight at room temperature, and SDS-PAGE to determine the completion of biotinylation.

e. Purification of biotinylated complexes

Biotinylated-labeled pMHC molecules were concentrated to 1ml using Millipore ultrafiltration tubes, biotinylated pMHC was purified by gel filtration chromatography, HiPrepTM16/60S200 HR column (GE universal electrical) was pre-equilibrated with filtered PBS using an Akta purifier (GE universal electrical), loaded with 1ml of concentrated biotinylated pMHC molecules, and then eluted with PBS at a flow rate of 1 ml/min.

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

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

The person skilled in the art is familiar with a variety of methods for obtaining TCRs, including, but not limited to, isolating the sequences of TCR α and β chains from T cell clones that are stimulated by cells presenting the pMHC complex of the invention the TCR sequences obtained can be cloned onto a suitable vector and then expressed in e.

T cell clones were obtained by stimulating peripheral blood lymphocytes of healthy volunteers with the short peptides of the present invention, sorting, and then performing monoclonal culture by limiting dilution, and the results of double positive staining for CD8+ and tetramer-PE are shown in FIG. 4.

Example 4 function of T cell cloning

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

Methods for detecting cell function using the ELISPOT assay are well known to those skilled in the art. The effector cells used in the IFN-gamma ELISPOT experiment in this example were T cell clones obtained in the present invention, and the target cells were T2(A2402) cells (transfected with HLA A2402 to T2) loaded with the short peptide of the present invention, T2(A2402) cells loaded with other short peptides, positive tumor cell lines SNU398 (transfected with AFP antigen) and HEPG2, and negative tumor cell lines K562(A24) (transfected with HLA A2402) and NCI-H266.

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

As shown in FIG. 3, the obtained T cell clone specific to a specific antigen reacted specifically with T2 cells loaded with the short peptide of the present invention, but did not react substantially with T2 cells loaded with other unrelated peptides. In addition, T cell clones obtained by using the short peptide of the invention have specific reaction on positive tumor cell lines and have no reaction on negative tumor cell lines.

Example 5 TCR binding the pMHC Complex of the invention

Using Quick-RNA^TMMiniPrep (ZYMO research) extracts the total RNA of the above T cell clone and obtains the TCR sequence. To further verify that the TCRs obtained were able to bind to the pMHC complexes of the invention, this example expressed soluble TCR proteins in e. It should be noted that soluble TCRs can be obtained according to the prior art, including, but not limited to, that described in patent document PCT/CN 2015/093806.

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

Kinetic parameters were calculated using BIAcore Evaluation software, and the kinetic profile of the binding of soluble TCR molecules of the invention to pMHC complexes of the invention is shown in FIG. 5, which shows the binding of the two. Meanwhile, the method is used for detecting the binding activity of the soluble TCR molecule and the short peptides of other unrelated antigens and the HLA complex, and the result shows that the TCR molecule is not bound with other unrelated antigens.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it will be appreciated that various alterations and modifications of the invention will become apparent to those skilled in the art after reading the above teachings of the invention, and that such equivalents are intended to be encompassed by the appended claims of this application.

Sequence listing

<110> Guangdong Xiangxue accurate medical technology Limited

<120> short peptides derived from AFP antigen

<130>P2019-1917

<150>CN201811314576.X

<151>2018-11-06

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>10

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>1

Lys Trp Val Glu Ser Ile Phe Leu Ile Phe

1 5 10

Claims

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

wherein the peptide consists of 10 or 11 amino acids.

2. A pMHC complex comprising the peptide of claim 1.

3. An isolated cell that presents the pMHC complex of claim 2 on the surface of the cell.

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

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 the peptide of claim 1 and/or the pMHC complex of claim 2.

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

9. Use of a peptide according to claim 1, a pMHC complex according to claim 2, for screening a library of T cell receptors or antibodies.

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.