Targeted DEC-205 oral vaccine as well as construction method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, discloses a construction method and application of a drug carrier targeting DEC-205, and particularly discloses a DEC-205 affinity peptide which comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 3 or a modification form of the polypeptide. The DEC-205 targeted polypeptide (namely DEC-205 affinity peptide) resistant to gastrointestinal tract enzyme degradation is obtained by taking DEC-205 as a drug delivery target and through a series of modification, the targeting property of a modified carrier can be improved, so that the modified carrier can efficiently target a DEC-205 receptor on the surface of DC, the antigen is promoted to be endocytosed by DC, and the antigen can be presented to CD4 + T and CD8 + T cells in a cross manner.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a DEC-205 targeted oral vaccine and a construction method and application thereof. Background Art

[0002] DC cells are widely present in the intestinal lamina propria and the intestinal associated lymphatic system. Targeting DC cells in the intestine enables more antigens to be directly delivered to antigen-presenting cells, thereby inducing subsequent specific immune responses. In addition, existing targeted vaccine carriers or targeted oral vaccine carriers often use modified targeting polypeptides or targeting antibodies to achieve targeting. However, using a carrier to encapsulate the antigen and then take it orally can protect the antigen from degradation by gastrointestinal enzymes, but it cannot protect the targeting polypeptide or antibody attached to the carrier, especially the antibody cannot be delivered orally, and the natural configuration amino acids cannot resist degradation by gastrointestinal enzymes. Therefore, it is necessary to provide a method that can target a higher proportion of antigen-presenting cells in the intestine and is more efficient to induce an immune response, while also being able to protect against gastrointestinal pH environmental damage and various enzyme degradation. Summary of the invention

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

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

[0005] The third aspect of the present invention aims to provide biological materials 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 purpose of the fourth aspect of the present invention is to provide the use of the 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.

[0007] The fifth aspect of the present invention aims to provide a reagent, a kit or a medicine.

[0008] The sixth aspect of the present invention aims to provide a nano vaccine.

[0009] The purpose of the seventh aspect of the present invention is to provide a method for preparing the nanovaccine of the fifth aspect of the present invention.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

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

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

[0013] In some embodiments of the present invention, the amino acids at positions 3 and 4 in 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 capital letters represent the abbreviations of natural amino acids and lowercase letters represent the abbreviations of D-configuration amino acids.

[0014] In some embodiments of the present invention, the amino acids at positions 5 and 6 in 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 capital letters represent the abbreviations of natural amino acids and lowercase letters represent the abbreviations of 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 modified form is an artificial drug, which is carried out under the premise of retaining the activity of the polypeptide. The purpose of modification includes extending half-life, increasing water solubility, reducing or eliminating toxic side effects, etc.

[0016] In some preferred embodiments of the present invention, the modified forms 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, monosulfide cyclization, etc.;

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

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

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

[0021] (5) Coupling protein or other carrier:

[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, maleimidation, chloroacetylation, bromoacetylation, succinylation, palmitation, malication, fatty acidation, formalization, chelation reaction (such as Hynic, DTPA, DOTA, NOTA modification), chloro, fluoro, bromo, 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, aldehyde formation, alcoholization, 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-phenylpropanylation, N-allylation, etc.;

[0027] (9) Radionuclide modification: 125I, 131I, 18F, 99mTc, 68Ga, 64Cu, 67Ga, 90Y, 111In, 177Lu, 89Zr and other radionuclide modification;

[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 Fmoc method), liquid phase synthesis, solid-liquid phase synthesis. Synthesis strategies include C-terminal synthesis, N-terminal synthesis, and segmented synthesis. If conventional modification is performed at the C-terminal or N-terminal during synthesis, the polypeptide contains a modified group.

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

[0032] The 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 coupling the target antigen with the DEC-205 affinity peptide. The target antigen is derived from humans, mice or pathogenic microorganisms (including viruses, bacteria, mycoplasmas, chlamydia, fungi, etc.). The target antigen can be an endogenous or exogenous antigen.

[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 may be (GGGS)nC, (GGSC)n or (GGG)n, wherein n≥1 and n is an integer.

