Screening method of polypeptide derived from n-glycosylated protein and polypeptide vaccine

Abstract

To provide a screening method of polypeptides derived from glycosylated proteins and a polypeptide vaccine combination, having an effective and strong specific anti-tumor immune response effect.SOLUTION: The method comprises: a step 1 of screening N-mannan oligosaccharide-modified proteins and modification sites; a step 2 of identifying a sequence of the modified proteins, cellular localization, immunogenic regions, conservation of glycosylation modification sites, expression levels in tissue cells, site mutations, and effect thereof on protein stability; a step 3 of identifying conservation of residue expression of the modification sites; a step 4 of predicting antigen polypeptides having high HLA affinity and B cell linear epitopes from polypeptides containing the modification sites; and a step 5 of scanning the modification sites of the antigen polypeptides, simulating binding between HLA-A*24:02 molecules and the antigen polypeptides, and identifying parameters of binding stability.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Priority claims This invention claims priority to Chinese patent application having a filing date of February 5, 2024 and application number 2024101647525, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of polypeptides derived from glycoproteins, and in particular to methods for screening polypeptides derived from glycosylated proteins and polypeptide vaccines. [Background technology]

[0003] Polysaccharides, carbohydrates covalently linked to biological molecules, are coated on the surfaces of human cells and pathogens, forming the basis for cell or organism identification. They are sensed and recognized by various carbohydrate-binding proteins (GBPs) expressed by immune cells, such as galectins, siglecs, and C-type lectins, and direct pro- and anti-inflammatory responses (Ref. 1). Cytotoxic T lymphocyte (CTL) immune responses in the tumor microenvironment (TME) are significantly suppressed by tumor glycosylation and / or the expression of different galectins, promoting immune escape (Ref. 2). Specific glycan modes expressed in melanoma correlate with patient clinical outcomes; GlcNAc, NeuAc, TF-Ag, and Fuc modes are associated with poor survival, while Man and Glc glycans promote favorable survival (Ref. 3). High mannose is most common in breast cancer (BC) metastases, followed by fucose and complex glycans (Ref. 4).

[0004] Protein N-linked glycosylation is one of the post-translational modifications (PTMs) that protect tumor or viral antigens from immune attack (Refs. 5 and 6). N-linked glycosylation inhibitor-1 (NGI-1), a cell-permeable small molecule inhibitor with low cytotoxicity, has become an important tool for modulating N-glycosylation in mammalian cells. It inhibits the glycosylation expression of lysosome-associated membrane protein 2 (LAMP2), causing lysosomal defects and autophagy (Ref. 7). NGI-1 targets oligosaccharyltransferase (OST) to remove N-glycosylation from herpes simplex virus type 1 (HSV-1), improving its sensitivity to inhibitory drugs (Ref. 8). NGI-1 enhances the radiosensitivity of malignant gliomas by reducing the glycosylation, protein expression, and activation levels of receptor tyrosine kinases (RTKs) (Ref. 9). Inhibition of OST removes N-linked glycosylation from the epidermal growth factor receptor (EGFR), reducing its activation and leading to synthetic lethality in cells resistant to EGFR tyrosine kinase inhibitor (TKI) therapy (Reference 10).

[0005] The mannose receptor (CD206, MR) is an endogenous lectin receptor that plays an important role in innate immunity (Ref. 11). Man3GlcNAc2, also known as mannan oligosaccharide (PMG) (Ref. 12), strongly prefers N-glucans with a core of PMG and Man3 trisaccharide (Ref. 13). The Man1-3GlcNAc2Fuc0-1 glycoform is rarely found in mammalian tissues, but is significantly more frequently found in tumors and viruses (Refs. 14-16). Designing a glycovaccine with dual C-type lectin-like receptor (CLR) targeting properties using DC-SIGN expressed by DCs and Langerin expressed by Langerhans cells (LCS) can enhance anti-tumor CD8+ T cell responses (Ref. 17).

[0006] When NGI-1 is used to inhibit N-glycosylation of tumor cells or / and DC cells, there is a possibility that the activation of anti-tumor immune responses induced by specific glycans will be affected due to the lack of specificity. Currently, there are very few reports on antibodies to specific glycan-modified proteins and specific glycan-modified antigen peptides both in and outside of China (limited by manufacturing technology, etc.). Based on this background, the present application hopes to solve the above-mentioned difficult problem by using a polypeptide vaccine combination derived from glycosylated proteins to exert an effective and potent specific anti-tumor immune response effect. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Alves I,Fernandes A,Santos-Pereira B,Azevedo CM,Pinho SS.Glycans as a key factor in self and nonself discrimination:impact on the breach of immune tolerance.FEBS Lett.2022 Jun;596(12):1485-1502. [Non-patent document 2] Makandar AI, Jain M, Yuba E, Sethi G, Gupta RK.Canvassing Prospects of Glyco-Nanovaccines for Developing Cross-Presentation Mediated Anti-Tumor Immunotherapy.Vaccines(Basel).2022 Nov 30;10(12):2049. [Non-patent document 3] Sosa Cuevas E,Roubinet B,Mouret S,Thepaut M,de Fraipont F,Charles J,Fieschi F,Landemarre L,Chaperot L,Aspord C.The melanoma tumor glyco-code impacts human dendritic cells'functionality and dictates clinical outcomes.Front Immunol.2023 Feb 20;14:1120434.

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Summary of the Invention

[0008] To address the technical problems and deficiencies of the prior art, the present invention provides a method for screening polypeptides derived from glycosylated proteins and a polypeptide vaccine combination. First, a screening system for antigen polypeptides derived from N-mannan oligosaccharide-modified proteins is established, and the immunogenicity and killing function of the antigen polypeptides are optimized and verified through virtual screening and in vivo / in vitro immunological testing. Based on this, the antigen polypeptide is combined with a saccharide adjuvant and NGI-1 to form a vaccine combination. This vaccine combination significantly improves the antitumor immune killing activity in mice in vivo and in vitro compared to combinations such as the antigen polypeptide, polypeptide + adjuvant, and polypeptide + NGI-1, while lacking killing effect on normal epithelial cells. This enhances the specific immune killing function of polypeptide vaccines and expands the functional modulation of immunoinhibitory glycoproteins based on NGI-1.

