CN116162138A - Dendritic polypeptides and uses thereof - Google Patents

Dendritic polypeptides and uses thereof Download PDF

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CN116162138A
CN116162138A CN202310163775.XA CN202310163775A CN116162138A CN 116162138 A CN116162138 A CN 116162138A CN 202310163775 A CN202310163775 A CN 202310163775A CN 116162138 A CN116162138 A CN 116162138A
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vaccine
dendritic
polypeptide
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杨莉
张瑞
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Sichuan University
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Sichuan University
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Abstract

The invention belongs to the field of biological medicine, and in particular relates to a dendronized polypeptide and application thereof. The invention aims to solve the technical problem of improving the efficiency of antigen transportation from an injection site to lymph nodes, thereby enhancing the immune effect of a vaccine. The technical problem is solved by the invention by providing the dendritic polypeptide DP7 and further providing the application of the dendritic polypeptide DP7 in the aspects of promoting vaccine immunity effect and the like. The dendritic DP7 has the characteristic of lymph node targeting, can be used as a drug delivery system to carry antigen into lymph nodes, improves the immune efficacy of the vaccine, can promote migration of DC cell vaccine to lymph nodes, improves the immune effect of the DC cell vaccine, and has good application prospect.

Description

Dendritic polypeptides and uses thereof
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a dendritic polypeptide and application thereof.
Background
The tumor vaccine has the characteristics of high specificity and small toxic and side effects. And is therefore a good choice to replace or assist traditional therapies to improve efficacy. The tumor vaccine has the main action mode of transmitting tumor antigen to Antigen Presenting Cell (APC), so as to activate inherent immune response and antigen specific adaptive immune response and play a systemic and specific anti-tumor role. Vaccine-based anti-tumor therapies, while having a superior theoretical basis, have not achieved satisfactory clinical therapeutic results. Therefore, increasing the in vivo immunostimulatory effect of a vaccine is one of the important research directions in the field of tumor immunotherapy. Adaptive immune responses are initiated primarily in secondary lymphoid organs, and therefore efficient accumulation of vaccine in Lymph Nodes (LNs) is a prerequisite for its induction of powerful antigen-specific immune responses. LN is the primary site of antigen presentation in which a large number of APCs colonize, these cells being adjacent to the naive T cells, enabling rapid antigen presentation upon antigen uptake. Furthermore, APCs colonizing LNs are immature phenotypes with a strong antigen uptake capacity. Thus, efficient delivery of tumor vaccines to LNs is one of the important strategies to improve vaccine efficiency.
Disappointing, because of the relatively limited tendency of endolymphatic drainage of antigens (e.g. proteins) and adjuvants (e.g. aluminium adjuvants), this suggests that a specialized targeted delivery platform with high affinity for antigens and adjuvants is required. Currently, the effective delivery of tumor vaccines to LNs is largely dependent on nano-delivery systems. The use of nanotechnology for cancer vaccine design holds great promise due to the inherent nature of nanodelivery systems that are captured by APCs. However, current nanovaccine systems still have obstacles in achieving effective tumor treatment. In part, because of unsatisfactory design of vaccine vectors, most vectors are single-acting, do not possess the efficacy of an immunoadjuvant, or complex synthetic procedures are not conducive to subsequent large-scale use.
DP7 (VQWRIRVAVIRK) is a novel cationic hydrophilic antibacterial peptide developed by the applicant on the basis of the previously studied amino acid activity prediction method, and then is modified by cholesterol, so that Chol-DP7 (DP 7-C) has the dual functions of a carrier and an immunoadjuvant. It can effectively transfer small RNAs and polypeptides to tumor cells and immune cells through the way that the small cell proteins and clathrins depend, and can be used as a delivery vehicle. As an immunoadjuvant, it can stimulate DC maturation by activating TLR2-MyD88-IKK-IκB-NF- κB signaling pathway and enhance the anti-tumor effect of tumor antigen loaded DC vaccine. While previous experiments have demonstrated that DP7-C can be used to enhance the anti-tumor effect of tumor antigen loaded DC vaccines, direct incubation of DP7-C with tumor antigen for subcutaneous administration is believed to allow patients to receive more convenient and faster treatment with lower costs due to the complexity and time-consuming preparation of DC vaccines in vitro. However, there is room for improvement in the lymph node targeting effect after the complexing of DP7-C with the antigen.
Therefore, effectively improving the lymph node drainage efficiency of the antigen is a problem to be solved in the current vaccine preparation field.
Disclosure of Invention
The invention aims to solve the technical problem of improving the lymph node drainage efficiency of an antigen, thereby improving the immune effect of an antigen-based vaccine.
The technical scheme for solving the technical problems is that the dendritic polypeptide DP7 is provided for enhancing antigen lymph node targeting efficiency, and the dendritic polypeptide DP7 has the efficacy of an immune adjuvant, so that the immune effect of the vaccine is enhanced. The polypeptide in the dendritic polypeptide is DP7 polypeptide, and the amino acid sequence of the polypeptide is VQWRIRVAVIRK (SEQ ID No. 1); the dendritic polypeptide formed by coupling DP7 polypeptides is formed by coupling 2-16 DP7 polypeptide molecules.