[0037] The third aspect of the present invention provides a biological material 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, wherein the biological material is any one of a1) to a12):

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

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

[0040] a3) a recombinant vector containing the nucleic acid molecule described in a1);

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

[0042] a5) a recombinant microorganism containing the nucleic acid molecule described in a1);

[0043] a6) a recombinant microorganism containing the expression cassette described in a2);

[0044] a7) a recombinant microorganism containing the recombinant vector described in a3);

[0045] a8) a recombinant microorganism containing the recombinant vector described in a4);

[0046] a9) a transgenic animal cell line containing the nucleic acid molecule described in a1);

[0047] a10) a transgenic animal cell line containing the expression cassette described in a2);

[0048] a11) a transgenic animal cell line containing the recombinant vector described in a3);

[0049] a12) A transgenic animal cell line containing the recombinant vector described in a4).

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

[0051] In some embodiments of the present invention, the vector comprises a promoter, and the promoter is operably linked to the nucleic acid molecule.

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

[0053] In some embodiments of the present invention, the non-pathogenic viral vector comprises an adenoviral vector or a retroviral vector.

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

[0055] In some embodiments of the present invention, the vector is a plasmid vector, a phagemid, a viral vector, a cell vector, a bacteriophage, a cosmid, a F cosmid, 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] The fourth aspect of the present invention provides the use 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 in at least one of (1) to (10):

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

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

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

[0061] (4) qualitatively and / or quantitatively detecting whether the DEC-205 protein is expressed, its expression location or its expression level;

[0062] (5) preparing products for qualitatively and / or quantitatively detecting whether DEC-205 protein is expressed, where it is expressed, or how much it is expressed;

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

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

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

[0066] (9) Improve the ability of T cells to kill 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) can be a wild-type protein of human or mouse origin, 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, colon adenocarcinoma, rectal adenocarcinoma, pancreatic cancer, lung adenocarcinoma, esophageal cancer, breast cancer, low-grade brain glioma, renal papillary cell carcinoma, myofibroblastoma, neuromuscular aberrant tumor, desmoid tumor, juvenile angiofibroma, enamel-type craniopharyngiocarcinoma, hepatoblastoma, pancreatic ductal carcinoma, biliary tract cancer, cerebral hemangioma, skin Merkel cell carcinoma, skin squamous cell carcinoma, melanoma in situ, and skin basal cell carcinoma sarcoma.

[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] The fifth aspect of the present invention provides a reagent, a kit or a medicine, 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 be affinity-bound to the DEC-205 protein, and used to detect the affinity of the test substance to the DEC-205 protein, or to qualitatively, quantitatively or positionally detect whether the DEC-205 protein is expressed, the expression amount or the expression position in the biological sample.

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

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

[0075] In some embodiments of the present invention, the DEC205 affinity peptide or immunogenic conjugate in the reagent, kit or drug may exist 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 DEC205 protein in vivo or in vitro, deliver antigens (and other combined drugs), enhance the immune response caused by antigens, 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 a DNA vaccine and an RNA vaccine; or, the vaccine is a polypeptide vaccine, a recombinant protein vaccine, a synthetic long peptide vaccine, a mixed peptide pool vaccine, a nano vaccine, etc.

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

[0079] In some embodiments of the present invention, the drug may further contain other pharmacological ingredients, and combined therapy may be achieved by combining the drug with other drugs.

[0080] In some embodiments of the present invention, the drug 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, and the dosage administered is a pharmaceutically acceptable dosage.

[0082] In some embodiments of the present invention, the dosage form of the drug is selected from tablets (such as ordinary tablets, double-layer tablets, multi-layer tablets, sustained-release tablets, single-chamber controlled-release tablets, double-chamber controlled-release tablets, microporous controlled-release tablets, sublingual tablets, orally rapidly 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 micropellets or small tablets, pH-dependent capsules containing micropellets or small tablets, gastrointestinal composite capsules), suppositories, injections, sprays and injections.

[0083] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0084] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of fillers, disintegrants, diluents, dispersants, excipients, stabilizers, lubricants, binders, wetting agents, flavoring agents, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, flavors 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; the binder is selected from cellulose derivatives, alginate, starch, water, dextrin, gelatin or polyvinyl pyrrolidone; the disintegrant is selected from microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose or cross-linked sodium carboxymethyl cellulose; the lubricant is selected from stearic acid, polyethylene glycol, calcium carbonate, sodium bicarbonate, micro-powder silica gel, talc or magnesium stearate; the suspending agent is selected from micro-powder silica gel, beeswax, cellulose, solid polyethylene glycol : The wetting agent is selected from glycerol, Tween-80, hydrogenated castor oil or lecithin; the solvent is selected from ethanol, liquid polyethylene glycol, isopropanol, Tween-80, glycerol, propylene glycol or vegetable oil, and the vegetable oil is selected from soybean oil, castor oil, peanut oil, blending oil, etc.; the surfactant is selected from sodium dodecylbenzene sulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, fatty acid sorbitan or polysorbate (Tween), etc.; the flavoring agent is selected from aspartame, sucralose, flavor, stevioside, acesulfame potassium, citric acid or saccharin sodium; the preservative is selected from at least one of methyl paraben or propyl paraben.

