Targeted dec-205 oral vaccine and construction method and application thereof

Abstract

This invention belongs to the field of biomedical technology and discloses a method for constructing and applying a drug carrier targeting DEC-205. Specifically, it discloses a DEC-205 affinity peptide, which includes a polypeptide or its modified form with an amino acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:3. This invention proposes using DEC-205 as a drug delivery target and, through a series of modifications, obtains a DEC-205-targeting polypeptide (i.e., a DEC-205 affinity peptide) resistant to gastrointestinal enzyme degradation. This improves the targeting ability of the modified carrier, enabling it to efficiently target the DEC-205 receptor on the surface of dendritic cells (DCs), promoting antigen endocytosis by DCs, and enabling cross-presentation of antigens to CD4. + T and CD8 + T cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an oral vaccine targeting DEC-205, its construction method, and its application. Background Technology

[0002] Dendritic cells (DCs) are widely distributed in the lamina propria of the intestine and the gut-associated lymphoid system. Targeting DCs in the intestine allows for the direct delivery of more antigens to antigen-presenting cells, thereby triggering a subsequent specific immune response. Furthermore, existing targeted vaccine vectors or oral targeted vaccine vectors often employ modified targeting peptides or antibodies to achieve targeting. While encapsulating antigens in a vector and then administering them orally can protect the antigens from degradation by gastrointestinal enzymes, it cannot protect the targeting peptides or antibodies attached to the vector. In particular, antibodies cannot be delivered orally, and naturally occurring amino acids are also unable to resist gastrointestinal enzyme degradation. Therefore, there is a need for a method that can target a higher proportion and more efficient number of antigen-presenting cells in the intestine to trigger an immune response, while also protecting them from the disruptive pH environment and various enzymatic degradation in the gastrointestinal tract. Summary of the Invention

[0003] The first aspect of the present invention is to provide a DEC-205 affinity peptide.

[0004] A second aspect of the present invention is to provide an immunogenic conjugate.

[0005] A third aspect of the present invention aims to provide biomaterials related to the DEC-205 affinity peptide of the first aspect of the present invention or the immunogenic conjugate of the second aspect of the present invention.

[0006] The fourth aspect of this invention aims to provide the application of the affinity peptide of the first aspect of this invention, the immunogenic conjugate of the second aspect of this invention, or the biomaterial of the third aspect of this invention.

[0007] The fifth aspect of this invention aims to provide a reagent, kit, or drug.

[0008] The sixth aspect of this invention aims to provide a nano-vaccine.

[0009] The seventh aspect of this invention is to provide a method for preparing a nano-vaccine according to the fifth aspect of this invention.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] In a first aspect, the present invention provides a DEC-205 affinity peptide, said affinity peptide comprising a polypeptide or a modified form thereof with an amino acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:3.

[0012] In some embodiments of the present invention, the amino acid configuration of the affinity peptide is independently selected from D-type or L-type.

[0013] In some embodiments of the present invention, the amino acids at positions 3 and 4 of the amino acid sequence shown in SEQ ID NO:2 are L-type, and the rest are D-type. That is, the amino acid sequence is hlHkHwlr( D His- D Leu- L His- D Lys- L His- D Trp- D Leu- D Arg), where uppercase letters represent the abbreviations for naturally occurring amino acids and lowercase letters represent the abbreviations for D-configuration amino acids.

[0014] In some embodiments of the present invention, the amino acids at positions 5 and 6 of the amino acid sequence shown in SEQ ID NO:3 are L-type, and the rest are D-type. That is, the amino acid sequence is hlwkHHlr( D His- D Leu- D Trp- D Lys- L His- L His- D Leu- D Arg), where uppercase letters represent the abbreviations for naturally occurring amino acids and lowercase letters represent the abbreviations for D-configuration amino acids.

[0015] In some embodiments of the present invention, the modification sites of the modified form include N-terminal modification, C-terminal modification, backbone modification, side chain modification, and / or amino acid modification. The modification is an artificially induced drug, performed while preserving peptide activity. The purposes of the modification include prolonging half-life, increasing water solubility, and reducing or eliminating toxic side effects.

[0016] In some preferred embodiments of the present invention, the modifications include, but are not limited to, one or more of the following aspects:

[0017] (1) Cyclic peptide synthesis: head-to-tail cyclization, side chain cyclization (lactone, lactam, ether bond, etc.), multiple disulfide bonds, monothioether cyclization, etc.