Preferably, the dendritic polypeptide is formed by coupling 2, 4 and 8 DP7 polypeptide molecules.
Wherein, the DP7 polypeptide molecules in the dendritic polypeptide of the present invention are coupled by using lysine as a connecting medium.
Wherein the DP7 polypeptide molecule of the dendritic polypeptide of the present invention takes part in coupling as the carbon-terminal and/or nitrogen-terminal amino acid residue. Further, the structural formula of the dendritic polypeptide of the present invention is at least one of the following:
Figure BDA0004095151730000021
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Figure BDA0004095151730000031
II type
Or alternatively
Figure BDA0004095151730000032
The invention provides application of the dendritic polypeptide in preparing a drug delivery carrier, a drug or a vaccine.
Wherein the drug delivery vehicle, drug or vaccine described in the above uses is a lymph node targeted drug delivery vehicle, drug or vaccine.
Wherein the vaccine used in the above application refers to at least one of an infectious disease vaccine, an autoimmune disease vaccine or a tumor vaccine.
Further, the infectious disease vaccine in the above use is at least one of a viral vaccine or a bacterial vaccine.
The invention also provides application of the dendritic polypeptide in preparing dendritic cell migration promoter or immunoadjuvant. Further, the above promotion of migration of dendritic cells means promotion of migration of dendritic cells to lymph nodes.
Further, the dendritic polypeptide can be incubated with dendritic cells in vitro to improve the migration capacity of the dendritic cells, or can be directly matched with the dendritic cells to further improve the migration capacity of the dendritic cells. Wherein, the direct matching use refers to injection.
The invention also provides a dendritic cell vaccine which takes dendritic cells treated by the dendritic polypeptide as a main active ingredient. Wherein the dendritic cells are myeloid DC cells or lymphoid DC cells. Further, the dendritic cells are isolated dendritic cells obtained from the patient.
Wherein, the treatment in the dendritic cell vaccine is to prepare the vaccine after incubating with the dendritic cells; alternatively, the vaccine can be prepared by mixing with dendritic cells.
Further, the dendritic cell vaccine can be prepared by the following method:
a. taking immature dendritic cells (imDCs) after induction culture;
b. adding the dendritic polypeptide into a culture medium, adding an agent for stimulating the maturation of dendritic cells and an antigen, and incubating to obtain mature dendritic cells loaded with the antigen;
c. preparing the dendritic cells loaded with the antigen and mature from the step b into dendritic cell vaccines.
Furthermore, the dendritic cell vaccine also comprises pharmaceutically acceptable auxiliary components.
Further, the pharmaceutically acceptable auxiliary component is at least one of a protective agent, an excipient, an immunoadjuvant, a dispersing agent or a cell culture medium.
The invention also provides a vaccine, which comprises an antigen and an immune adjuvant; the immune adjuvant is the dendritic polypeptide.
Wherein the antigen in the vaccine is an antigen component which can directly or indirectly provide immunogenicity in a recombinant protein vaccine, a polypeptide vaccine, an mRNA vaccine and a cell vaccine. Further, the cell vaccine is a dendritic cell vaccine.
Further, the antigen and the immunoadjuvant in the vaccine are in the same package or separate and distinct packages.
Furthermore, the vaccine also comprises pharmaceutically acceptable auxiliary components.
Wherein the pharmaceutically acceptable auxiliary component in the vaccine is at least one of a protective agent, an excipient, an immunoadjuvant, a dispersing agent or a cell culture medium.
The invention has the beneficial effects that: the invention creatively synthesizes dendritic DP7 and finds that the dendritic DP7 has special excellent performance. Firstly, the dendritic DP7 polypeptide and the active substance are co-incubated and then injected, so that the lymph node targeting efficiency of the active substance antigen can be improved, and the dendritic DP7 polypeptide can be used as a lymph node specific targeting delivery carrier. In addition, the antigen can activate cell immune response to assist antigen specific immune response, can be used as an immune adjuvant, and can enhance the immune effect of the vaccine taking the antigen as an active ingredient. In the vaccine preparation process, dendritic polypeptide and antigen are incubated for 5min and then injected subcutaneously, so that the preparation method is simple, low in cost and favorable for subsequent popularization and application, and has good application prospect. In addition, the dendritic DP7 polypeptide can promote migration of dendritic cells, can efficiently and specifically target and deliver antigens to lymph nodes through the transport function of the dendritic cells, induces anti-tumor immune response, and has particular application advantages in dendritic cell vaccines.
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Fig. 1: structure of KK2DP7 and HPLC and MS data.