[0009] In order to achieve the above object, the present invention provides a method for screening a polypeptide derived from an N-glycosylation-modified protein, the method comprising: Step S1: analyzing and identifying the sugar chain types and modification sites of N-glycosylated modified proteins in multiple types of tumor cell lines and normal cell lines, and screening for N-mannan oligosaccharide modified proteins and N-mannan oligosaccharide modification sites; Step S2: clarifying the sequence, cellular localization, immunogenic region, conservation of glycosylation modification site, expression level in tissue cells, site mutation and its effect on protein stability of the N-mannan oligosaccharide-modified protein; Step S3: detecting the DNA expression and mRNA transcription status of the N-mannan oligosaccharide modification site to clarify the conservation of residue expression of the N-mannan oligosaccharide modification site; a step S4 of predicting an antigen polypeptide having a high HLA affinity and an epitope that is a B cell linear epitope from the polypeptide containing the N-mannan oligosaccharide modification site; Step S5: further performing site scanning on the N-mannan oligosaccharide modification site of the antigen polypeptide to obtain a site-scanned antigen polypeptide, and simulating the binding of an HLA-A*24:02 molecule to the site-scanned antigen polypeptide in vitro to clarify the parameters of the binding stability between these antigen polypeptides and the HLA-A*24:02 molecule; and step S6 of synthesizing the site-scanned antigen polypeptides in vitro, verifying the level of IFN-γ release by activating CTL cells of these antigen polypeptides, clarifying the immunokilling effect of the antigen polypeptides on tumor cells and normal cells, and verifying the anti-tumor activity of the antigen polypeptides in vivo.

[0010] In some embodiments, in step S1, the glycan of the N-mannan oligosaccharide-modified protein comprises HexNAc(2)Hex(3)Fuc(1), and the N-mannan oligosaccharide-modified protein and the N-mannan oligosaccharide modification site are the 160th residue N(160N) on the adipocyte membrane-associated protein and the APMAP, respectively.

[0011] In some embodiments, in step S2, the cellular localization is the cell membrane, the immunogenic region is the amino acid sequence from positions 69 to 186 of the APMAP, the expression in tissue cells is widespread, the N-mannan oligosaccharide modification site 160N can be mutated to 160S, and mutation of the site is predicted to be harmless to the stability of the APMAP.

[0012] In some embodiments, in step S3, the 160th residue, N, of the APMAP is a conserved residue, and no mutation is observed at either the DNA or mRNA level.

[0013] In some embodiments, in step S4, the polypeptide containing the N-mannan oligosaccharide modification site comprises two 17 peptides, each consisting of 8 amino acids at the N-terminus and C-terminus, with the 160th residue N of the APMAP and the 160th mutation S of the APMAP as its midpoint, and the sequences are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively; or comprises 31 peptides, each consisting of 15 amino acids at the N-terminus and C-terminus, with the 160th residue N of the APMAP protein sequence and the 160th mutation S as its midpoint, and the amino acid sequences are shown in SEQ ID NO:23 and SEQ ID NO:24, respectively.

[0014] In some embodiments, in step S4, the sequences of the antigen polypeptides having an epitope that has high HLA affinity and is a B-cell linear epitope are shown in SEQ ID NO:3 and SEQ ID NO:4.

[0015] In some embodiments, the amino acid sequences of the site-scanned antigen polypeptides are set forth in SEQ ID NO:3 to SEQ ID NO:22, respectively.

[0016] In some embodiments, step S5 further includes simulating the binding between mannan oligosaccharide and N-160 or S-160 of the APMAP in vitro to clarify the stability of the binding between mannan oligosaccharide and N- or S-160 of the APMAP.

[0017] The present invention further provides a polypeptide vaccine, which comprises a polypeptide whose sequence is set forth in any of SEQ ID NO:3 to SEQ ID NO:22, mannan oligosaccharide, and NGI-1.

[0018] In some embodiments, the sequence of the polypeptide is set forth in SEQ ID NO:3 or SEQ ID NO:4.

[0019] The polypeptide for cancer treatment has a sequence set forth in any of SEQ ID NO:3 to SEQ ID NO:22.

[0020] The present invention has the following advantageous effects compared to the prior art.

[0021] 1. The present invention combines various means, such as mass spectrometry and DNA and mRNA high-throughput sequencing, to analyze and identify specific N-glycosylated sites and the conservation of their residues, and further predict T cell and B cell epitopes. This makes epitope screening more effective and accurate than many previous studies that directly use databases to predict protein epitopes.

[0022] 2. The present invention patent combines and analyzes virtual screening methods using multiple drugs (molecular docking, molecular dynamics simulation, protein binding stability prediction, etc.) with various databases (UniProt, Provean, HPA, NetMHCpan4.1, IEDB, etc.), further screening for polypeptide epitopes (including N>X) that contain specific N-glycosylation modifications and have immunogenicity, and verifies the immune activation effects and antitumor effects of the screened polypeptide epitopes (including N>X) and their vaccine combinations through in vivo / in vitro tests. Compared with the methods used in current research, the screening and verification processes are more complete and effective.

[0023] 3. The polypeptide vaccine combination sought to be protected by the present invention patent has been predicted in vitro and demonstrated through in vivo / in vitro experiments, and has a more significant anti-tumor immune-enhancing effect than the combination of polypeptide antigen and adjuvant or NGI-1 alone as discovered in current research.