[0018] (2) Isotope labeling: 13 C, 15 N, 18 O isotope labeling;

[0019] (3) Polyethylene glycol (PEG) modification: PEG2, PEG4, PEG8, PEG12, PEG24, PEG36, PEG2000, PEG5000, PEG3400, PEG20K, PEG40K and other modifications;

[0020] (4) Phosphorylation modification: phosphorylation modification of L-configuration or D-configuration amino acids (such as threonine T, serine S), phosphorylation modification of single or multiple amino acids;

[0021] (5) Coupling proteins or other carriers:

[0022] a. Peptide-protein coupling: KLH coupling, albumin coupling (such as bovine serum albumin BSA, chicken ovalbumin OVA, mouse serum albumin MSA, human serum albumin HSA), albumin affinity peptide coupling, tumor homing peptide coupling, membrane-penetrating peptide coupling, antibody Fc fragment coupling, elastin-like coupling, etc.

[0023] b. Peptide-nanocarrier coupling;

[0024] (6) Modification of N-terminal or side chain amino acids: acetylation, formylation, biotin labeling, trifluoroacetylation, benzoylation, 2-aminobenzoylation, maleimideation, chloroacetylation, bromoacetylation, succinylation, palmitation, malication, fatty acidation, formaldehydeation, chelation reactions (such as Hynic, DTPA, DOTA, Nota modification), chlorination, fluorination, bromination, nitro or methoxy substitution, fluorescent labeling (such as Cy series, Texas series, Alexa series, Rhodamine, Bodipy, Rox, FAM, FITC, MCA, TAMRA, Dnp), PAS modification, etc.

[0025] (7) C-terminal modification: amidation, esterification, aldehydeation, alcoholation, succinylation, fluorescent labeling (such as Cy series, rhodamine, AMC, AFC, PNA, CMK, FMK, etc.)

[0026] (8) Alkylation modification: N-methylation, side chain methylation, N-ethylation, N-phenylpropylation, N-allylation, etc.;

[0027] (9) Radionuclide modification: modification with radionuclides such as 125I, 131I, 18F, 99mTc, 68Ga, 64Cu, 67Ga, 90Y, 111In, 177Lu, 89Zr, etc.

[0028] (10) Other special modifications: glycopeptides, sulfonation, MAPS, etc.

[0029] In some preferred embodiments of the present invention, the modification is C-terminal PEG4 modification and N-terminal -NH2 acetylation modification.

[0030] In some embodiments of the present invention, the DEC-205 affinity peptide is prepared by chemical synthesis. Chemical synthesis methods include solid-phase synthesis (such as the Fmoc method), liquid-phase synthesis, and solid-liquid-phase synthesis. Synthetic strategies include C-terminal synthesis, N-terminal synthesis, and segmented synthesis. If conventional modifications are performed at the C-terminus or N-terminus during synthesis, the peptide contains modifying groups.

[0031] The DEC-205 affinity peptide provided by this invention can target a higher proportion and more efficient antigen-presenting cells in the gut, namely DEC-205. + DCs can efficiently deliver antigens to DCs in the gut. The endocytic receptor DEC-205 promotes the endocytosis of antigens by mature DCs and their presentation to T cells, triggering an immune response.

[0032] A second aspect of the present invention provides an immunogenic conjugate comprising the DEC-205 affinity peptide of the first aspect of the present invention.

[0033] In some preferred embodiments of the present invention, the immunogenic conjugate is an antigen conjugate, obtained by conjugating a target antigen with a DEC-205 affinity peptide. The target antigen is derived from humans, mice, or pathogenic microorganisms (including viruses, bacteria, mycoplasma, chlamydia, fungi, etc.). The target antigen can be endogenous or exogenous.

[0034] In some preferred embodiments of the present invention, the target antigen is a tumor-associated (including tumor-specific) antigen protein or antigen peptide.

[0035] In some preferred embodiments of the present invention, the target antigen is located at the N-terminus or C-terminus of the DEC-205 affinity peptide.

[0036] In some embodiments of the present invention, the target antigen is coupled to the DEC-205 affinity peptide via a linker. The linker can be (GGGS)nC, (GGSC)n, or (GGG)n, where n ≥ 1 and n is an integer.