Fig. 2: the efficiency of OVA uptake by DCs can be improved after incubation of KDP7, KK2DP7, KK2K4DP7 and OVA. (p < 0.0001)
Fig. 3: KDP7, KK2DP7, KK2K4DP7 were able to stimulate DC maturation. (p <0.0001 and p < 0.01)
Fig. 4: incubation with DC after incubation of KDP7, KK2DP7, KK2K4DP7 and OVA can increase the efficiency of presentation of DC to OVA
Fig. 5: KDP7, KK2DP7, KK2K4DP7 and DC after incubation can improve the migration efficiency of DC. (p <0.0001 and p < 0.01)
Fig. 6: fluorescence intensity comparison in subcutaneously administered lymph nodes after incubation of KDP7, KK2DP7, KK2K4DP7 and cy 7-OVA. (p <0.0001 and p < 0.01)
Fig. 7: and (3) detecting the immune effect of the KK2DP7/OVA complex vaccine by subcutaneous injection. a-b) CD8 + T cell proliferation efficiency; c-d) CD8 + Detecting the tetramer proportion; e-f) ELISPOT detection of antigen-specific lymphocytesAnd (3) cell reaction. (/ x:) represents p<0.0001)
Fig. 8: anti-tumor effects of KK2DP7/OVA complex vaccine subcutaneous injection. (/ x:) representation of p < 0.0001).
Detailed Description
The present invention will be specifically described below by way of description of specific embodiments.
In the previous studies of the present invention, a polypeptide having an antibacterial effect was obtained, the sequence of which was VQWRIRVAVIRK (SEQ ID No. 1), and which was designated as DP7. In further researches, the DP7 polypeptide is subjected to hydrophobic modification to form an amphiphilic compound DP7-C with the ability of self-assembling into micelles, so that on one hand, the cytotoxicity of the DP7 polypeptide can be reduced, and the antibacterial activity can be maintained; on the other hand, the polymer can be used as a delivery carrier of some medicaments after being assembled into nano particles. In addition, hydrophobically modified DP7 has the efficacy as an immunoadjuvant, which can stimulate DC maturation by activating TLR2-MyD 88-IKK-ikb-NF- κb signaling pathway and enhance the anti-tumor effect of the neoantigen loaded DC vaccine. While previous experiments have demonstrated that DP7-C can be used to enhance the anti-tumor effect of antigen-loaded DC vaccines, due to the complexity and time-consuming preparation of DC vaccines in vitro, it is believed that direct incubation of DP7-C with antigen for subcutaneous administration allows patients to receive more convenient and faster treatment at lower cost. However, further studies have found that the effect of DP7-C on targeting lymph nodes following antigen complexing is not yet satisfactory.
Therefore, the invention creatively designs the dendritic DP7 polypeptide with the beneficial characteristics of enhancing lymph node targeting and the like, and is particularly suitable for developing a nanometer vaccine system.
First, the present invention provides a dendritic polypeptide which is a dendritic polypeptide formed by coupling a plurality of DP7 polypeptide molecules to each other. The coupling may be made by coupling a plurality of DP7 polypeptide molecules, and generally may be made by coupling 2 to 16 DP7 polypeptide molecules. In the examples of the present invention, dendritic DP7 polypeptides were prepared and examined in which 2, 4 and 8 DP7 polypeptide molecules were coupled, respectively, as branched DP7 (hereinafter abbreviated as KDP 7), tetra-branched DP7 (hereinafter abbreviated as KK2DP 7) and octabranched DP7 (hereinafter abbreviated as KK2K4DP 7).
The DP7 polypeptide molecules are coupled by taking lysine as a connecting medium. The site of DP7 polypeptides involved in coupling may be a carbon-terminal or nitrogen-terminal amino acid residue.
The dendrimers of the present invention are capable of delivering drugs as drug delivery vehicles. In particular as a lymph node targeted drug delivery vehicle. The dendritic polypeptide can be used for loading and delivering protein polypeptide drugs, nucleic acid drugs and small molecule chemical drugs.
Meanwhile, the dendritic polypeptide can be used for preparing vaccines. For example, it can be used for preparing infectious disease vaccine, autoimmune disease vaccine or tumor vaccine.
The dendritic polypeptide can activate cell immune response to assist antigen specific immune response, and can be used as an immune adjuvant for preparing various vaccines.
The dendritic polypeptides of the present invention can also be used to prepare dendritic cell migration promoters. The promotion of migration of dendritic cells means promotion of migration of dendritic cells to lymph nodes.
In particular applications, the dendritic polypeptides of the present invention may be co-incubated with dendritic cells in vitro to increase their ability to migrate, or used in direct association with dendritic cells to further increase their ability to migrate. For example, the injection can be directly matched with dendritic cells.
The invention also provides a dendritic cell vaccine, which takes the dendritic cells treated by the dendritic polypeptide as a main active ingredient. The dendritic cells can be myeloid DC cells or lymphoid DC cells. In daily operations, isolated dendritic cells obtained from the patient's body may be selected.
The treatment is to incubate the dendritic cells and then take the incubated dendritic cells to prepare vaccines for use; alternatively, the vaccine can be prepared by mixing with dendritic cells.