[0024] In order to more clearly describe the specific embodiments of the present invention or the technical means in the prior art, the drawings necessary for describing the specific embodiments or the prior art will be briefly described below. Obviously, the drawings described below are only some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative work. [Brief explanation of the drawings]

[0025] [Figure 1] FIG. 1 is a schematic diagram showing the steps of the method of screening for polypeptides derived from glycosylated proteins of the present invention. [Figure 2] 1 is a schematic diagram of the expression of the APMAP protein of the present invention in normal and tumor tissues of the human body. [Figure 3] FIG. 1 is a schematic representation of the prediction of B-cell linear epitopes of the polypeptide sequences of the APMAP glycosylation sites of the present invention. [Figure 4] FIG. 1 is a schematic diagram of protein binding between the mannan oligosaccharide-modified APMAP(160N) and APMAP(N160S) of the present invention. [Figure 5] FIG. 1 is a schematic diagram of IFN-γ release levels (spots) after induction of CTL activation by the polypeptides of the present invention. [Figure 6] FIG. 1 is a schematic representation of IFN-γ release levels after induction of CTL activation by a glycosylated protein polypeptide vaccine combination of the present invention and co-culture with tumors. [Figure 7] 1 is a schematic diagram of the tumor growth inhibition rate of the glycosylated protein polypeptide vaccine combination of the present invention in resisting in vivo tumors in mice. DETAILED DESCRIPTION OF THE INVENTION

[0026] To clarify the objectives, technical means, and advantages of the embodiments of the present invention, the following clearly and completely describes the technical means in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Typically, the components of the embodiments of the present invention described and shown in the drawings herein may be arranged and designed in various configurations. Therefore, the detailed description of the embodiments of the present invention provided below in the drawings is not intended to limit the scope of the claimed invention, but represents only selected embodiments of the present invention. All other embodiments obtained based on the embodiments of the present invention without the need for creative efforts by those skilled in the art are within the scope of protection of the present invention.

[0027] According to one aspect of the present invention, there is provided a method for screening polypeptides derived from N-glycosylated proteins, which can yield 20 antigen polypeptides that can be verified through in vivo / in vitro experiments to exert effective antitumor effects.

[0028] The method includes the following steps S1 to S6.

[0029] In step S1, the glycan types and modification sites of N-glycosylated proteins in multiple tumor cell lines and normal cell lines are analyzed and identified, and N-mannan oligosaccharide-modified proteins and N-mannan oligosaccharide modification sites are screened.

[0030] In some examples, N-glycosylation modifications may include N-mannan oligosaccharide modifications, N-acetylglucosamine modifications, and the like.

[0031] In some embodiments, the tumor cell line may be a cell line of any tumor type (e.g., cervical cancer, colon cancer, etc.). The sugar chains of the N-mannan oligosaccharide (paucimannose)-modified protein mainly contain HexNAc(2)Hex(3)Fuc(1). The N-mannan oligosaccharide-modified protein and the N-mannan oligosaccharide modification site are adipocyte plasma membrane-associated protein (APMAP) and the 160th residue N (160N) of the APMAP, respectively.

[0032] Although the present invention screens only for N-mannan oligosaccharide-modified proteins and their sites, the method of the present invention may also be used to select other N-glycosylation-modified proteins and sites, and the present invention is not limited thereto.

[0033] In step S2, the sequence, cellular localization, immunogenic region, conservation of glycosylation modification sites, expression level in tissue cells, site mutations, and their effects on protein stability of the N-mannan oligosaccharide-modified protein are clarified.

[0034] In some embodiments, the cellular localization is the cell membrane, the immunogenic region is the amino acid sequence from positions 69 to 186 of the APMAP, the expression in tissue cells is widespread, the N-mannan oligosaccharide modification site 160N can be mutated to 160S, and mutation of the site does not affect the predicted stability of the APMAP.

[0035] In step S3, the DNA expression and mRNA transcription status of the N-mannan oligosaccharide modification site are detected to clarify the conservation of residue expression of the N-mannan oligosaccharide modification site.

[0036] In some embodiments, the 160th residue, N, of the APMAP detected in step S3 is a conserved residue, and no mutation is observed at either the DNA or mRNA level.

[0037] In step S4, an antigen polypeptide having high HLA affinity and an epitope that is a B cell linear epitope is predicted from polypeptides containing an N-mannan oligosaccharide modification site.

[0038] In step S4, the polypeptide containing the N-mannan oligosaccharide modification site may comprise two 17 peptides, each consisting of 8 amino acids at the N-terminus and C-terminus, with the 160th residue N of the APMAP and the 160th mutation S of the APMAP as midpoints, the sequences of which are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively, or may comprise 31 peptides, each consisting of 15 amino acids at the N-terminus and C-terminus, with the 160th residue N of the APMAP protein sequence and the 160th mutation S as midpoints, the amino acid sequences of which are shown in SEQ ID NO:23 and SEQ ID NO:24, respectively.

[0039] The sequences of the antigenic polypeptides described above, which have predicted high HLA affinity and epitopes that are B-cell linear epitopes, are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

[0040] In step S5, further site scanning is performed on the N-mannan oligosaccharide modification site of the antigen polypeptide to obtain a site-scanned antigen polypeptide, and the binding of HLA-A*24:02 molecules to the site-scanned antigen polypeptide is simulated in vitro to clarify the parameters of the stability of the binding between these antigen polypeptides and the HLA-A*24:02 molecules.

[0041] The amino acid sequences of the site-scanned antigen polypeptides are shown in SEQ ID NO:3 to SEQ ID NO:22, respectively.

[0042] Step S5 further includes simulating the binding between mannan oligosaccharide and the 160th N or 160th S residue of the APMAP in vitro to clarify the stability of the binding between mannan oligosaccharide and the N or S residue of the APMAP.

[0043] In some embodiments, N-site mannan oligosaccharide modifications are more advantageous for APMAP protein stability than S-site mannan oligosaccharide modifications.

[0044] Step S6: The site-scanned antigen polypeptides are synthesized in vitro, and the level of IFN-γ release by these antigen polypeptides in activating CTL cells is verified to clarify the immunokilling effect of the antigen polypeptides on tumor cells and normal cells, and the anti-tumor activity of the antigen polypeptides is verified in vivo.