[0037] A third aspect of the invention provides a biomaterial related to the DEC-205 affinity peptide of the first aspect of the invention or the immunogenic conjugate of the second aspect of the invention, said biomaterial being any one of a1) to a12):

[0038] a1) A nucleic acid molecule encoding the DEC-205 affinity peptide of the first aspect of the present invention or the immunogenic conjugate of the second aspect of the present invention;

[0039] a2) An expression cassette containing the nucleic acid molecule described in a1);

[0040] a3) A recombinant vector containing the nucleic acid molecules described in a1);

[0041] a4) A recombinant vector containing the expression cassette described in a2);

[0042] a5) Recombinant microorganisms containing the nucleic acid molecules described in a1);

[0043] a6) Recombinant microorganisms containing the expression cassette described in a2);

[0044] a7) Recombinant microorganisms containing the recombinant vector described in a3);

[0045] a8) Recombinant microorganisms containing the recombinant vector described in a4);

[0046] a9) Transgenic animal cell lines containing the nucleic acid molecules described in a1);

[0047] a10) Transgenic animal cell lines containing the expression cassette described in a2);

[0048] a11) Transgenic animal cell lines containing the recombinant vector described in a3);

[0049] a12) Transgenic animal cell lines containing the recombinant vector described in a4).

[0050] In some embodiments of the present invention, the transgenic animal cell line does not contain reproductive material.

[0051] In some embodiments of the present invention, the vector includes a promoter that is operatively linked to the nucleic acid molecule.

[0052] In some embodiments of the present invention, the vector is independently selected from non-pathogenic viral vectors and non-viral vectors.

[0053] In some embodiments of the present invention, the non-pathogenic viral vector includes an adenovirus vector or a retrovirus vector.

[0054] In some embodiments of the present invention, the non-viral vector includes a plasmid vector.

[0055] In some embodiments of the present invention, the vector is a plasmid vector, a phage particle, a viral vector, a cell vector, a bacteriophage, a sclerotium, an F sclerotium, or an artificial chromosome.

[0056] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, and the viral vector may be an optional virus.

[0057] A fourth aspect of the present invention provides the use of at least one of (1) to (10) of the DEC-205 affinity peptide of the first aspect of the present invention, the immunogenic conjugate of the second aspect of the present invention, or the biomaterial of the third aspect of the present invention:

[0058] (1) Affinity to DEC-205 protein;

[0059] (2) Prepare products for the prevention and / or treatment of tumors, pathogenic microbial infections or autoimmune diseases;

[0060] (3) Prepare products for in vitro and in vivo targeting of DEC-205;

[0061] (4) Qualitative and / or quantitative detection of DEC-205 protein expression, expression location or expression level;

[0062] (5) Prepare products for qualitative and / or quantitative detection of whether DEC-205 protein is expressed, its expression location, or its expression level;

[0063] (6) Prepare products that promote the endocytosis of drugs by dendritic cells;

[0064] (7) Promotes the maturation and / or cross-presentation of dendritic cells;

[0065] (8) Prepare products that promote the maturation and / or cross-presentation of dendritic cells;

[0066] (9) Enhance the killing ability of T cells against tumor cells;

[0067] (10) Prepare products that enhance the ability of T cells to kill tumor cells.

[0068] In some embodiments of the present invention, the DEC-205 protein described in (1) may be a wild-type human or mouse protein or a mutant protein that still retains its activity.

[0069] In some embodiments of the present invention, the tumor described in (2) is a solid tumor, including endometrial cancer, hepatocellular carcinoma, colonic adenocarcinoma, rectal adenocarcinoma, pancreatic cancer, lung adenocarcinoma, esophageal cancer, breast cancer, low-grade glioma, renal papillary cell carcinoma, myofibroblastoma, neuromuscular vagus tumor, desmoidoma, juvenile angiofibroma, enamel craniopharynx carcinoma, hepatoblastoma, pancreatic ductal carcinoma, biliary tract cancer, cerebral hemangioma, Merkel cell carcinoma of the skin, squamous cell carcinoma of the skin, melanoma in situ, and basal cell carcinoma sarcoma of the skin.

[0070] In some embodiments of the present invention, the product includes at least one of a drug, a reagent, a kit, and a probe.

[0071] A fifth aspect of the present invention provides a reagent, kit, or drug comprising the DEC-205 affinity peptide of the first aspect of the present invention, the immunogenic conjugate of the second aspect of the present invention, or the biomaterial of the third aspect of the present invention.

[0072] In some embodiments of the present invention, the reagents include immunoassay and / or diagnostic reagents, molecular assay and / or diagnostic reagents, etc. The reagents can bind to the DEC-205 protein and are used to detect the affinity of the analyte for the DEC-205 protein, or to qualitatively, quantitatively, or locally detect the expression, level, or location of the DEC-205 protein in biological samples.