Specific preparations can be referred to as follows:
a. taking immature dendritic cells (imDCs) after induction culture;
b. adding the dendritic polypeptide into a culture medium, adding an agent for stimulating the maturation of dendritic cells and an antigen, and incubating to obtain mature dendritic cells loaded with the antigen;
c. preparing the dendritic cells loaded with the antigen and mature from the step b into dendritic cell vaccines.
Naturally, the vaccine may also include pharmaceutically acceptable auxiliary ingredients. Such as at least one of a protective agent, an excipient, an immunoadjuvant, a dispersing agent, or a cell culture medium.
The invention further provides a vaccine comprising an antigen and an immunoadjuvant; the immune adjuvant is the dendritic polypeptide.
Wherein the antigen is an antigen component capable of directly or indirectly providing immunogenicity in recombinant protein vaccines, polypeptide vaccines, mRNA vaccines and cell vaccines. Such as antigen proteins, antigen peptides, mRNA for expressing antigen proteins or antigen peptides, antigens for preparing dendritic cell vaccines, and the like.
The antigen and the dendritic cell migration promoting agent may be in the same package or in separate and distinct packages. Pharmaceutically acceptable auxiliary components are also included.
It will be appreciated by those skilled in the art that the antigens described above in the present invention may be tumour antigens, including specific proteins or polypeptides expressed by tumour cells. Such as WT1, MUC1, EGFRvIII, HER-2, MAGE-A3, NY-ESO-1, PSMA, GD2 or MART1, or a combination of individual mutant neoantigens based on patient tumor sequence determination.
The antigen may also be a viral antigen, such as a protein or polypeptide that constitutes a viral part, or a specific protein or polypeptide that is expressed in a virus-infected cell under control of a viral expression mechanism. Viral associated antigens such as EBV, LMP2, HPV E6E 7, adenovirus 5Hexon or HCMV pp 65.
The antigen may also be a bacterial antigen, including proteins or polypeptides expressed by bacteria. Such as Pseudomonas aeruginosa antigen, tetanus bacillus antigen, streptococcus pneumoniae, salmonella and the like.
It will also be appreciated by those skilled in the art that the antigen may also be a disease-associated antigen, including autoimmune-associated antigens, such as those involved in or over-expressed during autoimmune diseases or conditions. For example, ppIAPP, IGRP, GAD or myelin basic protein antigen.
The present invention is described in further detail below by way of examples.
The experimental materials and equipment mainly used in the examples are as follows:
1. cell strain for experiment and experimental animal
EG7-OVA cell lines were purchased from American type culture Collection (American Type Culture Collection, ATCC). The culture was performed with RPMI-1640 (Gibico) medium containing 10% fetal bovine serum (Fetal bovine serum, FBS, gibico). C57/BL6J female mice of 6-8 weeks of age used in the experiments were purchased from Experimental animals Inc. of Lewa, beijing and were kept in SPF-grade environment.
2. Main reagent material and kit
Cell culture medium for experiments: 1640 medium (RPMI-1640) and fetal bovine serum (Fetal bovine serum, FBS) were purchased from Gibco corporation, USA.
Cytokines CCL19, CCL21 were purchased from absin biotechnology limited.
24-well Transwell Small Kongzi Corning Biotechnology Co., ltd, 0.5 μm.
GM-CSF is purchased from Shanghai Biotechnology Co., ltd.
ELISPOT kit and CFDA-SE cell labelling kit were purchased from Biyun Biotech Co.
3. Main instrument and equipment
Flow cytometer: FACSCalibur.
EXAMPLE 1 Synthesis of the branched Polypeptides KDP7, KK2DP7, KK2K4DP7
Branched DP7 polypeptides are synthesized by standard solid phase peptide synthesis. Briefly described as follows: fmoc-Lys (Fmoc) -OH was hung on the resin, 2 Fmoc was removed, and 2 NH2 groups were exposed, which was condensed according to the polypeptide sequence VQWRIRVAVIRK from right to left, to obtain a 2-branched polypeptide.
By attaching Fmoc-Lys (Fmoc) -OH to the resin, removing 2 Fmocs, exposing 2 NH2, connecting 2 exposed NH2 with 2 Fmoc-Lys (Fmoc) -OH, removing all Fmoc, exposing 4 amino groups, condensing according to the polypeptide sequence VQWRIRVAVIRK from right to left, thus obtaining the 4-branch polypeptide.
By attaching Fmoc-Lys (Fmoc) -OH to the resin, 2 Fmoc were removed, 2 NH2 were exposed, 2 Fmoc-Lys (Fmoc) -OH was attached to 2 exposed NH2, all Fmoc were removed, 4 amino groups were exposed, and 4 Fmoc-Lys (Fmoc) -OH was attached. After all Fmoc were removed, 8 amino groups were exposed and condensed according to the polypeptide sequence VQWRIRVAVIRK from right to left, thus obtaining an 8-branched polypeptide.