[0045] According to another aspect of the present invention, there is provided a polypeptide vaccine comprising a polypeptide whose sequence is set forth in any of SEQ ID NO:3 to SEQ ID NO:22, mannan oligosaccharides, and NGI-1.

[0046] In some embodiments, the polypeptide vaccine comprises a polypeptide having SEQ ID NO:3 or SEQ ID NO:4, mannan oligosaccharide, and NGI-1. In some embodiments, the amount of the polypeptide added in the polypeptide vaccine is 10 μg to 10 mg, the amount of the mannan oligosaccharide added in the polypeptide vaccine is 10 μg to 10 mg, and the amount of NGI-1 added in the polypeptide vaccine is 5 to 5000 mM.

[0047] As can be seen from in vivo / in vitro experiments, the polypeptide vaccine of the present invention can effectively inhibit tumor growth and reduce tumor volume.

[0048] According to another aspect of the present invention, there is provided a polypeptide for use in treating cancer, or use of a polypeptide in preparing a medicament for treating cancer. The sequence of the polypeptide is set forth in any of SEQ ID NO: 3 to SEQ ID NO: 22. The cancer may include any of colon cancer, gastric cancer, lung cancer, etc.

[0049] Example 1: Screening method for polypeptides derived from glycosylated proteins This example provides a method for screening polypeptides derived from glycosylated proteins, and the specific steps are as follows:

[0050] Using liquid chromatography-mass spectrometry, we analyzed and identified the glycan types and modification sites of N-glycosylated proteins from multiple types of tumor cells and normal epithelial cells, screened for residue sites with specific sugar modifications, and used various databases such as UniPro to investigate parameters such as protein sequence, conservation of glycosylation modification sites, and the effect of site mutations on protein stability, thereby clarifying polypeptide sequences containing specific sugar modification sites (including N>X mutations). DNA whole-exome sequencing and eukaryotic mRNA transcriptome sequencing were used to detect DNA mutations and mRNA transcription status of specific sugar modification sites, and clarify the conservation of site residue expression. Furthermore, we used various databases, such as NetMHCpan4.1, to predict the affinity of polypeptide sequences (including N>X mutations) for HLA-I molecules and their B cell linear epitopes. We then identified nonapeptide antigens (containing N>X glycosylation sites) with predicted high HLA-I affinity and B cell linear epitopes. We then used various methods, such as virtual docking, to predict important parameters, such as the stability of binding between HLA-I molecules and nonapeptide antigens (containing N>X glycosylation sites). Finally, we used ELISpot to verify the level of IFN-γ release by the above nonapeptides and their nonapeptide vaccine combinations in inducing and activating CTL cells (or by co-culture with human tumor cell microspheres and human normal epithelial cells). We also used tumor-bearing mouse models to verify the antitumor immune effects of the polypeptide vaccine combinations in vivo.

[0051] Specifically, as shown in FIG. 1, the method includes: Step S1: Analyzing and identifying the sugar chain types and modification sites of N-glycosylated proteins from multiple types of tumor cells and normal epithelial cells using liquid chromatography-mass spectrometry, and screening for residue sites with specific sugar modifications; Step S2: Using various databases such as UniProt and Provean, related parameters such as protein sequence, conservation of glycosylation modification sites, and the effect of site mutations on protein stability are investigated to identify polypeptide sequences containing specific glycosylation sites (including N>X mutations); Step S3: detecting DNA mutations and mRNA transcription status at specific glycosylation sites by DNA whole exome sequencing and eukaryotic mRNA transcriptome sequencing, and clarifying the conservation of site residue expression; Step S4: predicting the affinity of HLA-I molecules and polypeptide sequences (including N>X mutations) and B cell linear epitopes using various databases such as NetMHCpan4.1 to identify nonapeptide antigens (including glycosylation sites N>X) that have predicted high affinity for HLA-I and contain B cell linear epitopes; Step S5: predicting important parameters such as the stability of binding between HLA-I molecules and nonapeptide antigens (containing glycosylation sites N>X) using various means such as virtual screening, and synthesizing the nonapeptides (containing glycosylation sites N>X) in vitro; and step S6, in which the nonapeptide and its vaccine combination in step S5 are used to verify the level of IFN-γ release by inducing and activating CTL cells (or by co-culturing with human tumor cell microspheres / human normal epithelial cells) and verifying the anti-tumor immune effect of the polypeptide vaccine combination in vivo using a tumor-bearing mouse model.

[0052] [Example 2] Prediction of immunogenicity of antigens derived from N-mannan oligosaccharide (paucimannose)-modified proteins Using liquid chromatography-mass spectrometry, we analyzed and identified the glycan types and modification sites of N-glycosylated proteins in multiple tumor cell lines (e.g., CaSki, Hela, SiHa, etc.) and corresponding normal epithelial cell lines (e.g., HcerEpic, H8, etc.), and screened for proteins and modification sites with N-mannan oligosaccharide (paucimannose, mainly containing HexNAc(2)Hex(3)Fuc(1)). Specific N-glycosylation modifications of proteins, their residue sites, and subcellular localization are shown in Table 1 below.

[0053] Table 1. Specific glycosylation of proteins, their residue sites, and subcellular localization [Table 1]

[0054] Using UniProt (https: / / www.uniprot.org / ) and Provean (https: / / provean.jcvi.org / index.php), we investigated various parameters of the screened N-mannan oligosaccharide glycosylated proteins, including their immunogenic sequences, reported residue mutations (N>X), and the effect of the mutations on protein function. The relevant characteristic parameters of N-glycosylated protein sites are listed in Table 2.

[0055] Table 2. Related characteristic parameters of N-mannan oligosaccharide-modified protein sites [Table 2]

[0056] In the present invention, X represents any amino acid unless otherwise specified.