[0073] In some embodiments of the present invention, the immunoassay and / or diagnostic reagent further comprises one or more of the following: sample diluent, ELISA plate, blocking solution, washing solution, substrate, stop solution, negative control, positive control, etc.

[0074] In some embodiments of the present invention, the molecular detection and / or diagnostic reagent further comprises one or more of polymerase (RNA- and / or DNA-dependent polymerase), amplification reaction buffer, positive control, negative control, etc.

[0075] In some embodiments of the present invention, the DEC205 affinity peptide or immunogenic conjugate in the reagent, kit, or drug may be present in a free form or in the form of a pharmaceutically acceptable salt thereof.

[0076] In some embodiments of the present invention, the drug includes preventive and / or therapeutic vaccine drugs, cell preparations, etc. The drug can target the DEC205 protein in vitro and in vivo, deliver antigens (and other combined drugs), enhance the immune response induced by the antigen, and be used for anti-tumor or anti-pathogenic microbial infection.

[0077] In some embodiments of the present invention, the vaccine is a nucleic acid vaccine, including DNA vaccines and RNA vaccines; or, the vaccine is a polypeptide vaccine, recombinant protein vaccine, synthetic long peptide vaccine, mixed peptide pool vaccine, nanovaccine, etc.

[0078] In some embodiments of the present invention, the cell preparation is a DC cell preparation, an antigen-specific T cell preparation, etc.

[0079] In some embodiments of the present invention, the drug may also contain other pharmacologically active ingredients, thereby achieving combined treatment by using it in combination with other drugs.

[0080] In some embodiments of the present invention, the medicament may further comprise a pharmaceutically acceptable adjuvant or immunomodulator, selected from poly-ICLC, 1018ISS, Amplivax, MF59, AS03, AS04, AS15, BCG, CP-870, CP-893, CpG7909, CyaA, cyclic dinucleotides (such as STING), dSLIM, GM-CSF, IL-2, IC30, IC31, MontanideISA51, etc.

[0081] In some embodiments of the present invention, the drug can be administered topically at a pharmaceutically acceptable dose.

[0082] In some embodiments of the present invention, the dosage form of the drug is selected from tablets (such as ordinary tablets, bilayer tablets, multilayer tablets, sustained-release tablets, single-compartment controlled-release tablets, dual-compartment controlled-release tablets, microporous controlled-release tablets, sublingual tablets, orally disintegrating tablets, dispersible tablets, enteric-coated tablets), pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules (such as ordinary capsules, sustained-release capsules, controlled-release capsules, capsules containing microcapsules or small pieces, pH-dependent capsules containing microcapsules or small pieces, gastrointestinal compound capsules), suppositories, injections, sprays, and injections.

[0083] In some embodiments of the present invention, the drug further includes pharmaceutically acceptable excipients.

[0084] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one selected from fillers, disintegrants, diluents, dispersants, excipients, stabilizers, lubricants, binders, humectants, flavoring agents, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, fragrances, and solvents.

[0085] In some embodiments of the present invention, the filler is selected from starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, or glucose, etc.; the binder is selected from cellulose derivatives, alginate, starch, water, dextrin, gelatin, or polyvinylpyrrolidone, etc.; the disintegrant is selected from microcrystalline cellulose, sodium carboxymethyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, or croscarmellose sodium; the lubricant is selected from stearic acid, polyethylene glycol, calcium carbonate, sodium bicarbonate, micronized silica gel, talc, or magnesium stearate; and the suspending agent is selected from micronized silica gel, beeswax, cellulose, or solid polyethylene glycol. The wetting agent is selected from glycerin, Tween-80, hydrogenated castor oil, or lecithin; the solvent is selected from ethanol, liquid polyethylene glycol, isopropanol, Tween-80, glycerin, propylene glycol, or vegetable oil, wherein the vegetable oil is selected from soybean oil, castor oil, peanut oil, blended oil, etc.; the surfactant is selected from sodium dodecylbenzenesulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, fatty acid sorbitan, or polysorbate (Tween), etc.; the flavoring agent is selected from aspartame, sucralose, flavoring, stevia, acesulfame potassium, citric acid, or sodium saccharin; the preservative is selected from at least one of methylparaben or propylparaben.

[0086] A sixth aspect of the present invention provides a nanovaccine comprising the DEC-205 affinity peptide of the first aspect of the present invention, chitosan, and a protein carrier.

[0087] In some embodiments of the present invention, the affinity peptide and chitosan are linked by coupling and encapsulated in a protein carrier.

[0088] In some embodiments of the present invention, the protein carrier includes at least one of hemocyanin, bovine serum albumin, ovalbumin, human serum albumin, and mouse serum albumin.