HPLC and MS detection of the synthesized branched peptides was performed to verify the synthesized end products. The structure and HPLC and MS results are shown in figure 1. The results show that branched polypeptides KDP7, KK2DP7 and KK2K4DP7 are synthesized.
Example 2 detection of the efficiency of DC uptake of KDP7/OVA, KK2DP7/OVA, KK2K4DP7/OVA Complex 1, preparation of KDP7, KK2DP7, KK2K4DP7 and OVA Complex
KDP7(VQWRIRVAVIRKK)、KK2DP7((VQWRIRVAVIRK) 2 KK) and KK2K4DP7 (((VQWRIRVAVIRK) 2 K) 2 KK) was synthesized by solid phase peptide synthesis from shanghai Chu peptide biotechnology limited. The final product was purified by HPLC and identified by mass spectrometry.
Directly adding deionized water into KDP7, KK2DP7 and KK2K4DP7 freeze-dried powder for dissolution, and sub-packaging at-20deg.C for preservation. When preparing their complexes with OVA, they were incubated with an aqueous solution of OVA in deionized water and medium for 5 min.
2. Acquisition and culture of DC cells
(1) Taking tibia and fibula of an adult C57BL/6J female mouse with age of about 6 weeks, placing the tibia and fibula in 75% ethanol for soaking for 5min to kill bacteria, removing muscle tissues, and soaking the fibula in a culture medium of RPMI 1640+1% PS; cutting both ends of the leg bones by using sterilized scissors, and sucking fresh RPMI 1640+1% PS culture medium by using a syringe to blow out bone marrow cells until the bone marrow cells are completely blown out;
(2) filtering the collected culture medium containing bone marrow cells with 70 μm sieve, centrifuging at 1200rpm for 3min, discarding supernatant, dissolving with erythrocyte lysate (1.3 g Tris-base and 3.74g NH4Cl, dissolving with 490ml ultra-pure water, adjusting pH of the solution to 7.2-7.4 with concentrated hydrochloric acid, adding ultra-pure water to 500ml, removing bacteria with 0.22 μm filter, preserving at 4deg.C, and preparing for use), re-suspending cells, standing at room temperature for 3min, centrifuging at 1200rpm for 3min, washing erythrocyte lysate with RPMI1640+10% FBS+1% PS culture medium, and re-suspending cells;
(3) the resuspended cells were separated into culture dishes, 2X 10 per dish 6 -3×10 6 For each cell, 10ml of RPMI1640+10% FBS+1% PS medium was added to each dish, and 20ng/ml GM-CSF cytokine was added, and the dishes were placed in a 37℃cell incubator to culture, and fresh RPMI1640+10% FBS+1% PS medium containing 20ng/ml GM-CSF was added on the third day of culture until the 8 th day of culture to obtain immature DCs (imDCs). Taking DCs cultured until the 8 th day, washing the culture medium by using 1ml PBS, re-suspending the cells by using 100 mu l PBS, adding 1 mu l APC Hamster Anti-Mouse CD11c streaming antibody, slightly mixing, and then placing the mixture at 4 ℃ for light-shielding incubation for 40min; after the incubation, the excess antibody was washed off with PBS, and the cells were resuspended in 200. Mu.l of PBS, and CD11c was detected using a flow cytometer + The proportion of DCs. When the CD11c ratio is greater than 80%, then DC induction is indicated to be successful.
3. Efficiency of transfection of DC with KDP7/OVA, KK2DP7/OVA, KK2K4DP7/OVA Complex
Immature DCs from day 8 of culture were plated in 24 well plates at 5X 10 wells per well 5 Individual cells. Mu.l of KDP7/OVA, KK2DP7/OVA, KK2K4DP7/OVA complex incubated for 5min in 1640 complete medium, with FITC-labeled use concentration of 10. Mu.g/ml and KDP7, KK2DP7, KK2K4DP7 use concentrations of 5, 10, 20, 40. Mu.g/ml, were each added. After further culturing for 24 hours, the cells were harvested, washed off with excess medium, resuspended in 200. Mu.l PBS, and the proportion of fluorescent cells was detected using a flow cytometer.
From the results, it was found that the transfection efficiencies of KDP7, KK2DP7, KK2K4DP7 into OVA and DC were all 90% or more from the viewpoint of transfection efficiency (FIG. 2).
Example 3 detection of the efficiency of KDP7, KK2DP7, KK2K4DP7 to stimulate DC maturation
Immature DCs from day 8 of culture were plated in 24 well plates at 5X 10 wells per well 5 Individual cells. Treated with 10, 20, 40, 80. Mu.g/ml KDP7, KK2DP7, KK2K4DP7, respectively. After 24h, cells were collected and stained for CD11c, CD80 and CD86 to detect maturation of BMDCs.
From the results of flow cytometry, KDP7, KK2DP7, KK2K4DP7 all induced maturation of DCs, and wherein KK2DP7 was more advantageous in inducing maturation of DCs than KDP7 and KK2K4DP7 (FIG. 3).
