Dendrimer polypeptides and their uses

Abstract

The present invention belongs to the field of biomedicine, specifically to dendritic polypeptides and their uses. The technical problem addressed by the present invention is to improve the efficiency of antigen transport from the injection site to lymph nodes, thereby enhancing the immune efficacy of vaccines. The present invention addresses this technical problem by providing a dendritic polypeptide, DP7, and further provides uses of the dendritic polypeptide, DP7, for promoting the immune efficacy of vaccines. The dendritic DP7 of the present invention possesses lymph node targeting properties and can be used as a drug delivery system to carry antigens into lymph nodes, thereby enhancing the immune efficacy of vaccines. Furthermore, it can promote the migration of DC cell vaccines to lymph nodes, thereby enhancing their immune efficacy, and thus has promising application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a dendritic polypeptide and its use. Background Art

[0002] Tumor vaccines offer high specificity and minimal side effects. Therefore, they are a promising alternative to or supplement traditional therapies to enhance their efficacy. The primary mode of action of tumor vaccines is to deliver tumor antigens to antigen-presenting cells (APCs), thereby activating both innate and antigen-specific adaptive immune responses, exerting both systemic and specific anti-tumor effects. Despite their superior theoretical foundation, vaccine-based anti-tumor therapies have yet to achieve satisfactory clinical efficacy. Therefore, enhancing the in vivo immune-stimulating effects of vaccines is a key research direction in the field of tumor immunotherapy. Adaptive immune responses are primarily initiated in secondary lymphoid organs, and therefore, effective vaccine accumulation in lymph nodes (LNs) is a prerequisite for inducing robust antigen-specific immune responses. LNs are the primary site of antigen presentation, home to numerous APCs, which reside adjacent to naive T cells and enable rapid antigen presentation following antigen uptake. Furthermore, APCs residing in LNs exhibit an immature phenotype and possess robust antigen uptake capacity. Therefore, effective delivery of tumor vaccines to LNs is a key strategy for improving vaccine efficacy.

[0003] Disappointingly, the endolymphatic drainage of antigens (such as proteins) and adjuvants (such as aluminum adjuvants) is relatively limited, indicating the need for specialized targeted delivery platforms with high affinity for antigens and adjuvants. Currently, the effective delivery of tumor vaccines to LNs mainly relies on nanodelivery systems. Since nanodelivery systems have the inherent property of being captured by APCs, the use of nanotechnology for cancer vaccine design has great prospects. However, current nanovaccine systems still have obstacles in achieving effective tumor treatment. This is partly due to the unsatisfactory design of vaccine carriers. Most carriers have a single function, do not have the efficacy of immune adjuvants, or the complex synthesis process is not conducive to subsequent large-scale use.

[0004] DP7 (VQWRIRVAVIRK) is a novel cationic hydrophilic antimicrobial peptide developed by the applicant based on a previously developed amino acid activity prediction method. It was then modified with cholesterol, resulting in the discovery that Chol-DP7 (DP7-C) possesses dual functions as a carrier and immune adjuvant. It can effectively deliver small RNAs and peptides to tumor cells and immune cells through caveolin- and clathrin-dependent pathways, making it a promising delivery vehicle. As an immune adjuvant, it can stimulate DC maturation by activating the TLR2-MyD88-IKK-IκB-NF-κB signaling pathway and enhance the antitumor effects of DC vaccines loaded with tumor antigens. Although previous experiments have demonstrated that DP7-C can enhance the antitumor effects of DC vaccines loaded with tumor antigens, due to the complexity and time-consuming in vitro preparation of DC vaccines, it is believed that direct incubation of DP7-C with tumor antigens and subcutaneous administration could provide patients with more convenient and faster treatment, while also reducing costs. However, there is room for improvement in the lymph node targeting of DP7-C complexed with antigens.

[0005] Therefore, effectively improving the lymph node drainage efficiency of antigens is a problem that needs to be solved in the current field of vaccine preparation. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to improve the lymph node drainage efficiency of antigens, thereby improving the immune effect of vaccines based on antigens.

[0007] The present invention addresses the aforementioned technical problems by providing a dendritic polypeptide, DP7, for enhancing the efficiency of antigen targeting to lymph nodes. Because the dendritic polypeptide, DP7, itself acts as an immune adjuvant, it enhances the immune efficacy of vaccines. The polypeptide described in the dendritic polypeptide of the present invention is a DP7 polypeptide having the amino acid sequence VQWRIRVAVIRK (SEQ ID No. 1). The dendritic polypeptide, formed by coupling DP7 polypeptides to each other, is composed of 2 to 16 coupled DP7 polypeptide molecules.

[0008] Preferably, the dendrimer polypeptide is formed by coupling 2, 4, or 8 DP7 polypeptide molecules.