[0089] In some embodiments of the present invention, the nanovaccine also includes a drug for treating tumors.

[0090] In some embodiments of the present invention, the drug is conjugated to a protein carrier.

[0091] In some embodiments of the present invention, the immunization of the vaccine is achieved by injection via nasal cavity, oral cavity, subcutaneous, intramuscular, intravenous, intraperitoneal or dermal cavity; preferably oral (i.e., oral administration).

[0092] In some embodiments of the present invention, the tumors include endometrial cancer, hepatocellular carcinoma, colonic adenocarcinoma, rectal adenocarcinoma, pancreatic cancer, lung adenocarcinoma, esophageal cancer, breast cancer, low-grade glioma, renal papillary cell carcinoma, myofibroblastoma, neuromuscular vagus tumor, desmoidoma, juvenile angiofibroma, enamel craniopharyngioma, hepatoblastoma, pancreatic ductal carcinoma, biliary tract cancer, cerebral aneurysm, Merkel cell carcinoma of the skin, squamous cell carcinoma of the skin, melanoma in situ, and basal cell carcinoma sarcoma of the skin.

[0093] In some embodiments of the present invention, the nanovaccine may further comprise a pharmaceutically acceptable adjuvant or immunomodulator, selected from poly-ICLC, 1018ISS, Amplivax, MF59, AS03, AS04, AS15, BCG, CP-870, CP-893, CpG7909, CyaA, cyclic dinucleotides (such as STING), dSLIM, GM-CSF, IL-2, IC30, IC31, Montanide ISA51, etc.

[0094] A seventh aspect of the present invention provides a method for preparing a nano-vaccine according to the sixth aspect of the present invention, comprising the following steps:

[0095] Affinity peptides and / or drugs are activated in solutions containing NHS and EDC;

[0096] The activated product was mixed with chitosan and reacted to obtain an affinity peptide coupling carrier.

[0097] A nano-vaccine is obtained by mixing an acetic acid solution containing an affinity peptide-coupled carrier with a sodium tripolyphosphate solution containing a protein carrier.

[0098] In some embodiments of the present invention, the activation time is 1 to 2 hours; more specifically, it is 1 hour.

[0099] In some embodiments of the present invention, the mass ratio of the DEC-205 affinity peptide to the chitosan is 1:2 to 3; more specifically, it is 1:2.

[0100] In some embodiments of the present invention, the reaction time is 10-13 hours; further, 10-12 hours; and even further, 12 hours.

[0101] In some embodiments of the present invention, the solvent of the solution containing NHS and EDC is MES buffer.

[0102] In some embodiments of the present invention, the mass ratio of the affinity peptide coupling vector to the protein vector is 2 to 3:1.

[0103] In some embodiments of the present invention, the mixing is a stirring mixture, and the stirring time is 15 minutes.

[0104] The beneficial effects of this invention are:

[0105] This invention proposes using DEC-205 as the oral vaccine delivery target. Through a series of modifications, an oral vaccine targeting peptide (i.e., the DEC-205 affinity peptide) resistant to the pH environment of the gastrointestinal tract and degradation by various enzymes was obtained, thus providing protection for the targeting vector. This DEC-205 affinity peptide can efficiently target the DEC-205 receptor on the surface of dendritic cells (DCs), promoting in situ endocytosis of antigens by intestinal DCs, enabling antigen presentation and cross-presentation to CD4. + T and CD8 + T cells.

[0106] This invention conjugates an immunogenic conjugate by coupling the DEC-205 affinity peptide with a target antigen, and then prepares it into a drug for the prevention and treatment of tumors, pathogenic microorganism infections or autoimmune diseases, or a detection and diagnostic reagent, which has high clinical application value in the field of immunoprophylaxis and treatment.

[0107] Modifying the DEC-205 affinity peptide provided by this invention onto an oral carrier can construct a highly efficient and practical oral targeted vaccine platform. Encapsulating different antigens can stimulate the body to have a highly efficient immune response to the antigen. For example, this invention conjugates DEBP-8 with CS to obtain a simple and efficient oral targeted vaccine material (nano-vaccine). This nano-vaccine is protected from damage by the pH environment of the gastrointestinal tract and degradation by various enzymes. It can deliver antigens orally and provide protection to the body. Attached Figure Description

[0108] Figure 1The data shows the cleavage of the DEC-205 targeting peptide in simulated intestinal and gastric fluids. In A, the original DEC-205 targeting peptide cleaved at the lysine (K) site after being treated in simulated intestinal fluid for half an hour. In B, the D-hr8 peptide obtained by replacing the natural conformation lysine (K) with the D conformation lysine (k) cleavage in simulated intestinal fluid is shown.