[0057] Based on the related properties of the gene / protein, such as its expression in the cell membrane, the presence of the N-glycosylation site in the immunogenic region, and the known mutation (N>S) not affecting the protein function, APMAP(160N) and APMAP(N160S) were selected as the research subject of the present invention patent. APMAP, whose Chinese name is adipose membrane-associated protein (Adipose Membrane Associated Protein) and ID is Q9HDC9, is an N-glycosylation modification at the 160th residue.

[0058] As shown in Figure 2, the expression of APMAP in human normal tissues and tumor tissues was investigated using HPA (https: / / www.proteinatlas.org / ). Panel A shows the expression of APMAP protein in human normal tissues, and Panel B shows the expression of APMAP protein in human tumor tissues. This suggests that APMAP protein is widely expressed in human tissues and tumor tissues.

[0059] Using UniProt, we investigated the nociceptin sequence consisting of eight amino acids at the N-terminus and C-terminus of the APMAP protein sequence, centered on the 160th N-glycosylation residue, and found that it was PLGIRAGPNGTLFVADA (SEQ ID NO: 1, the sequence before mutation) and PLGIRAGPSGTLFVADA (SEQ ID NO: 2, the sequence in which N was mutated to S, i.e., N>S).

[0060] DNA whole-exome sequencing and eukaryotic mRNA transcriptome sequencing were used to detect DNA mutations and mRNA transcription status of N-glycosylation modification protein sites. The glycosylation site N-160 was a conserved site, and no gene mutations were detected in either.

[0061] Using NetMHCpan4.1 (https: / / services.healthtech.dtu.dk / services / NetMHCpan-4.1 / ), we identified potential polypeptides presented by the HLA-I molecule HLA-A*24:02 for the above two amino acid sequences based on affinity prediction. Cytotoxic T lymphocyte (CTL) epitopes are short peptides in antigenic proteins that are processed by antigen-presenting cells (APCs), bind to MHC-I molecules, and are specifically recognized by T cell antigen receptors (TCRs) to induce corresponding CTL clones and generate immune responses. These epitopes generally consist of 8–10 consecutive amino acid residues and play a crucial role in the CTL activation process, determining the specific killing effect of CTLs. Cytotoxic T lymphocyte (CTL) epitopes are high-affinity HLA-I antigen epitopes. The predicted affinity of the polypeptide sequences for binding to HLA-A*24:02 is shown in Table 3 below.

[0062] Table 3. Polypeptide sequences of APMAP glycosylation sites and predicted affinity for HLA-A*24:02 [Table 3]

[0063] As shown in Table 3, the predicted results show that RAGPNGTLF (SEQ ID NO: 3) and RAGPSGTLF (SEQ ID NO: 4) are presented by HLA-A*24:02 molecules. Furthermore, site scanning was performed on the N residues of the RAGPNGTLF (SEQ ID NO: 3) antigen sequence to predict the change in affinity between the site-scanned antigen sequence and HLA-A*24:02. The results are shown in Table 4.

[0064] Table 4. Predicted affinity of polypeptides to HLA-A*24:02 after scanning N-mannan oligosaccharide glycosylation sites [Table 4]

[0065] As shown in Table 4, in vitro prediction suggested that all of the N-glycosylation site scanned polypeptides were capable of binding to HLA-A*24:02 molecules.

[0066] As shown in Figure 3, the IEDB database (http: / / tools.iedb.org / bcell / ) was used to predict whether the polypeptide sequences DEPVCGRPLGIRAGPNGTLFVADAYKGLFEV (SEQ ID NO: 23) and DEPVCGRPLGIRAGPSGTLFVADAYKGLFEV (SEQ ID NO: 24) (31 peptide sequences consisting of 15 amino acids at the N-terminus and C-terminus, with the 160th N-glycosylated residue in the APMAP protein sequence as the midpoint) contain a B-cell linear epitope. The above-mentioned SEQ ID NO: 1 and SEQ ID NO: 2 may also be used.

[0067] The light gray areas above the abscissa in Figures 3(A) and 3(B) represent predicted B cell linear epitopes, which are DEPVCG (SEQ ID NO:25), GIRAGPNG (SEQ ID NO:26), and GIRAGPSG (SEQ ID NO:27), respectively. GIRAGPNG (SEQ ID NO:26) and GIRAGPSG (SEQ ID NO:27) overlap with the HLA-A*24:02 restricted polypeptides RAGPNGTLF (SEQ ID NO:3) and RAGPSGTLF (SEQ ID NO:4), respectively, suggesting that the latter may induce both specific T cell and B cell immune responses. RAGPNGTLF (SEQ ID NO:3) and GIRAGPNG (SEQ ID NO:26), and RAGPSGTLF (SEQ ID NO:4) and GIRAGPSG (SEQ ID NO:27), each share one epitope, and the minimal motifs of the epitopes are RAGPNG (SEQ ID NO:28) and RAGPSG (SEQ ID NO:29), respectively. These two motifs are the minimal motifs shared by the CTL cell epitope and the B cell linear epitope (i.e., the overlapping portion described above). Therefore, RAGPNGTLF (SEQ ID NO:3) and RAGPSGTLF (SEQ ID NO:4) can induce both specific T cell and B cell immune responses.

[0068] Molecular docking and molecular dynamics simulations were performed (using Rosetta 3.8 to generate the 3D structure of the polypeptide and perform energy optimization using the relax module. The HLA-A*24:02 structure is PDB ID: 7JYU (https: / / www.rcsb.org / structure / 7JYU). Molecular docking was performed using autodock vina 1.1.2, and the protein-polypeptide complex obtained by molecular docking was subjected to molecular dynamics simulation using the amber18 software package). The HLA-A*24:02 molecule and the 20 antigen nonapeptides (SEQ ID NO: 3 to SEQ ID NO: 4) were analyzed. The binding of the N-modified RAGP[N(mannan oligosaccharide)]GTLF polypeptide and the S-modified RAGP[S(mannan oligosaccharide)]GTLF polypeptide of the 22 different sequence / modified antigens mentioned above with the HLA-A*24:02 molecule was simulated in vitro to clarify the stability parameters of the virtual binding between the 22 different sequence / modified antigens and the HLA-A*24:02 molecule. The results are shown in Table 5.