[0009] The DP7 polypeptide molecules in the dendrimer polypeptide of the present invention are coupled to each other using lysine as a linking medium.

[0010] The sites of the DP7 polypeptide molecules involved in the coupling in the dendrimer polypeptides of the present invention are the amino acid residues at the carbon terminal and / or nitrogen terminal. Furthermore, the structural formula of the dendrimer polypeptides of the present invention is at least one of the following:

[0011]

[0012]

[0013] Formula II

[0014] or

[0015]

[0016] The present invention provides use of the above-mentioned dendrimer polypeptide in preparing drug delivery vectors, drugs or vaccines.

[0017] Wherein, the drug delivery vector, drug or vaccine described in the above uses is a lymph node-targeted drug delivery vector, drug or vaccine.

[0018] The vaccine mentioned in the above-mentioned use refers to at least one of an infectious disease vaccine, an autoimmune disease vaccine or a tumor vaccine.

[0019] Furthermore, the infectious disease vaccine described in the above use is at least one of a viral vaccine or a bacterial vaccine.

[0020] The present invention also provides the use of the above-mentioned dendritic polypeptide in the preparation of a dendritic cell migration promoter or an immune adjuvant. Furthermore, the above-mentioned promotion of dendritic cell migration refers to promoting the migration of dendritic cells to lymph nodes.

[0021] Furthermore, the above-mentioned dendritic polypeptide can be co-incubated with dendritic cells in vitro to enhance their migration ability, or can be directly combined with dendritic cells to further enhance their migration ability, wherein the direct combination refers to injection.

[0022] The present invention also provides a dendritic cell vaccine comprising dendritic cells treated with the above-mentioned dendritic polypeptide as the primary active ingredient. The dendritic cells are myeloid DCs or lymphoid DCs. Furthermore, the dendritic cells are ex vivo DCs obtained from the patient.

[0023] The treatment described in the above dendritic cell vaccine is to prepare the vaccine after co-incubation with dendritic cells; or to prepare the vaccine after mixing with dendritic cells.

[0024] Furthermore, the dendritic cell vaccine can be prepared using the following method:

[0025] a. Take immature dendritic cells (imDCs) after induction culture;

[0026] b. adding the above-mentioned dendritic polypeptide, as well as reagents and antigens that stimulate dendritic cell maturation, to the culture medium, and incubating to obtain antigen-loaded and mature dendritic cells;

[0027] c. The antigen-loaded and mature dendritic cells obtained in step b are prepared as dendritic cell vaccines.

[0028] Furthermore, the dendritic cell vaccine mentioned above also includes pharmaceutically acceptable auxiliary components.

[0029] Furthermore, the pharmaceutically acceptable auxiliary component is at least one of a protective agent, an excipient, an immune adjuvant, a dispersant or a cell culture medium.

[0030] The present invention also provides a vaccine comprising an antigen and an immune adjuvant; the immune adjuvant is the above-mentioned dendrimer polypeptide.

[0031] The antigens in the above vaccines are antigenic components that can directly or indirectly provide immunogenicity in recombinant protein vaccines, peptide vaccines, mRNA vaccines, and cell vaccines. Furthermore, the cell vaccine is a dendritic cell vaccine.

[0032] Furthermore, the antigen and immune adjuvant in the above vaccine are in the same package or in separate packages.

[0033] Furthermore, the above-mentioned vaccine also includes pharmaceutically acceptable auxiliary ingredients.

[0034] Wherein, the pharmaceutically acceptable auxiliary component in the above-mentioned vaccine is at least one of a protective agent, an excipient, an immune adjuvant, a dispersant or a cell culture medium.

[0035] The beneficial effects of the present invention are as follows: the present invention creatively synthesizes dendritic DP7 and discovers that it possesses exceptional properties. First, the dendritic DP7 polypeptide, after co-incubation with an active substance and subsequent injection, can improve the lymph node targeting efficiency of the active substance antigen, making it useful as a lymph node-specific targeted delivery vehicle. Furthermore, it can activate cellular immune responses to assist antigen-specific immune responses, making it useful as an immune adjuvant, enhancing the immune efficacy of vaccines containing antigens as active ingredients. During vaccine preparation, the dendritic polypeptide and antigen need only be co-incubated for 5 minutes before subcutaneous injection. This simple and low-cost preparation method facilitates subsequent widespread use and has promising application prospects. Furthermore, the dendritic DP7 polypeptide of the present invention can promote dendritic cell migration and, through the transport function of dendritic cells, efficiently and specifically deliver antigens to lymph nodes, inducing anti-tumor immune responses, offering particular advantages in dendritic cell vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 : Structure and HPLC and MS data of KK2DP7.