[0109] Figure 2 These are important amino acid sites for interaction between the polypeptide and mDEC-205.

[0110] Figure 3 The results of affinity assays were used to screen mutant peptides.

[0111] Figure 4 The figure shows the endocytosis capacity of BMDC for different peptides. In the figure, ## indicates p < 0.01.

[0112] Figure 5 This is a schematic diagram of the synthesis of DEBP-8-CS.

[0113] Figure 6 The figure shows the effect of DEBP-8 on the internalization of nanoparticles in mature BMDC. In the figure, * represents p < 0.05 and *** represents p < 0.001.

[0114] Figure 7 The enrichment of OVA@DEBP-8-CNP in the lamina propria and Pierre's knots of the mouse intestine.

[0115] Figure 8 OVA@DEBP-8-CNP can prevent tumor growth in mice. Detailed Implementation

[0116] The present invention will be further described in detail below through specific embodiments.

[0117] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0118] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0119] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0120] anti-DEC-205 (anti-CD205), source: eBioscience, catalog number: 12-2051-82; anti-CD11c, source: eBioscience, catalog number: 17-0114-82; DAPI: using anti-fluorescence quenching mounting medium (containing DAPI), source: Servicebio, catalog number: G1407-25ML.

[0121] Example 1: Peptide Modification

[0122] In this embodiment, the DEC-205 targeting peptide (sequence: hlHKHHlr (SEQ ID NO:1), where the lowercase single letter represents the corresponding amino acid with D configuration) was modified into a polypeptide DEBP-8 that is resistant to gastrointestinal enzyme degradation and has high affinity for DEC-205.

[0123] The specific process is as follows:

[0124] 1. Liquid chromatography-mass spectrometry (LC-MS) analysis of peptide breakpoints:

[0125] (1) In order to enable the peptide to be coupled with chitosan, the C-terminus of the original DEC-205 target peptide (i.e. the peptide shown in SEQ ID NO:1) was modified with PEG4 and the N-terminus of -NH2 was acetylated during the solid-phase synthesis of the peptide.

[0126] (2) Dissolve the above peptides in 2 mL of simulated gastrointestinal fluid to about 500 μM, take 80 μL at different time points, add 360 μL of acetonitrile and 360 μL of 0.5% glacial acetic acid solution to precipitate excess protein in the simulated gastrointestinal fluid.

[0127] (3) Centrifuge the sample in a centrifuge (12000 rpm / min, 15 min), take the supernatant, filter it through a 0.22 μm filter membrane, and analyze the sample by UPLC-MS;

[0128] (4) After finding the break site, K was replaced with a D-configured k amino acid during the solid-phase synthesis of the polypeptide, and the above experiment was repeated.

[0129] The results showed that the original DEC-205 targeting peptide ① (SEQ ID NO:1) was cleaved at the lysine (K) site after half an hour of treatment in simulated intestinal fluid, breaking into two parts ② and ③, indicating that the original targeting peptide could not be taken orally. Figure 1(A). The liquid chromatography (LC) results show that the peptide eluted at 7.86 min in simulated gastric juice (SGF) at both 0 h and 12 h, indicating no degradation in gastric juice. In simulated intestinal juice (SIF), the peptide eluted at 7.85 min at 0 min, and after 30 min, peaks were observed at 2.15 min and 6.91 min. Analysis of the molecular weight using the corresponding mass spectrometry results leads to the conclusion that the peptide does not degrade in simulated gastric juice, but cleaves at the lysine (K) site in simulated intestinal juice. Replacing the native lysine with the D-configuration lysine (K) resulted in the D-hr8 peptide (amino acid sequence hlHkHHlr), which no longer cleaved in simulated intestinal juice. Figure 1 (B)

[0130] 2. To improve the affinity of the peptide for DEC-205, the D-hr8 peptide was further modified. Specifically,

[0131] (1) Synthesis of C-terminal biotin-labeled alanine scanning peptides: During the synthesis of the peptides, the amino acids at each site were replaced with alanine to obtain 8 alanine scanning peptides.

[0132] (2) Affinity experiment: 293T cells and 293T-mDEC-205 cells with high mDEC-205 expression obtained by transient transfection were collected and divided into 2×10 groups. 5 Each tube contained 10 cells and was divided into different groups, with 3 replicates per group. The control peptide and alanine scanning peptide were incubated with PBS or GA for 30 min (4℃), then washed three times with ice-cold PBS. SA-PE was added to fluorescently label the peptides bound to the cells, and the level of peptides bound to the cells was detected by flow cytometry.