[0069] Table 5. Predicted binding of N-glycosylation site scanned polypeptide sequences to HLA-A*24:02 molecules [Table 5]

[0070] In addition, we simulated the binding of mannan oligosaccharides to the 160th residue of N or 160th residue of S (N>S) of the protein APMAP in vitro to clarify the stability of the binding between mannan oligosaccharides and the N or S residue of APMAP protein. (Molecular dynamics simulations of membrane proteins were performed using the amber18 software package, with the ff14SB force field parameters for proteins and the gaff general force field parameters for mannose. The AM1-BCC atomic charges were calculated using the ANTECHAMBER module. PDB format files of the wild-type protein and its mutants were uploaded to the DynaMut online server (https: / / biosig.lab.uq.edu.au / dynamut / prediction) to analyze the effect of mutations on stability.)

[0071] As shown in Table 5, P5 is an N-glycosylation site, and the binding energy (P5 (kcal / mol)) between the residue at this site and the HLA-A*24:02 molecule is as follows, from highest to lowest: RAGPIGTLF(SEQ ID NO:5)>RAGPRGTLF(SEQ ID NO:19)>RAGPLGTLF(SEQ ID NO:10)>RAGPFGTLF(SEQ ID NO:6)>RAGPMGTLF(SEQ ID NO:11)>RAGPHGTLF(SEQ ID NO:13)>RAGPVGTLF(SEQ ID NO:7)>RAGPQGTLF(SEQ ID NO:18)>RAGPGTLF(SEQ ID NO:9)>RAGPWGTLF(SEQ ID NO:8)>RAGPTGTLF(SEQ ID NO:14)>RAGPNGTLF(SEQ ID NO:3)>RAGPCGTLF(SEQ ID NO:22)>RAGPKGTLF(SEQ ID NO:16)>RAGPYGTLF(SEQ ID NO:12)>RAGPGGTLF(SEQ ID NO:17)>RAGPAGTLF(SEQ ID NO:15)>RAGPSGTLF (SEQ ID NO:4)>RAGPEGTLF (SEQ ID NO:20)>RAGPDGTLF (SEQ ID NO:21).

[0072] The total binding energy (DELTA TOTAL (kcal / mol)) between the above polypeptide and the HLA-A*24:02 molecule is as follows, from highest to lowest: RAGPWGTLF(SEQ ID NO:8)>RAGPQGTLF(SEQ ID NO:18)>RAGPRGTLF(SEQ ID NO:19)>RAGPGTLF(SEQ ID NO:9)>RAGPKGTLF(SEQ ID NO:16)>RAGPTGTLF(SEQ ID NO:14)>RAGPYGTLF(SEQ ID NO:12)>RAGPFGTLF(SEQ ID NO:6)>RAGPVGTLF(SEQ ID NO:7)>RAGPHGTLF(SEQ ID NO:13)>RAGPNGTLF(SEQ ID NO:3)>RAGPLGTLF(SEQ ID NO:10)>RAGPDGTLF(SEQ ID NO:21)>RAGPIGTLF(SEQ ID NO:5)>RAGPCGTLF(SEQ ID NO:22)>RAGPAGTLF(SEQ ID NO:15)>RAGPSGTLF(SEQ ID No. 4) > RAGPGGTLF (SEQ ID No. 17) > RAGPMGTLF (SEQ ID No. 11) > RAGPEGTLF (SEQ ID No. 20).

[0073] When RAGP[N(mannan oligosaccharide)]GTLF bound to HLA-A*24:02 molecules, both the P5 energy and total energy were significantly higher than those of the polypeptide not modified with mannan oligosaccharides, suggesting that the mannan oligosaccharide modification facilitated the binding of the polypeptide to HLA molecules. When RAGP[S(mannan oligosaccharide)]GTLF bound to HLA-A*24:02 molecules, both the P5 energy and total energy were significantly lower than those of the polypeptide not modified with mannan oligosaccharides, suggesting that the mannan oligosaccharide modification did not contribute to the binding of the polypeptide to HLA-A molecules. As shown in Figure 4, Panel A is a schematic diagram of the binding between mannan oligosaccharides and APMAP(160N) protein, and Panel B is a schematic diagram of the binding between mannan oligosaccharides and APMAP(N160S) protein. As shown in Table 6 below, DynaMut analysis showed that after N-160 was mutated to S, the protein stability decreased (ΔΔG DynaMut=-0.106 kcal / mol), i.e., the mannan oligosaccharide modification at the N-site is beneficial to the stability of APMAP protein.

[0074] Table 6. Prediction of protein stability of APMAP(160N) and APMAP(N160S) modified with mannan oligosaccharides [Table 6]

[0075] [Example 3: In vivo / in vitro validation experiment of antigen polypeptide] Twenty polypeptides were identified, namely, RAGPWGTLF (SEQ ID NO:8), RAGPQGTLF (SEQ ID NO:18), RAGPRGTLF (SEQ ID NO:19), RAGPPGTLF (SEQ ID NO:9), RAGPKGTLF (SEQ ID NO:16), RAGPTGTLF (SEQ ID NO:14), RAGPYGTLF (SEQ ID NO:12), RAGPFGTLF (SEQ ID NO:6), RAGPVGTLF (SEQ ID NO:7), RAGPHGTLF (SEQ ID NO:13), RAGPNGTLF (SEQ ID NO:3) (before mutation), RAGPLGTLF (SEQ ID NO:10), RAGPDGTLF (SEQ ID NO:21), RAGPIGTLF (SEQ ID NO:5), RAGPCGTLF (SEQ ID NO:22), RAGPAGTLF (SEQ ID NO:13). SEQ ID NO:15), RAGPSGTLF (SEQ ID NO:4), RAGPGGTLF (SEQ ID NO:17), RAGPMGTLF (SEQ ID NO:11), and RAGPEGTLF (SEQ ID NO:20) (average purity 98%, acetate salt) were synthesized in vitro. PBMC cells from HLA-A*24:02 homozygotes were cultured in vitro and induced to differentiate into imDCs. The 20 polypeptides were loaded onto the imDCs, resulting in polypeptide-loaded mDCs, which were then used to induce T cell differentiation into CTL cells. As shown in Figure 5, the level of IFN-γ release by the polypeptides inducing CTL cells was examined using enzyme-linked immunospot (ELISpot). The results are shown in Table 7 below.