[0037] Figure 2: Incubation of KDP7, KK2DP7, and KK2K4DP7 with OVA can enhance the efficiency of OVA uptake by DCs. (**** indicates p<0.0001)

[0038] Figure 3 : KDP7, KK2DP7, and KK2K4DP7 can stimulate DC maturation. (**** indicates p < 0.0001, ** indicates p < 0.01)

[0039] Figure 4 : KDP7, KK2DP7, KK2K4DP7 and OVA incubated with DC can improve the efficiency of DC presentation of OVA

[0040] Figure 5 : KDP7, KK2DP7, and KK2K4DP7 can enhance DC migration efficiency after incubation with DC. (**** indicates p<0.0001, ** indicates p<0.01)

[0041] Figure 6 Comparison of fluorescence intensity in lymph nodes after subcutaneous administration of KDP7, KK2DP7, KK2K4DP7, and cy7-OVA. (**** indicates p < 0.0001, ** indicates p < 0.01)

[0042] Figure 7 : Detection of the immune effect of subcutaneous injection of KK2DP7 / OVA complex vaccine. ab) CD8 + T cell proliferation efficiency; cd) CD8 + Tetramer ratio test; ef) ELISPOT test for antigen-specific lymphocyte response. (**** indicates p < 0.0001)

[0043] Figure 8 : Antitumor effect of subcutaneous KK2DP7 / OVA complex vaccine. (**** indicates p < 0.0001). DETAILED DESCRIPTION

[0044] The present invention is described in detail below through the introduction of specific implementation methods.

[0045] In preliminary research for this invention, an antibacterial polypeptide with the sequence VQWRIRVAVIRK (SEQ ID No. 1) was obtained and named DP7. Further research revealed that hydrophobic modification of the DP7 polypeptide resulted in an amphiphilic compound, DP7-C, capable of self-assembly into micelles. This, while reducing the cytotoxicity of the DP7 polypeptide while maintaining its antibacterial activity, also enabled its assembly into nanoparticles, making it a suitable delivery vehicle for certain drugs. Furthermore, the hydrophobic modification of DP7 has been shown to be effective as an immune adjuvant, stimulating DC maturation by activating the TLR2-MyD88-IKK-IκB-NF-κB signaling pathway and enhancing the anti-tumor effects of DC vaccines loaded with neoantigens. Although previous experiments have demonstrated the potential for enhancing the anti-tumor effects of antigen-loaded DC vaccines, due to the complex and time-consuming in vitro preparation of DC vaccines, we believe that direct subcutaneous administration of DP7-C incubated with antigens would allow for more convenient and rapid treatment for patients, while also reducing costs. However, further studies found that the lymph node targeting effect of DP7-C complexed with antigens was not satisfactory.

[0046] Therefore, the present invention creatively designs a dendritic DP7 polypeptide with beneficial properties such as enhanced lymph node targeting, which is particularly suitable for the development of nano-vaccine systems.

[0047] First, the present invention provides a dendritic polypeptide, which is a dendritic polypeptide formed by coupling multiple DP7 polypeptide molecules. The coupling can be formed by coupling multiple DP7 polypeptide molecules, generally 2 to 16 DP7 polypeptide molecules. In the examples of the present invention, dendritic DP7 polypeptides formed by coupling 2, 4, and 8 DP7 polypeptide molecules were prepared and investigated, namely, a two-branched DP7 (hereinafter abbreviated as KDP7), a four-branched DP7 (hereinafter abbreviated as KK2DP7), and an eight-branched DP7 (hereinafter abbreviated as KK2K4DP7).

[0048] The DP7 polypeptide molecules of the present invention are coupled using lysine as a linking medium. The site of the DP7 polypeptide involved in the coupling can be the carbon-terminal or nitrogen-terminal amino acid residue.

[0049] The dendrimer polypeptides of the present invention can be used as drug delivery vehicles to deliver drugs, particularly as lymph node-targeted drug delivery vehicles. The dendrimer polypeptides of the present invention can be used to load and deliver protein peptide drugs, nucleic acid drugs, and small molecule chemical drugs.

[0050] Furthermore, the dendrimer polypeptides of the present invention can also be used to prepare vaccines, for example, vaccines for infectious diseases, autoimmune diseases or tumors.

[0051] The dendrimer polypeptide of the present invention can activate cellular immune response to assist antigen-specific immune response and can be used as an immune adjuvant in the preparation of various vaccines.

[0052] The dendritic polypeptide of the present invention can also be used to prepare a dendritic cell migration promoter. The promoter of dendritic cell migration refers to promoting the migration of dendritic cells to lymph nodes.

[0053] In specific applications, the dendritic polypeptides of the present invention can be co-incubated with dendritic cells in vitro to enhance their migration ability, or can be used directly with dendritic cells to further enhance their migration ability, for example, by direct injection with dendritic cells.