[0086] The sixth aspect of the present invention provides a nano vaccine, 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 are 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 coupled to a protein carrier.

[0091] In some embodiments of the present invention, the immunization of the vaccine includes nasal, oral, subcutaneous, intramuscular, intravenous, intraperitoneal or intradermal injection; preferably oral (ie, oral).

[0092] In some embodiments of the present invention, the tumor includes endometrial cancer, hepatocellular carcinoma, colon adenocarcinoma, rectal adenocarcinoma, pancreatic cancer, lung adenocarcinoma, esophageal cancer, breast cancer, low-grade brain glioma, renal papillary cell carcinoma, myofibroblastoma, neuromuscular aberrant tumor, desmoid tumor, juvenile angiofibroma, enamel-type craniopharyngiocarcinoma, hepatoblastoma, pancreatic ductal carcinoma, biliary tract cancer, cerebral hemangioma, skin Merkel cell carcinoma, skin squamous cell carcinoma, melanoma in situ, and skin basal cell carcinoma sarcoma.

[0093] In some embodiments of the present invention, the nanovaccine may also contain 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] The seventh aspect of the present invention provides a method for preparing the nanovaccine of the sixth aspect of the present invention, comprising the following steps:

[0095] The affinity peptide and / or drug is activated in a solution containing NHS and EDC;

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

[0097] The 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; further, 1 hour.

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

[0100] In some embodiments of the present invention, the reaction time is 10 to 13 hours; further 10 to 12 hours; 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 carrier to the protein carrier is 2 to 3:1.

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

[0104] The beneficial effects of the present invention are:

[0105] The present invention proposes to use DEC-205 as the oral vaccine delivery target, and through a series of modifications, obtains an oral vaccine targeting polypeptide (i.e., DEC-205 affinity peptide) that is resistant to gastrointestinal pH environment and various enzyme degradation, thereby providing protection for the targeting carrier. The DEC-205 affinity peptide can efficiently target the DEC-205 receptor on the surface of DC, promote the in situ endocytosis of antigens by intestinal DCs, and enable the antigens to be presented and cross-presented to CD4 + T and CD8 + T cells.

[0106] The present invention couples the DEC-205 affinity peptide with the target antigen to prepare an immunogenic conjugate, and prepares the conjugate into a drug for preventing and treating tumors, pathogenic microorganism infections or autoimmune diseases, or a detection and diagnostic reagent, which has high clinical application value in the field of immune prevention and treatment.

[0107] Modifying the DEC-205 affinity polypeptide provided by the present invention on an oral carrier can construct an efficient and practical oral targeted vaccine platform. Encapsulating different antigens can stimulate the body's efficient immunity to the antigen. For example, the present invention couples DEBP-8 with CS to obtain a simple and efficient oral targeted vaccine material (nano vaccine). The nano vaccine can protect it from gastrointestinal pH environment damage and various enzyme degradations, and can deliver antigens orally to provide protection to the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Figure 1The figure shows the breakage of the DEC-205 targeting peptide in simulated intestinal fluid and gastric fluid; wherein A shows the breakage of the original DEC-205 targeting peptide at the lysine (K) site after being treated in simulated intestinal fluid for half an hour, and B shows the breakage of the D-hr8 polypeptide obtained by replacing the natural configuration lysine (K) with the D configuration lysine (k) in simulated intestinal fluid.

[0109] Figure 2 It is an important amino acid site for interaction between the peptide and mDEC-205.

[0110] Figure 3 Results of affinity screening experiments for mutant peptides.

[0111] Figure 4 The endocytosis ability of BMDC to different polypeptides. In the figure, ## indicates p<0.01.

[0112] Figure 5 Schematic diagram of the synthesis of DEBP-8-CS.

[0113] Figure 6 This is the promotion of DEBP-8 in the endocytosis of nanoparticles in mature BMDCs. In the figure, * represents p < 0.05, *** represents p < 0.001.