[0133] result Figure 2 As shown, alanine scanning of the peptide reveals important interaction sites between the peptide and mDEC-205. Replacing amino acids at different sites on the peptide with alanine significantly reduces the affinity of the peptide for the target, indicating these are crucial interaction sites. Specifically, replacing amino acids at positions 4, 5, 6, and 8 greatly decreases the specific affinity of the peptide, demonstrating that these four amino acids are important binding sites for mDEC-205.

[0134] (3) MOE running molecular dynamics: First, the structure of the peptide was predicted in PEP-FOLD. The peptide was then molecularly docked with hDEC-205 protein (PDB ID: 7jpt) using MOE and molecular dynamics was run. Finally, the binding mode closest to the experimental results was selected to run the single-point amino acid mutation.

[0135] (4) MOE performs single amino acid site mutation, that is, MOE replaces the amino acid at each site with the remaining 19 amino acids and their D conformation, and scores the binding of the mutant peptide to the target site. Finally, the peptide with the higher score is selected and synthesized for affinity experiment (Table 1) to screen out our final high affinity and specific endocytosis-resistant anti-gastrointestinal enzyme degradation peptide.

[0136] Table 1. Software affinity scores (dAffinity) and molecular weights of mutant peptides.

[0137]

[0138] See results Figure 3 As shown, the sixth amino acid was changed from the L-configuration His to the D-configuration Trp, which significantly improved its affinity.

[0139] In summary, through two steps of modification—replacing the 4th amino acid K with k and changing the 6th amino acid from the L configuration H to the D configuration w—a high-affinity mDEC-205 affinity peptide was finally obtained: hlHkHwlr (SEQ ID NO:2), denoted as DEBP-8.

[0140] 3. To verify the endocytic activity of peptides in BMDCs, an endocytosis experiment was performed using BMDC cells. Specifically, the leg bones of 6-week-old C57BL / 6 mice were expelled, and GM-CSF (20 ng / mL) and IL-4 (10 ng / mL) were added to induce BMDCs. The BMDCs induced on day 5 were expelled and seeded in confocal dishes at a density of 2 × 10⁶ cells / mL. 5 After the cells adhered to the culture medium, the DMEM complete medium was replaced with serum-free DMEM. 50 μM peptide was added and co-incubated with the cells (37℃, 30 min). After half an hour, the culture medium was removed, and the peptide on the cell surface was washed off with PBS. Then, DAPI was stained, the cells were fixed and perforated, and SA-PE labeled peptide was added. Then, confocal microscopy was used to observe the endocytosis of peptides in the cells.

[0141] Experimental results are as follows Figure 4 As shown, BMDC can rapidly and massively endocytose the modified peptide DEBP-8, while it can only endocytose a very small amount of the original DEC-205 peptide within half an hour.

[0142] In summary, through modification, an orally administered, high-affinity mDEC-205 affinity peptide, DEBP-8, was obtained.

[0143] Example 2: Construction of an oral chitosan nanovaccine

[0144] In this embodiment, an oral chitosan nanovaccine was constructed by conjugating peptides with chitosan and encapsulating OVA protein using an iontophoresis method. The experimental procedure is as follows:

[0145] (1) Solid-phase synthesis of C-terminal PEG4-modified and N-terminal acetylated DEBP-8.

[0146] (2) Dissolve 5 mg of the polypeptide synthesized in step (1) together with NHS and EDC in MES buffer, activate the carboxyl group for 1 h, then add the reaction product to 10 mg of chitosan (CS) aqueous solution and react for 12 h (process as follows). Figure 5 ), thus obtaining DEBP-8-CS solution.

[0147] (3) The above reaction product was ultrafiltered with an ultrafiltration tube with a molecular weight cutoff of 10kD to remove unreacted peptides and other molecules. The product was then lyophilized and used for infrared spectroscopy scanning to characterize the peptide coupling.

[0148] (4) Dissolve 5 mg of CS or DEBP-8-CS in 2.5 mL of 0.5% acetic acid solution. Mix 2 mg of OVA protein with 1 mL of 1.67 mg / mL sodium tripolyphosphate (TPP) solution in advance. Then, while stirring, drop the TPP and OVA protein mixture into the CS or DEBP-8-CS solution and stir for 15 min to obtain OVA@CNP and OVA@DEBP-8-CNP.