[0054] The present invention also provides a dendritic cell vaccine comprising dendritic cells treated with the above-mentioned dendritic polypeptide as the primary active ingredient. The dendritic cells can be myeloid DCs or lymphoid DCs. In routine practice, ex vivo dendritic cells obtained from the patient can be used.

[0055] The above treatment is to incubate the dendritic cells together with the dendritic cells and then prepare the vaccine for use; or to mix the dendritic cells with the dendritic cells and then prepare the vaccine for use.

[0056] The specific preparation method can be referred to as follows:

[0057] a. Take immature dendritic cells (imDCs) after induction culture;

[0058] b. adding the above-mentioned dendritic polypeptide, as well as reagents and antigens that stimulate dendritic cell maturation, to the culture medium, and incubating to obtain antigen-loaded and mature dendritic cells;

[0059] c. The antigen-loaded and mature dendritic cells obtained in step b are prepared as dendritic cell vaccines.

[0060] Naturally, the above-mentioned vaccine may also include pharmaceutically acceptable auxiliary components, such as at least one of a protective agent, an excipient, an immune adjuvant, a dispersant or a cell culture medium.

[0061] The present invention further provides a vaccine comprising an antigen and an immune adjuvant; the immune adjuvant is the above-mentioned dendrimer polypeptide.

[0062] The above-mentioned antigens are antigenic components that can directly or indirectly provide immunogenicity in recombinant protein vaccines, peptide vaccines, mRNA vaccines, and cell vaccines, such as antigenic proteins, antigenic peptides, mRNA for expressing antigenic proteins or antigenic peptides, antigens for preparing dendritic cell vaccines, dendritic cell vaccines, etc.

[0063] The antigen and dendritic cell migration promoter can be in the same package or in separate packages, and also include pharmaceutically acceptable auxiliary components.

[0064] Those skilled in the art will appreciate that the antigens described herein may be tumor antigens, including specific proteins or polypeptides expressed by tumor cells, such as WT1, MUC1, EGFRvIII, HER-2, MAGE-A3, NY-ESO-1, PSMA, GD2, or MART1, among other reported tumor antigens, or personalized combinations of mutated neoantigens derived from patient tumor sequencing.

[0065] The aforementioned antigens may also be viral antigens, such as proteins or polypeptides that constitute part of a virus, or specific proteins or polypeptides that are expressed in virus-infected cells under the control of viral expression machinery, such as EBV, LMP2, HPV E6 E7, adenovirus 5 Hexon, or HCMV pp65, among other virus-associated antigens.

[0066] The above-mentioned antigens may also be bacterial antigens, including proteins or polypeptides expressed by bacteria, such as Pseudomonas aeruginosa antigens, Clostridium tetani antigens, Streptococcus pneumoniae, Salmonella and other bacterial antigens.

[0067] Those skilled in the art will also understand that the antigens may also be disease-related antigens, including autoimmune-related antigens, such as antigens involved in autoimmune diseases or disorders or overexpressed during autoimmune diseases, for example, autoimmune-related antigens such as ppIAPP, IGRP, GAD65, or myelin basic protein antigens.

[0068] The present invention is described in further detail below through examples.

[0069] The main experimental materials and equipment used in the examples are as follows:

[0070] 1. Experimental cell lines and experimental animals

[0071] The EG7-OVA cell line was purchased from the American Type Culture Collection (ATCC). Cells were cultured in RPMI-1640 (Gibico) medium supplemented with 10% fetal bovine serum (FBS). Six- to eight-week-old female C57 / BL6J mice were purchased from Beijing Weitonglihua Laboratory Animal Co., Ltd. and maintained in an SPF environment.

[0072] 2. Main reagent materials and kits

[0073] Experimental cell culture medium: RPMI-1640 culture medium and fetal bovine serum (FBS) were purchased from Gibco, USA.

[0074] Cytokines CCL19 and CCL21 were purchased from Absin Biotechnology Co., Ltd.

[0075] 0.5 μm 24-well Transwell microwells were purchased from Corning Biotechnology Co., Ltd.

[0076] GM-CSF was purchased from Shanghai Puxin Biotechnology Co., Ltd.

[0077] ELISPOT kit and CFDA-SE cell labeling kit were purchased from Beyotime Biotechnology Co., Ltd.

[0078] 3. Main instruments and equipment

[0079] Flow cytometer: FACSCalibur.

[0080] Example 1 Synthesis of branched polypeptides KDP7, KK2DP7, and KK2K4DP7

[0081] The branched DP7 peptide was synthesized using a standard solid-phase peptide synthesis method. A brief description follows: Fmoc-Lys(Fmoc)-OH was attached to the resin, and the two Fmoc groups were removed, exposing the two NH2 groups. Following the peptide sequence VQWRIRVAVIRK, condensation was performed from right to left to produce the two-branched peptide.