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

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

[0116] The present invention is further described in detail below through specific examples.

[0117] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0118] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0119] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

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

[0121] Example 1 Polypeptide modification

[0122] In this example, the DEC-205 targeting peptide (sequence: hlHKHHlr (SEQ ID NO: 1), lowercase single letters represent that the corresponding amino acid is in D configuration) is transformed into a polypeptide, DEBP-8, which is resistant to gastrointestinal enzyme degradation and has high affinity for DEC-205.

[0123] The specific process is as follows:

[0124] 1. LC-MS analysis of peptide cleavage sites:

[0125] (1) In order to enable the polypeptide to be coupled to chitosan, during the solid phase synthesis of the polypeptide, the C-terminal PEG4 modification and the N-terminal -NH2 acetylation of the original DEC-205 targeting peptide (i.e., the polypeptide shown in SEQ ID NO: 1) were performed;

[0126] (2) dissolving the above polypeptide into about 500 μM in 2 mL of simulated gastrointestinal fluid, taking out 80 μL at different time points, adding 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) The sample was centrifuged in a centrifuge (12000 rpm / min, 15 min), the supernatant was filtered through a 0.22 μm filter membrane, and the sample was analyzed by UPLC-MS;

[0128] (4) After finding the cleavage site, replace K with a D-configuration k amino acid during the solid-phase synthesis of the peptide and repeat the above experiment.

[0129] The results showed that the original DEC-205 targeting peptide ① (SEQ ID NO: 1) was broken at the lysine (K) site after being treated in simulated intestinal fluid for half an hour, breaking into two parts ② and ③, indicating that the original targeting peptide could not be taken orally ( Figure 1A in the figure). It can be seen from the liquid phase diagram that the polypeptide has a chromatographic peak at 7.86 minutes at both 0h and 12h in simulated gastric fluid (SGF), proving that it has not been degraded in gastric fluid; in simulated intestinal fluid (SIF), at 0min, the polypeptide has a chromatographic peak at 7.85min, and after 30min, it has peaks at 2.15min and 6.91min. By analyzing the molecular weight according to the corresponding mass spectrometry results, it can be concluded that the polypeptide is not degraded in simulated gastric fluid, and the lysine (K) site will break in simulated intestinal fluid. After replacing the natural configuration lysine with the D configuration lysine (k), the obtained D-hr8 polypeptide (amino acid sequence is hlHkHHlr) will no longer break in simulated intestinal fluid ( Figure 1 (middle B).

[0130] 2. In order 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 peptide synthesis process, the amino acid at each site was replaced with alanine to obtain 8 alanine scanning peptides;

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

[0133] result Figure 2 As shown in the figure, alanine scanning peptide proves the important interaction sites between the peptide and mDEC-205. After the amino acids at different sites of the peptide are replaced with alanine, the sites where the affinity with the target decreases are the important interaction sites between the peptide and the target. That is, after replacing the 4th, 5th, 6th, and 8th amino acids, the specific affinity of the peptide is greatly reduced, proving that the 4th, 5th, 6th, and 8th amino acids are the important binding sites between the peptide and mDEC-205.

[0134] (3) Molecular dynamics (MDE): First, the structure of the peptide was predicted in PEP-FOLD. The peptide was then docked with the 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 single-site amino acid mutations.

[0135] (4) MOE performed single amino acid site mutation, that is, MOE replaced the amino acid at each site with the remaining 19 amino acids and their D-configuration forms, and scored the binding of the mutant peptides to the target. Finally, the peptides with higher scores were selected for synthesis and affinity experiments were performed again (Table 1) to screen out our final high-affinity and specific endocytosis-resistant peptides that are resistant to gastrointestinal enzyme degradation.

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

[0137]

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

[0139] In summary, through two steps of transformation: replacing the K amino acid at position 4 with k, and transforming the amino acid at position 6 from L-configuration H to D-configuration w, we finally obtained a high-affinity mDEC-205 affinity peptide: hlHkHwlr (SEQID NO: 2), denoted as DEBP-8.