Example 4 detection of the efficiency of the DC presentation of KDP7/OVA, KK2DP7/OVA, KK2K4DP7/OVA complexes
Immature DCs from day 8 of culture were plated in 24 well plates at 5X 10 wells per well 5 Individual cells. Mu.l of the KDP7/OVA, KK2DP7/OVA, KK2K4DP7/OVA complex incubated in 1640 complete medium for 5min, at a concentration of 10ug/ml for OVA and 40 ug/ml for KDP7, KK2DP7, KK2K4DP7, respectively, were added. Culturing for 24 hr, 48 hr, and 72 hr, collecting cells, washing off excessive culture medium, and washing with SIINFEKL-H2K b+ Antibody 4 ℃ staining 40min, adding PBS washing once. The fraction of cells with fluorescence was then detected by flow cytometry, resuspended in 200 μl PBS.
From the results, it was found that the antigen presenting efficiency of KDP7, KK2DP7, KK2K4DP7 was significantly higher in cells after transfection of OVA to DC than in KDP7 and KK2DP7 groups at 72h (FIG. 4).
Example 5 detection of the efficiency of KDP7, KK2DP7, KK2K4DP7 in vitro stimulation of DC migration
Immature DCs from day 8 of culture were plated in 24 well plates at 5X 10 wells per well 5 Individual cells. KDP7, KK2DP7 and KK2K4DP7 were added at 10, 20 and 40. Mu.g/ml, respectively, and treated for 24 hours. Cells were digested and washed 3 times with 1640 double medium and resuspended with 1640 medium. Cells were plated in 24 wellsIn the transwell wells of the plates (pore size 5.0 μm), 1X 10 per well 5 The individual cell volume was 100. Mu.l. The following chamber is added: 500 μl1640 complete medium+CCL19 (250 ng/ml) +CCL21 (250 ng/ml); after 24 hours, the cells of the lower chamber were counted and the efficiency of migration of DCs was calculated.
The results show that KDP7, KK2DP7, KK2K4DP7 all improved DC migration efficiency in vitro to some extent compared to the control group, and that KK2DP7 was the most efficient in promoting DC migration (fig. 5).
Example 6 lymph node targeting efficiency detection of branched polypeptide/OVA complexes
Mu.g of KDP7, KK2DP7, KK2K4DP7 and 20ug of Cy7-OVA were incubated in 100. Mu.l of PBS for 5min and injected beside the subcutaneous lymph nodes of the mice. After 4h, the proximal and distal lymph nodes of the mice were subjected to fluorescence imaging, and fluorescence intensity in the lymph nodes was detected and analyzed statistically.
The results showed a significant increase in fluorescence intensity in both proximal and distal lymph nodes of the KK2DP7/OVA group compared to the OVA group alone and the KDP7/OVA combination KK2K4DP7/OVA group (fig. 6). The subsequent experiments mainly select KK2DP7 with strong lymph node targeting ability as a research object.
Example 7 detection of the immune Effect of KK2DP7/OVA Complex
Female mice of 6-8 weeks of age C57BL/6J were randomly divided into 3 groups (PBS group, OVA group, KK2DP7/OVA group) of 3 mice each. Mu.l of OVA (20. Mu.g) or KK2DP7 (60. Mu.g)/OVA (20. Mu.g) was injected beside the lymph nodes of the mice on days-21, -14, -7. Mice were sacrificed on day 0 and their spleens were taken for subsequent experiments.
(1) Flow cytometry detection of T cell proliferation-CFSE division method
(1) 7 days after the last immunization, spleen lymphocytes of the mice were isolated and taken 3X 10 6 Cells were suspended with 1ml cfdase cell marker fluid and placed in 15ml bd tubes;
(2) diluting the CFDASE storage solution to 2X by using the CFDASE cell marking solution, and uniformly mixing;
(3) 1ml of CFDA stock solution (2X) was added to (1) and gently mixed;
(4) incubating for 30min at 37 ℃ in dark;
(5) adding 10ml1640+10% FBS culture medium, mixing uniformly, and stopping marking;
(6) centrifuging to remove supernatant, and repeating (5);
(7) cells were diluted to 2X 10 with 1640+10% FBS medium 6 Mu.l of cells (1X 10) were added per well per ml in 96-well plates 5 Individual per well) with 10 μg/ml OVA, respectively 257-264 Polypeptide stimulation, 3 multiple wells were placed in each group, total volume was made up to 200 μl with 1640 complete medium containing 10% FBS, and after mixing, 96 well plates were placed in CO 2 Culturing in an incubator for 4 days.
(8) Cultured cells were collected into flow tubes, 100. Mu.l of PBS was added to resuspend the cells, 1. Mu.l of surface marker (PE-CD 8) antibody of T cells was added to each tube, and flow analysis was performed.
The experimental results show that: the mouse T cell proliferation efficiency was significantly higher in the KK2DP7/OVA immunized group than in the OVA and PBS groups (FIGS. 7a-7 b).