[0082] By hanging Fmoc-Lys(Fmoc)-OH on the resin, removing two Fmoc groups, and then exposing two NH2 groups, the two exposed NH2 groups are then connected to two Fmoc-Lys(Fmoc)-OH groups. After all Fmoc groups are removed, four amino groups are exposed. According to the polypeptide sequence VQWRIRVAVIRK, condensation is carried out from right to left to obtain a four-branched polypeptide.

[0083] By attaching Fmoc-Lys(Fmoc)-OH to the resin, removing two Fmoc groups, and then exposing two NH2 groups, the two exposed NH2 groups are then connected to two Fmoc-Lys(Fmoc)-OH groups. After all Fmoc groups are removed, four amino groups are exposed, and then four Fmoc-Lys(Fmoc)-OH groups are connected. After all Fmoc groups are removed, eight amino groups are exposed. According to the peptide sequence VQWRIRVAVIRK, condensation is carried out from right to left to obtain an 8-branched peptide.

[0084] The synthesized branched peptide was tested by HPLC and MS to verify the final product. The structure and HPLC and MS results are shown in Figure 1 The results showed that the branched polypeptides KDP7, KK2DP7, and KK2K4DP7 were synthesized.

[0085] Example 2 Detection of the efficiency of DC uptake of KDP7 / OVA, KK2DP7 / OVA, and KK2K4DP7 / OVA complexes 1. Preparation of KDP7, KK2DP7, KK2K4DP7 and OVA complexes

[0086] KDP7 (VQWRIRVAVIRKK), KK2DP7 ((VQWRIRVAVIRK)2KK), and KK2K4DP7 (((VQWRIRVAVIRK)2K)2KK) were synthesized by solid-phase peptide synthesis at Shanghai Chupeptide Biotechnology Co., Ltd. The final products were purified by HPLC and identified by mass spectrometry.

[0087] Lyophilized powders of KDP7, KK2DP7, and KK2K4DP7 were dissolved directly in deionized water and stored in aliquots at -20°C. To prepare complexes with OVA, simply incubate them with an aqueous solution of OVA in deionized water and culture medium for 5 minutes.

[0088] 2. Acquisition and Culture of DC Cells

[0089] ① Obtain the tibia and fibula from adult C57BL / 6J female mice (approximately 6 weeks old). Soak them in 75% ethanol for 5 minutes to kill bacteria. Then, remove the muscle tissue and soak the leg bones in RPMI 1640 + 1% PS medium. Use sterile scissors to cut off the ends of the leg bones. Use a syringe to draw up fresh RPMI 1640 + 1% PS medium and blow out the bone marrow cells until all the bone marrow cells are blown out.

[0090] ② Filter the collected culture medium containing bone marrow cells through a 70 μm mesh, centrifuge at 1200 rpm for 3 min, discard the supernatant, and resuspend the cells in red blood cell lysis buffer (weigh 1.3 g Tris-base and 3.74 g NH4Cl, dissolve in 490 ml ultrapure water, adjust the pH of the solution to 7.2-7.4 with concentrated hydrochloric acid, then add ultrapure water to 500 ml, remove bacteria through a 0.22 μm filter, store at 4°C, and prepare immediately before use). Let it stand at room temperature for 3 min, centrifuge at 1200 rpm for 3 min, and finally wash away the red blood cell lysis buffer with RPMI1640 medium + 10% FBS + 1% PS, and resuspend the cells.

[0091] ③ Divide the resuspended cells into culture dishes, 2×10 6 -3×10 6For each dish, add 10 ml of RPMI1640 + 10% FBS + 1% PS culture medium and 20 ng / ml GM-CSF cytokine. Place the culture dish in a 37 ° C cell culture incubator and culture on the third day of culture. Add fresh RPMI1640 + 10% FBS + 1% PS culture medium containing 20 ng / ml GM-CSF until the 8th day of culture to obtain immature DCs (imDCs). Take the DCs cultured on the 8th day, wash the culture medium with 1 ml PBS, resuspend the cells with 100 μl PBS, add 1 μl APC Hamster Anti-Mouse CD11c flow cytometry antibody, mix gently, and incubate at 4 ° C in the dark for 40 minutes; after the incubation is completed, wash off the excess antibody with PBS, resuspend the cells with 200 μl PBS, and use flow cytometry to detect CD11c. + When the CD11c ratio is greater than 80%, it indicates that DC induction is successful.