[0140] 3. In order to verify the endocytic activity of the peptide in BMDC, BMDC cells were used for endocytosis experiments. Specifically, the legs of 6-week-old C57BL / 6 mice were taken, the bone marrow was blown out, GM-CSF (20 ng / mL) and IL-4 (10 ng / mL) were added to induce them into BMDC, and the BMDC on the fifth day of induction were blown out and 2×10 5 After the cells adhered to the wall, the DMEM complete medium was replaced with serum-free DMEM, and 50 μM polypeptide was added to incubate with the cells (37°C, 30 min). After half an hour, the medium was removed, and the polypeptide on the cell surface was washed off with PBS, and then stained with DAPI. After the cells were fixed and the membrane was broken, SA-PE labeled polypeptide was added, and then the endocytosis of the polypeptide by the cells was observed by confocal microscopy.

[0141] The experimental results are as follows Figure 4 As shown, BMDCs can rapidly and massively internalize the modified peptide DEBP-8, whereas they can only internalize very little of the original DEC-205 polypeptide within half an hour.

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

[0143] Example 2 Construction of oral chitosan nano vaccine

[0144] In this example, the polypeptide was coupled to chitosan and the OVA protein was encapsulated by the ion gel method to construct an oral chitosan nanovaccine. The experimental process is as follows:

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

[0146] (2) 5 mg of the peptide synthesized in step (1) was dissolved in MES buffer together with NHS and EDC to activate the carboxyl group for 1 h. The reaction product was then added to 10 mg of chitosan (CS) aqueous solution and reacted for 12 h (the process was as follows Figure 5 ) to obtain a DEBP-8-CS solution.

[0147] (3) The reaction product was ultrafiltered using an ultrafiltration tube with a molecular weight cutoff of 10 kD to remove unreacted polypeptides and other molecules. The product was freeze-dried and used for infrared spectrum scanning to characterize the coupling of the polypeptide.

[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, and then drop the TPP and OVA protein mixed solution into the CS or DEBP-8-CS solution under stirring and stir for 15 min to obtain OVA@CNP and OVA@DEBP-8-CNP.

[0149] Example 3

[0150] This example is used to investigate whether DEBP-8 can effectively promote BMDC endocytosis of nanoparticles and promote endocytosis of antigens in Peyer's patches of the intestine. The details are as follows:

[0151] 1. BMDC internalization of nanoparticles

[0152] Rhodamine B was labeled on OVA protein by NHS / EDC reaction in Example 2, and then loaded into CNP and DEBP-CNP, and the specific method was the same as that in Example 2 to obtain OVA-Rhodamine@CNP and OVA-Rhodamine@DEBP-8-CNP.

[0153] The legs of C57BL / 6 mice were taken, the bone marrow was blown out, GM-CSF and IL-4 were added to induce them into BMDCs, and 1 μg / mL LPS was added on the 5th day of induction. Mature BMDCs were obtained on the 6th day, the cells were blown off and spread on a confocal glass dish, and OVA-rhodamine@CNP and OVA-rhodamine@DEBP-8-CNP containing 10 μg / mL OVA protein were added. After internalization for different periods of time, the non-internalized nanoparticles were washed away with PBS, and the nanoparticles internalized by BMDC were observed confocally after Hoechest staining. The statistical graph shows the fluorescence intensity statistics of OVA-rhodamine in BMDC cells.

[0154] The results are as follows Figure 6 As shown, after modification of DEBP-8, mature BMDCs were able to internalize more nanoparticles.

[0155] 2. Peyer's knot ligation experiment

[0156] C57BL / 6 mice were anesthetized, and the part of the intestine containing Peyer's patches was removed. One section was ligated with surgical thread, and the above-mentioned OVA-rhodamine@CNP and OVA-rhodamine@DEBP-8-CNP were injected into the small intestine. The other end was ligated, and the mice were killed after waiting for 1 hour. The above-mentioned intestinal part was removed and fixed in paraformaldehyde, and fixed sections were made. After staining with DAPI and UEA-1-Lectin antibodies (the secondary antibody was SA-APC), the phagocytosis of nanoparticles in the intestinal lamina propria and Peyer's patches was observed confocally.

[0157] The results are as follows Figure 7 As shown, OVA@DEBP-8-CNP can be enriched in the intestinal lamina propria and Peyer's patches of mice.

[0158] Example 4

[0159] This example is used to investigate whether OVA@DEBP-8-CNP can prevent C57BL / 6 mice from being attacked by B16-OVA. The details are as follows:

[0160] Six-week-old C57BL / 6 mice were randomly divided into four groups. The mice were immunized orally once a week. The first group was the NS control group, the second group was the 100 μg OVA protein group, the third group was the OVA@CNP group containing 100 μg OVA protein, and the fourth group was the OVA@DEBP-8-CNP group containing 100 μg OVA protein. After four oral immunizations, 2×10 5 B16-OVA cells were added to observe the tumor growth in mice. The results showed that OVA@DEBP-8-CNP can effectively prevent the growth of B16-OVA tumors in mice ( Figure 8 ).