[0076] Table 7. IFN-γ release levels after induction of CTL activation by polypeptides [Table 7]

[0077] As shown in Table 7 and Figure 5, all of the above polypeptides can be presented by DCs to activate CTL cells and generate spots, which is due to the antigen sequence (RAGP XThese results suggest that the immunogenicity of GTLF (X is any one of the 20 natural amino acids) is high, regardless of whether P5 is an N residue or not. The activation levels (mean spot values) are as follows, from highest to lowest: RAGPVGTLF(SEQ ID NO:7)>RAGPDGTLF(SEQ ID NO:21)>RAGPTGTLF(SEQ ID NO:14)>RAGPMGTLF(SEQ ID NO:11)=RAGPYGTLF(SEQ ID NO:12)>RAGPGGTLF(SEQ ID NO:17)>RAGPWGTLF(SEQ ID NO:8)>RAGPCGTLF(SEQ ID NO:22)>RAGPIGTLF(SEQ ID NO:5)>RAGPAGTLF(SEQ ID NO:15)>RAGPQGTLF(SEQ ID NO:18)>RAGPFGTLF(SEQ ID NO:6)>RAGPEGTLF(SEQ ID NO:20)>RAGPHGTLF(SEQ ID NO:13)=RAGPNGTLF(SEQ ID NO:3)>RAGPKGTLF(SEQ ID NO:16)>RAGPRGTLF(SEQ ID No. 19)>RAGPLGTLF (SEQ ID No. 10)>RAGPPGTLF (SEQ ID No. 9)>RAGPSGTLF (SEQ ID No. 4).

[0078] Of the 20 polypeptides, RAGPNGTLF (SEQ ID NO: 3) and RAGPSGTLF (SEQ ID NO: 4) were selected as representatives. The combined effects of these two polypeptides, mannan oligosaccharide (CAS No.: 70858-45-6) and NGI-1 (CAS No.: 790702-57-7), were examined using ELISpot. After inducing mDC vaccination into imDCs in each group, CTL cells were activated and the level of IFN-γ release was examined when co-cultured with microspheres formed from human colon cancer cells HT-29 and human colon epithelial cells HCoEpiC, respectively (see Figure 6). As shown in FIG. 6 , the TN-PEP group, the TN-PEP+M group, the TN-PEP+M+N group, the TS-PEP+M+N group, the TN-PEP+N group, and the T-NONE group were respectively added with RAGPNGTLF (SEQ ID NO: 3), RAGPNGTLF (SEQ ID NO: 3) + mannan oligosaccharides, RAGPNGTLF (SEQ ID NO: 3) + mannan oligosaccharides + NGI-1, RAGPSGTLF (SEQ ID NO: 3) + mannan oligosaccharides + NGI-1, RAGPNGTLF + NGI-1, and blank PBS control to induce activated CTLs and co-culture with human colon cancer cell HT-29 microspheres. The EN-PEP group, the EN-PEP+M group, the EN-PEP+M+N group, the ES-PEP+M+N group, the EN-PEP+N group, and the E-NONE group were respectively added with RAGPNGTLF (SEQ ID NO: 3) + mannan oligosaccharides, RAGPNGTLF (SEQ ID NO: 3) + mannan oligosaccharides + NGI-1, RAGPSGTLF (SEQ ID NO: 3) + mannan oligosaccharides + NGI-1, and blank PBS control to induce activated CTLs and co-culture with human colon cancer cell HT-29 microspheres. NO:4), RAGPNGTLF (SEQ ID NO:4) + mannan oligosaccharides, RAGPNGTLF (SEQ ID NO:4) + mannan oligosaccharides + NGI-1, RAGPSGTLF (SEQ ID NO:4) + mannan oligosaccharides + NGI-1, RAGPNGTLF (SEQ ID NO:4) + NGI-1, and blank PBS control were added to induce activated CTLs and co-culture them with human colon epithelial cell HCoEpiC microspheres.The results, as shown in Figure 6A, showed that after co-culture with human colon cancer cells, the IFN-γ release levels of CTLs were as follows: TS-PEP+M+N > TN-PEP+M+N > TN-PEP+M > T-NONE > TN-PEP > TN-PEP+N. The TS-PEP+M+N group was significantly higher than the other four groups, with the difference reaching statistical significance (P<0.05). For normal colon epithelium (EN-PEP), the difference between the groups shown in Figure 6B was not statistically significant (ns, P>0.05). As can be seen from the above results, the combined use of polypeptides, mannan oligosaccharides, and NGI-1 produced the most potent effects.

[0079] Subcutaneously transplanted tumor (mouse colon cancer CT-26 cells, 6x10E 5 The BALB / C mouse model was used (injected into the right armpit of each mouse at 10 μg / mouse). On days 7, 14, 18, and 20 after tumor cell inoculation, the BNP group received a polypeptide vaccine combination of RAGPNGTLF (SEQ ID NO: 3) (10 μg to 10 mg) + mannan oligosaccharide (CAS No.: 70858-45-6, 10 μg to 10 mg) + NGI-1 (CAS No.: 790702-57-7, 5 to 5000 mM); the RAGPSGTLF (SEQ ID NO: 4) group received a polypeptide vaccine combination of RAGPNGTLF (SEQ ID NO: 4) (10 μg to 10 mg) + mannan oligosaccharide (CAS No.: 70858-45-6, 10 μg to 10 mg) + NGI-1 (CAS No.: 790702-57-7, 5 to 5000 mM); Mice were injected with the BSP group (BNP, BSP, BSP-BNP ...