[0092] 3. Efficiency of DC transfection with KDP7 / OVA, KK2DP7 / OVA, and KK2K4DP7 / OVA complexes

[0093] Immature DCs were cultured on day 8 and plated in 24-well plates, with 5 × 10 5 Cells were incubated in 1640 complete medium for 5 minutes. 30 μl of KDP7 / OVA, KK2DP7 / OVA, or KK2K4DP7 / OVA complexes were added, respectively. FITC-labeled OVA was used at a concentration of 10 μg / ml, and KDP7, KK2DP7, and KK2K4DP7 were used at concentrations of 5, 10, 20, and 40 μg / ml. After 24 hours of culture, cells were harvested, excess medium was washed off, and cells were resuspended in 200 μl of PBS. The percentage of fluorescent cells was determined by flow cytometry.

[0094] From the results, we can see that the transfection efficiency of KDP7, KK2DP7, and KK2K4DP7 in transfecting OVA into DC can reach more than 90% ( Figure 2 ).

[0095] Example 3 Detection of the efficiency of KDP7, KK2DP7, and KK2K4DP7 in stimulating DC maturation

[0096] Immature DCs were cultured on day 8 and plated in 24-well plates, with 5 × 10 5 Cells were treated with 10, 20, 40, and 80 μg / ml of KDP7, KK2DP7, and KK2K4DP7, respectively. After 24 hours, cells were collected and stained for CD11c, CD80, and CD86 to assess BMDC maturation.

[0097] From the results of flow cytometry, KDP7, KK2DP7, and KK2K4DP7 can all induce DCs maturation, and KK2DP7 has more advantages than KDP7 and KK2K4DP7 in inducing DCs maturation ( Figure 3 ).

[0098] Example 4 Detection of the Efficiency of KDP7 / OVA, KK2DP7 / OVA, and KK2K4DP7 / OVA Complexes Presented by DCs

[0099] Immature DCs were cultured on day 8 and plated in 24-well plates, with 5 × 10 5 30 μl of KDP7 / OVA, KK2DP7 / OVA, and KK2K4DP7 / OVA complexes were added and incubated in 1640 complete medium for 5 min, with OVA at a concentration of 10 μg / ml and KDP7, KK2DP7, and KK2K4DP7 at a concentration of 40 μg / ml. Cells were harvested after culturing for 24 h, 48 h, and 72 h, and the excess medium was washed away and the cells were stained with SIINFEKL-H2K. b+ Antibody staining was performed at 4°C for 40 min, followed by washing with PBS and resuspending in 200 μl PBS. The percentage of fluorescent cells was determined using flow cytometry.

[0100] The results showed that the antigen presentation efficiency of DCs was significantly increased after KDP7, KK2DP7, and KK2K4DP7 were transfected with OVA. At 72 hours, the antigen presentation efficiency of the KK2DP7 group was significantly higher than that of the KDP7 group and the KK2K4DP7 group ( Figure 4 ).

[0101] Example 5 Detection of the efficiency of KDP7, KK2DP7, and KK2K4DP7 in stimulating DC migration in vitro

[0102] Immature DCs were cultured on day 8 and plated in 24-well plates, with 5 × 10 5 cells. 10, 20, and 40 μg / ml of KDP7, KK2DP7, and KK2K4DP7 were added and treated for 24 hours. After digestion, the cells were washed three times with 1640 double-free medium and resuspended in 1640 medium. The cells were plated in transwell wells of a 24-well plate (pore size 5.0 μm) at 1×10 cells per well. 5 The volume of each cell was 100 μl. 500 μl of 1640 complete medium + CCL19 (250 ng / ml) + CCL21 (250 ng / ml) were added to the lower chamber; after 24 hours, the cells in the lower chamber were counted to calculate the efficiency of DCs migration.

[0103] The results showed that compared with the control group, KDP7, KK2DP7, and KK2K4DP7 could improve the in vitro migration efficiency of DCs to a certain extent, and KK2DP7 had the best efficiency in promoting DC migration ( Figure 5 ).

[0104] Example 6 Detection of Lymph Node Targeting Efficiency of Branched Polypeptide / OVA Complex

[0105] 60 μg of KDP7, KK2DP7, KK2K4DP7, and 20 μg of Cy7-OVA were incubated in 100 μl of PBS for 5 minutes and then injected into the subcutaneous lymph nodes of mice. Four hours later, proximal and distal lymph nodes were harvested for fluorescence imaging, and fluorescence intensity in the lymph nodes was measured and statistically analyzed.

[0106] The results showed that the fluorescence intensity in the proximal and distal lymph nodes of the KK2DP7 / OVA group was significantly increased compared with the OVA group alone and the KDP7 / OVA combination KK2K4DP7 / OVA group ( Figure 6 In our subsequent experiments, we mainly used KK2DP7, which has strong lymph node targeting ability, as the research object.