[0161] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A DEC-205 affinity peptide, comprising a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 3 or a modified form thereof.

2. The affinity peptide according to claim 1, characterized in that The configuration of the amino acids in the affinity peptide is independently selected from D-type or L-type; Preferably, the 3rd and 4th amino acids in the amino acid sequence shown in SEQ ID NO: 2 are L-type, and the rest are D-type; Preferably, the 5th and 6th amino acids in the amino acid sequence shown in SEQ ID NO: 3 are L-type, and the rest are D-type.

3. The affinity peptide according to claim 2, characterized in that The modification sites of the modified forms include N-terminal modification, C-terminal modification, backbone modification, side chain modification and / or amino acid modification.

4. An immunogenic conjugate comprising the affinity peptide according to any one of claims 1 to 3.

5. A biological material related to the affinity peptide according to any one of claims 1 to 3 or the immunogenic conjugate according to claim 4, wherein the biological material is any one of a1) to a12): a1) a nucleic acid molecule encoding the affinity peptide according to any one of claims 1 to 3 or the immunogenic conjugate according to claim 4; a2) an expression cassette containing the nucleic acid molecule described in a1); a3) a recombinant vector containing the nucleic acid molecule described in a1); a4) a recombinant vector containing the expression cassette described in a2); a5) a recombinant microorganism containing the nucleic acid molecule described in a1); a6) a recombinant microorganism containing the expression cassette described in a2); a7) a recombinant microorganism containing the recombinant vector described in a3); a8) a recombinant microorganism containing the recombinant vector described in a4); a9) a transgenic animal cell line containing the nucleic acid molecule described in a1); a10) a transgenic animal cell line containing the expression cassette described in a2); a11) a transgenic animal cell line containing the recombinant vector described in a3); a12) A transgenic animal cell line containing the recombinant vector described in a4).

6. Use of the affinity peptide according to any one of claims 1 to 3, the immunogenic conjugate according to claim 4, or the biomaterial according to claim 5 in at least one of (1) to (10): (1) Affinity for DEC-205 protein; (2) Preparation of products for the prevention and / or treatment of tumors, pathogenic microbial infections or autoimmune diseases; (3) preparing products for targeting DEC-205 in vitro and in vivo; (4) qualitatively and / or quantitatively detecting whether the DEC-205 protein is expressed, its expression location or its expression level; (5) preparing products for qualitatively and / or quantitatively detecting whether DEC-205 protein is expressed, where it is expressed, or how much it is expressed; (6) preparing products that promote the endocytosis of drugs by dendritic cells; (7) promoting maturation and / or cross-presentation of dendritic cells; (8) preparing products that promote maturation and / or cross-presentation of dendritic cells; (9) Improve the ability of T cells to kill tumor cells; (10) Prepare products that enhance the ability of T cells to kill tumor cells.

7. A reagent, a kit or a medicine, comprising the affinity peptide according to any one of claims 1 to 3, the immunogenic conjugate according to claim 4 or the biomaterial according to claim 5; Preferably, the medicine also includes a medicine for treating tumors.

8. A nano vaccine comprising the affinity peptide according to any one of claims 1 to 3, chitosan and a protein carrier; Preferably, the affinity peptide and chitosan are linked by coupling and encapsulated in a protein carrier; Preferably, the protein carrier comprises at least one of hemocyanin, bovine serum albumin, ovalbumin, human serum albumin and mouse serum albumin.

9. The nanovaccine according to claim 8, characterized in that The nano-vaccine also includes drugs for treating tumors; Preferably, the drug is coupled to a protein carrier; Preferably, the vaccine is administered by nasal, oral, subcutaneous, intramuscular, intravenous, intraperitoneal or intradermal injection.

10. The method for preparing the nanovaccine according to claim 9, comprising the following steps: The affinity peptide and / or drug is activated in a solution containing NHS and EDC; The activated product is mixed with chitosan and reacted to obtain an affinity peptide coupling carrier; The nano vaccine is obtained by mixing an acetic acid solution containing an affinity peptide coupled carrier with a sodium tripolyphosphate solution containing a protein antigen.

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