[0080] Through in vivo / in vitro experiments, the effects of RAGPNGTLF (SEQ ID NO: 3) and RAGPSGTLF (SEQ ID NO: 4) were finally verified, and the other 18 polypeptides of the present invention can also achieve effective anti-tumor effects.

[0081] The beneficial effects of this patent are as follows: For the high-affinity HLA-I antigens SEQ ID NO:3 and SEQ ID NO:4, affinity was first predicted using a database, followed by analysis of the binding stability between the antigen and HLA-I using in vitro molecular docking and molecular dynamics simulation. Furthermore, two methods, in vitro ELISPOT testing and in vivo immunization in mice, were used to verify antigen epitope protection. Analysis of the binding stability between the antigen and HLA-I using in vitro molecular docking and molecular dynamics simulation provides evidence for the high affinity prediction. Second, the combination of mannan oligosaccharides as an immune adjuvant and NGI-1's immune cell function-enhancing effect (antigen + sugar + NGI-1) has not yet been reported in China or abroad, and the entire antigen screening process has not been reported.

[0082] It should be understood that the above are merely specific examples of the present invention and do not limit the scope of the present invention. Any equivalent changes or modifications made based on the structures, features and principles described in the claims of the present invention should be included in the claims of the present invention.

[0083] Finally, the above examples are merely specific embodiments of the present invention, and are intended to illustrate the technical means of the present invention and not to limit the present invention, and the scope of protection of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the above examples, it should be understood that those skilled in the art may still make modifications or obvious changes to the technical means described in the above examples, or make equivalent substitutions for some of the technical features thereof, within the technical scope disclosed in the present invention. These modifications, changes, or substitutions do not deviate the essence of the corresponding technical means from the spirit and scope of the technical means of the embodiments of the present invention, and all should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined based on the scope of protection of the claims.

Claims

1. Step S1: analyzing and identifying the sugar chain types and modification sites of N-glycosylated modified proteins of multiple types of tumor cell lines and normal cell lines, and screening for N-mannan oligosaccharide modified proteins and N-mannan oligosaccharide modification sites; Step S2: clarifying the sequence, cellular localization, immunogenic region, conservation of glycosylation modification site, expression level in tissue cells, site mutation and its effect on protein stability of the N-mannan oligosaccharide-modified protein; Step S3: detecting the DNA expression and mRNA transcription status of the N-mannan oligosaccharide modification site to clarify the conservation of residue expression of the N-mannan oligosaccharide modification site; a step S4 of predicting an antigen polypeptide having high HLA affinity and an epitope that is a B-cell linear epitope from the polypeptide containing the N-mannan oligosaccharide modification site; Step S5: performing site scanning on a single residue of the antigen polypeptide where the N-mannan oligosaccharide modification site is located to obtain a site-scanned antigen polypeptide, and simulating the binding of an HLA-A*24:02 molecule to the site-scanned antigen polypeptide in vitro to clarify the parameters of the binding stability between these antigen polypeptides and the HLA-A*24:02 molecule; and step S6 of synthesizing the site-scanned antigen polypeptides in vitro, verifying the level of IFN-γ release by activating CTL cells of these antigen polypeptides, clarifying the immunocidal effect of the antigen polypeptides on tumor cells and normal cells, and verifying the antitumor activity of the antigen polypeptides in vivo.

2. The method of claim 1, wherein in step S1, the sugar chain of the N-mannan oligosaccharide-modified protein contains HexNAc(2)Hex(3)Fuc(1), and the N-mannan oligosaccharide-modified protein and the N-mannan oligosaccharide modification site are an adipocyte membrane-associated protein (APMAP) and the 160th residue N (160N) of the APMAP, respectively.

3. The method of claim 1, wherein in step S2, the cellular localization is the cell membrane, the immunogenic region is the amino acid sequence from positions 69 to 186 of the APMAP, the expression in tissue cells is widespread, the N-mannan oligosaccharide modification site 160N can be mutated to 160S, and the mutation of the site is predicted to be harmless to the stability of the APMAP.

4. The method according to claim 3, wherein in step S3, the 160th residue N of the APMAP is a conserved residue, and no mutation is observed at either the DNA or mRNA level.

5. 4. The method of claim 3, wherein in step S4, the polypeptide containing the N-mannan oligosaccharide modification site comprises two 17 peptides, each consisting of 8 amino acids at the N-terminus and C-terminus, with the 160th residue N of the APMAP and the 160th mutation S of the APMAP as midpoints, and the sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; or comprises 31 peptides, each consisting of 15 amino acids at the N-terminus and C-terminus, with the 160th residue N of the APMAP protein sequence and the 160th mutation S as midpoints, and the amino acid sequences are shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively.

6. 4. The method of claim 3, wherein in step S4, the sequences of the antigen polypeptides having high HLA affinity and epitopes that are B-cell linear epitopes are shown in SEQ ID NO: 3 and SEQ ID NO:

4.

7. The method of claim 1, wherein the amino acid sequences of the site-scanned antigen polypeptides are set forth in SEQ ID NO: 3 to SEQ ID NO: 22, respectively.

8. The method of claim 1, characterized in that step S5 further comprises simulating in vitro the binding between the mannan oligosaccharide and the 160th N or 160th S residue of the APMAP to clarify the stability of the binding between the mannan oligosaccharide and the N or S residue of the APMAP.

9. A polypeptide having a sequence set forth in any one of SEQ ID NO: 3 to SEQ ID NO: 22; Mannan oligosaccharides, NGI-1.

10. The polypeptide vaccine of claim 9, wherein the sequence of the polypeptide is set forth in SEQ ID NO: 3 or SEQ ID NO:

4.

11. A polypeptide for use in treating cancer, characterized in that the sequence is set forth in any one of SEQ ID NO: 3 to SEQ ID NO: 22.

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