[0149] Example 3

[0150] This embodiment investigates whether DEBP-8 can effectively promote the endocytosis of nanoparticles by BMDCs and promote the endocytosis of antigens at the Peyre's knot site in the intestine. Specifically:

[0151] 1. BMDC internalization of nanoparticles

[0152] Rhodamine B was labeled onto the OVA protein using the NHS / EDC reaction described in Example 2, and then encapsulated into CNP and DEBP-CNP, following the same method as in Example 2. This yielded OVA-Rhodamine@CNP and OVA-Rhodamine@DEBP-8-CNP.

[0153] Bone marrow was expelled from the legs of C57BL / 6 mice and GM-CSF and IL-4 were added to induce BMDCs. On day 5 of induction, 1 μg / mL LPS was added, and mature BMDCs were obtained on day 6. The cells were blown off and plated in confocal glass dishes. OVA-Rhodamine@CNP and OVA-Rhodamine@DEBP-8-CNP containing 10 μg / mL LOVA protein were added. After endocytosis for different time periods, the un-endocytosed nanoparticles were washed away with PBS. After Hoechst staining, the endocytosis of nanoparticles in BMDCs was observed by confocal microscopy. The statistical graph shows the fluorescence intensity of OVA-Rhodamine in BMDC cells.

[0154] The results are as follows Figure 6 As shown, after modification with DEBP-8, mature BMDC is able to engulf more nanoparticles.

[0155] 2. Paecil Test

[0156] C57BL / 6 mice were anesthetized, and the portion of the intestine containing Parsley's nodes was removed. One end was ligated with surgical sutures, and OVA-Rhodamine@CNP and OVA-Rhodamine@DEBP-8-CNP were injected into the small intestine. The other end was ligated, and the mice were sacrificed after 1 hour. The intestinal portion was removed, fixed in paraformaldehyde, and fixed sections were prepared. After staining with DAPI and UEA-1-Lectin antibody (secondary antibody was SA-APC), confocal microscopy was used to observe the phagocytosis of nanoparticles in the intestinal lamina propria and Parsley's nodes.

[0157] The results are as follows Figure 7 As shown, OVA@DEBP-8-CNP can accumulate in the lamina propria and Pascal's knot of the mouse intestine.

[0158] Example 4

[0159] This example was used to investigate whether OVA@DEBP-8-CNP could prevent C57BL / 6 mice from being challenged with B16-OVA. Details are as follows:

[0160] Six-week-old C57BL / 6 mice were randomly divided into four groups. Initially, mice were orally immunized weekly. Group 1 was the NS control group; Group 2 received oral administration of 100 μg OVA protein; Group 3 received oral administration of OVA@CNP containing 100 μg OVA protein; and Group 4 received oral administration of OVA@DEBP-8-CNP containing 100 μg OVA protein. After four oral immunizations, 2 × 10⁻⁶ mg of OVA was subcutaneously injected into the right posterior back of each mouse. 5 We used B16-OVA cells to observe tumor growth in mice. The results showed that OVA@DEBP-8-CNP effectively prevented the growth of B16-OVA tumors in mice. Figure 8 ).

[0161] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A DEC-205 affinity peptide, wherein the amino acid sequence of the affinity peptide is hlHkHwlr, where uppercase letters represent abbreviations of native amino acid configurations and lowercase letters represent abbreviations of D-configuration amino acids.

2. The use of the affinity peptide of claim 1 in the preparation of products for qualitative and / or quantitative detection of whether DEC-205 protein is expressed, its expression location, or its expression level.

3. A reagent, kit, or drug comprising the affinity peptide of claim 1.

4. The reagent, kit, or drug according to claim 3, characterized in that, The drugs also include those used to treat tumors.

5. A nanovaccine comprising the affinity peptide, chitosan, and protein carrier as described in claim 1; The affinity peptide and chitosan are linked by coupling and encapsulated in a protein carrier; The protein carrier is ovalbumin; The nanovaccine also includes drugs for treating tumors; The drug is conjugated to a protein carrier; The vaccine is administered via nasal, oral, subcutaneous, intramuscular, intravenous, intraperitoneal, or intradermal injection.

6. The method for preparing the nano-vaccine according to claim 5, comprising the following steps: Affinity peptides and / or drugs are activated in solutions containing NHS and EDC; The activated product was mixed with chitosan and reacted to obtain an affinity peptide coupling carrier. A nano-vaccine is obtained by mixing an acetic acid solution containing an affinity peptide-coupled carrier with a sodium tripolyphosphate solution containing a protein carrier.

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