[0107] Example 7 Detection of the immune effect of KK2DP7 / OVA complex

[0108] 6-8 week old C57BL / 6J female mice were randomly divided into three groups (PBS, OVA, and KK2DP7 / OVA), with three mice per group. On days -21, -14, and -7, mice were injected with 100 μl of OVA (20 μg) or KK2DP7 (60 μg) / OVA (20 μg) adjacent to their lymph nodes. Mice were sacrificed on day 0 and their spleens were harvested for subsequent experiments.

[0109] (1) Flow cytometry detection of T cell proliferation-CFSE cleavage method

[0110] ① Seven days after the last immunization, separate the mouse spleen lymphocytes and take 3×10 6 cells, suspend the cells with 1 ml CFDASE cell labeling solution and place them in a 15 ml BD tube;

[0111] ② Dilute the CFDASE storage solution to 2× with CFDASE cell labeling solution and mix well;

[0112] ③ Add 1 ml of CFDA storage solution (2×) to ① and mix gently;

[0113] ④Incubate at 37℃ in the dark for 30 min;

[0114] ⑤ Add 10 ml of 1640 + 10% FBS culture medium, mix well, and terminate labeling;

[0115] ⑥ Centrifuge and remove the supernatant, repeat ⑤;

[0116] ⑦ Dilute the cells to 2×10 with 1640+10% FBS medium. 6 / ml, 50 μl of cells (1×10 5 per well), and 10 μg / ml OVA 257-264 For peptide stimulation, three replicate wells were set up in each group, and the total volume was made up to 200 μl with 1640 complete medium containing 10% FBS. After mixing, the 96-well plate was placed in a CO2 incubator and cultured for 4 days.

[0117] ⑧ Collect the cultured cells into flow cytometry tubes, add 100 μl PBS to each tube to resuspend the cells, add 1 μl T cell surface marker (PE-CD8) antibody to each tube, and then perform flow cytometry analysis.

[0118] The experimental results showed that the T cell proliferation efficiency of mice in the KK2DP7 / OVA immunization group was significantly higher than that in the OVA group and the PBS group ( Figure 7 a-7b).

[0119] (2) Flow cytometry detection of CD8 tetramers in the spleen after immunization

[0120] ① Seven days after the last immunization, separate the mouse spleen lymphocytes and take 1×10 6 Cells were cultured in 12-well plates using 1640 μg / mL + 10% FBS + PS.

[0121] ②Add 10 μg / ml OVA 257-264 Stimulate for 7 days;

[0122] ③ Collect the cultured cells into flow cytometry tubes, add 100 μl PBS to each tube to resuspend the cells, add 1 μl T cell surface marker (PE-CD8) antibody and tetramer dye respectively, and then perform flow cytometry analysis.

[0123] The experimental results showed that the CD8 + The proportion of T cell tetramers was significantly higher than that in the OVA group and PBS group ( Figure 7 c-7d).

[0124] (3) ELISPOT detection of antigen-specific lymphocyte response after immunization

[0125] Day 1:

[0126] ① Activation of pre-coated plates: Add 200 μl of RPMI-1640 serum-free medium to each well, let it stand at room temperature for 5-10 minutes, and then remove it;

[0127] ②Add cell suspension: resuspend the separated spleen lymphocytes to 5×10 6 cells / ml, add 100 μl of spleen lymphocyte suspension and control, i.e. 5×10 5 cells / well;

[0128] ③Add stimulant: OVA 257-264 10 μg / well, set up 3 replicates;

[0129] ④ Incubation: Cover the plate and place in a 37°C, 5% CO2 incubator for 48 hours;

[0130] Day 3:

[0131] ① Cell lysis: Pour out the cells and culture medium in the well, add ice-cold ddH2O, 200μl / well, and place in a 4℃ refrigerator for 10 minutes to hypotonic lyse the cells;

[0132] ② Wash the plate: Pour the liquid in the wells and add 1× Washing Buffer (200 μl / well) for 5-7 times, each time for 30-60 seconds. After the last wash, blot dry on absorbent paper.

[0133] ③ Detection antibody incubation: Add the diluted biotinylated antibody working solution to each experimental well, 100 μl / well; incubate at 37°C for 1 hour;

[0134] ④Wash the board: follow ②;

[0135] ⑤ Enzyme-linked avidin incubation: Add the diluted enzyme-linked avidin working solution to each experimental well, 100 μl / well; incubate at 37°C for 1 hour;

[0136] ⑥Wash the board: follow ②;

[0137] ⑦ Color development: Add the prepared AEC color development solution to each experimental well, 100 μl / well; incubate in a 37°C incubator in the dark for color development, and check every 5-10 minutes;

[0138] ⑧Terminate color development: Pour out the liquid in the wells, uncover the plate base, and wash the front and back surfaces and the base with ddH2O 5 times to terminate color development; place the plate in a cool place at room temperature, wait for it to dry naturally, and then close the base;

[0139] ⑨Count the spots on the ELISpot plate and record various parameters of the spots for statistical analysis.