(2) Detection of CD8 tetramers in post-immunization spleen by flow cytometry
(1) 7 days after the last immunization, spleen lymphocytes of the mice were isolated and 1X 10 were taken 6 Individual cells were cultured in 12-well plates using 1640+10% fbs+ps;
(2) add 10. Mu.g/ml OVA 257-264 Stimulating for 7 days;
(3) cultured cells were collected into flow tubes, 100. Mu.l of PBS was added to resuspend the cells, and 1. Mu. l T cell surface marker (PE-CD 8) antibody and tetramer dye were added, respectively, and then flow analysis was performed.
The experimental results show that: mouse CD8 of KK2DP7/OVA immunized group + T cell tetramer ratio was significantly higher than in the OVA and PBS groups (FIGS. 7c-7 d).
(3) Detection of antigen-specific lymphocyte responses after immunization by ELISPOT
The first day:
(1) activation of the pre-coated plates: adding 200 mu l of RPMI-1640 serum-free culture medium into each hole, standing at room temperature for 5-10min, and buckling out;
(2) adding a cell suspension: the isolated spleen lymphocytes were resuspended to 5X 10 6 Cells/ml, 100. Mu.l of spleen lymphocyte suspension and control,i.e. 5X 10 5 Individual cells/wells;
(3) adding a stimulus: OVA (OVA) 257-264 10 mug/well, 3 compound wells are arranged;
(4) incubation: covering the plate cover, and placing the plate cover into a 37 ℃ and 5% CO2 incubator for culturing for 48 hours;
third day:
(1) lysing the cells: the cells and medium in the wells were poured off and ice-cold ddH was added 2 O,200 μl/well, and placing in a refrigerator at 4deg.C for 10min to hypotonically lyse the cells;
(2) washing the plate: pouring the liquid in the hole, 1 XWash buffer, 200. Mu.l/hole, washing 5-7 times; each time stay for 30-60s; finally, buckling and drying on the absorbent paper;
(3) incubation of detection antibody: adding diluted biotin-labeled antibody working solution into each experimental hole to obtain 100 μl/hole; incubating for 1h at 37 ℃;
(4) washing the plate: according to (2);
(5) enzyme-linked avidin incubation: adding diluted enzyme-labeled avidin working solution into each experimental hole, wherein 100 μl/hole; incubating for 1h at 37 ℃;
(6) washing the plate: according to (2);
(7) color development: adding the working solution of the AEC color development liquid into each experimental hole, wherein the working solution of the AEC color development liquid is 100 mu l/hole; developing at 37 deg.C in a incubator in dark place, and checking every 5-10 min;
(8) terminating the color development: pouring the liquid in the hole, uncovering the base of the plate, and using ddH 2 O washing the front and back surfaces and the base for 5 times, and stopping color development; placing the plate at a shade place at room temperature, and closing the base after the plate is naturally dried;
(9) ELISPot plate spot counts, and various parameters of the spots were recorded for statistical analysis.
The experimental results show that: the number of IFN-gamma spots generated by the mouse antigen-specific lymphocytes of the KK2DP7/OVA immunized group was significantly higher than those of the OVA and PBS groups (FIGS. 7e-7 f).
Example 8 detection of anti-tumor Effect of KK2DP7/OVA Complex
Female 6-8 week old C57BL/6J mice were randomly assigned to 3 groups (PBS group, OVA group, KK2DP7/OVA group) of 6 mice each. Subcutaneous inoculation of the right back of each mouse on day 0 1X 10 6 EG7-OVA tumor cells were injected with 100. Mu.l KK2DP7 (60. Mu.g)/OVA (20. Mu.g) beside the lymph nodes of mice on days 4, 11 and 18, respectively, and tumor size was measured every 2 days after tumor growth, with a tumor volume calculation formula of 0.52X length X width 2 . Tumor growth curves and average tumor growth curves and tumor survival rates for each group were recorded for each mouse within each group and statistically analyzed.
The results showed that the KK2DP7/OVA immunized group had the slowest tumor growth and had significant differences compared to the OVA-combined PBS group (fig. 8).
In the above examples of the invention, various drug delivery systems based on dendritic polypeptides KDP7, KK2DP7, KK2K4DP7 with better lymph node targeting are provided. The dendritic polypeptide-based delivery system is capable of delivering OVA into DCs with high efficiency, is capable of stimulating DC maturation as an immunoadjuvant, increases the presentation efficiency of DCs to OVA, and has the ability to promote DC migration. The KK2DP7 has more excellent overall effect. The delivery system successfully improves the lymph node targeting of OVA and enhances the immune effect of the vaccine. In the vaccine preparation process, the effect can be achieved only by incubating the dendritic polypeptide and the antigen for 5min and then injecting subcutaneously, the preparation method is simple and low in cost, is beneficial to subsequent popularization and use, and has good application prospect.