[0140] The experimental results showed that the number of IFN-γ spots produced by antigen-specific lymphocytes in the KK2DP7 / OVA immunization group was significantly higher than that in the OVA and PBS groups ( Figure 7 e-7f).

[0141] Example 8 Detection of the anti-tumor effect of KK2DP7 / OVA complex

[0142] 6-8 week old C57BL / 6J female mice were randomly divided into 3 groups (PBS group, OVA group, KK2DP7 / OVA group), with 6 mice in each group. On day 0, 1×10 6 EG7-OVA tumor cells were injected into the lymph nodes of mice on days 4, 11, and 18, respectively. The tumor size was measured every 2 days after the tumor grew. The tumor volume was calculated as 0.52 × length × width. 2 The tumor growth curve of each mouse in each group, the average tumor growth curve of each group, and the tumor survival rate were recorded and statistically analyzed.

[0143] The results showed that the tumor growth in the KK2DP7 / OVA immunization group was the slowest, and there was a significant difference compared with the OVA combined with PBS group ( Figure 8 ).

[0144] The above-mentioned examples of the present invention provide multiple drug delivery systems based on the dendrimer peptides KDP7, KK2DP7, and KK2K4DP7, which exhibit excellent lymph node targeting. These dendrimer peptide-based delivery systems can efficiently deliver OVA to DCs. As immune adjuvants, they can stimulate DC maturation, increase DC presentation efficiency for OVA, and promote DC migration. KK2DP7, in particular, exhibits superior overall efficacy. This delivery system successfully enhances OVA lymph node targeting and strengthens the vaccine's immune efficacy. During vaccine preparation, the dendrimer peptides simply need to be incubated with the antigen for 5 minutes before subcutaneous injection to achieve efficacy. This simple and low-cost preparation method facilitates subsequent widespread use and holds great promise for future applications.

Claims

1. A dendritic polypeptide, characterized in that: The dendrimer polypeptide is formed by coupling 2, 4 or 8 DP7 polypeptide molecules to each other; the amino acid sequence of the DP7 polypeptide is VQWRIRVAVIRK; the structural formula of the dendrimer polypeptide is at least one of the following:

2. Use of the dendrimer polypeptide according to claim 1 in the preparation of an immune adjuvant.

3. A dendritic cell vaccine, characterized in that: Dendritic cells treated with the dendritic polypeptide according to claim 1 are used as the main active ingredient; the dendritic cells are also loaded with antigens.

4. The dendritic cell vaccine according to claim 3, characterized in that: The dendritic cells are myeloid DC cells or lymphoid DC cells.

5. The dendritic cell vaccine according to claim 4, characterized in that: The dendritic cells are in vitro dendritic cells obtained from the patient himself.

6. The dendritic cell vaccine according to any one of claims 3 to 5, characterized in that: The treatment is to prepare the vaccine after co-incubation with dendritic cells; or to prepare the vaccine after mixing with dendritic cells.

7. The dendritic cell vaccine according to claim 6, characterized in that Prepared using the following method: a. Take immature dendritic cells after induction culture; b. adding the dendritic polypeptide according to claim 1, as well as reagents and antigens that stimulate dendritic cell maturation, to a culture medium, and incubating to obtain antigen-loaded and mature dendritic cells; c. The antigen-loaded and mature dendritic cells obtained in step b are prepared as dendritic cell vaccines.

8. The dendritic cell vaccine according to claim 7, characterized in that: Pharmaceutically acceptable auxiliary ingredients are also included.

9. The dendritic cell vaccine according to claim 8, characterized in that: The pharmaceutically acceptable auxiliary component is at least one of a protective agent, an excipient, an immune adjuvant, a dispersant or a cell culture medium.

10. A vaccine characterized in that The method comprises an antigen and an immune adjuvant; the immune adjuvant is the dendrimer polypeptide according to claim 1.

11. The vaccine according to claim 10, characterized in that The antigen is an antigen component that can directly or indirectly provide immunogenicity in a recombinant protein vaccine, a polypeptide vaccine, an mRNA vaccine or a cell vaccine.

12. The vaccine according to claim 11, characterized in that: The cell vaccine is a dendritic cell vaccine.

13. The vaccine according to any one of claims 10 to 12, characterized in that: The antigen and immune adjuvant are in the same package or in separate packages.

14. The vaccine according to claim 13, characterized in that: Pharmaceutically acceptable auxiliary ingredients are also included.

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