Claims (25)

1. A dendritic polypeptide, characterized in that: the polypeptide is DP7 polypeptide, and the amino acid sequence of the polypeptide is VQWRIRVAVIRK; dendritic polypeptides formed by coupling the two polypeptides are formed by coupling 2-16 DP7 polypeptide molecules.
2. The dendritic polypeptide of claim 1, wherein: is formed by coupling 2, 4 and 8 DP7 polypeptide molecules.
3. The dendritic polypeptide according to any one of claims 1-2, characterized in that: the DP7 polypeptide molecules are coupled by using lysine as a connecting medium.
4. A dendritic polypeptide according to any of claims 1-3, characterized in that: the DP7 is involved in coupling at the carbon-terminal and/or nitrogen-terminal amino acid residues.
5. The dendritic polypeptide according to any of the claims 1-5, characterized in that the structural formula of the dendritic polypeptide is at least one of the following:
Figure FDA0004095151720000011
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Figure FDA0004095151720000021
6. use of a dendritic polypeptide according to any of claims 1 to 5 for the preparation of a drug delivery carrier, a drug, or a vaccine.
7. Use according to claim 6, characterized in that: the drug delivery carrier, drug or vaccine is lymph node targeted drug delivery carrier, drug or vaccine.
8. Use of a dendritic polypeptide according to any of claims 1-5 for the preparation of a vaccine.
9. Use according to claim 9, characterized in that: the vaccine refers to at least one of an infectious disease vaccine, an autoimmune disease vaccine or a tumor vaccine.
10. The use according to claim 9, characterized in that the infectious disease vaccine is at least one of a viral vaccine or a bacterial vaccine.
11. Use of a dendritic polypeptide according to any of claims 1 to 5 for the preparation of a dendritic cell migration promoting agent or an immunoadjuvant.
12. Use according to claim 11, characterized in that: the promotion of migration of dendritic cells means promotion of migration of dendritic cells to lymph nodes.
13. Use according to claim 12, characterized in that: the dendritic cell migration promoting agent can be incubated with the dendritic cells in vitro to enhance their migration ability, or can be directly conjugated with the dendritic cells to further enhance their migration ability.
14. Use according to claim 13, characterized in that: the direct matching use refers to injection.
15. A dendritic cell vaccine characterized in that: dendritic cells treated with the dendritic polypeptide according to any one of claims 1 to 5 as a main active ingredient.
16. The dendritic cell vaccine of claim 15, wherein: the dendritic cells are myeloid DC cells or lymphoid DC cells.
17. The dendritic cell vaccine of claim 15, wherein: the dendritic cells are isolated dendritic cells obtained from a patient.
18. The dendritic cell vaccine of any one of claims 15-17, wherein: the treatment is to prepare vaccine after incubating with dendritic cells; alternatively, the vaccine can be prepared by mixing with dendritic cells.
19. The dendritic cell vaccine according to any one of claims 15 to 18, characterized in that it is prepared using the following method:
a. taking immature dendritic cells after induction culture;
b. adding the dendritic polypeptide according to any one of claims 1 to 5 to a culture medium, and adding an agent for stimulating the maturation of dendritic cells and an antigen, and incubating to obtain mature dendritic cells loaded with the antigen;
c. preparing the dendritic cells loaded with the antigen and mature from the step b into dendritic cell vaccines.
20. The dendritic cell vaccine of any one of claims 15-19, wherein: pharmaceutically acceptable auxiliary components are also included.
21. The dendritic cell vaccine of claim 16, wherein: the pharmaceutically acceptable auxiliary component is at least one of a protective agent, an excipient, an immunoadjuvant, a dispersing agent or a cell culture medium.
22. The vaccine is characterized by comprising an antigen and an immunological adjuvant; the immunoadjuvant is the dendritic polypeptide according to any one of claims 1 to 5.
23. The vaccine of claim 22, wherein the antigen is an antigen component that provides immunogenicity directly or indirectly in a recombinant protein vaccine, polypeptide vaccine, mRNA vaccine, or cellular vaccine; further, the cell vaccine is a dendritic cell vaccine.
24. The vaccine of claim 22 or 23, wherein: the antigen and the immune adjuvant are in the same package or in separate and distinct packages.
25. The vaccine of any one of claims 22-24, wherein: pharmaceutically acceptable auxiliary components are also included.
CN202310163775.XA 2023-02-24 2023-02-24 Dendritic polypeptides and uses thereof Pending CN116162138A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113817677A (en) * 2021-09-29 2021-12-21 四川大学 Use of pantothenic acid or derivatives thereof and alpha-D-glucose-1, 6-bisphosphate or derivatives thereof for promoting DC migration

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113817677A (en) * 2021-09-29 2021-12-21 四川大学 Use of pantothenic acid or derivatives thereof and alpha-D-glucose-1, 6-bisphosphate or derivatives thereof for promoting DC migration
CN113817677B (en) * 2021-09-29 2023-08-18 四川大学 Use of pantothenic acid or derivatives thereof and alpha-D-glucose-1, 6-biphosphoric acid or derivatives thereof for promoting DC migration

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