CN111249453B - Nano vaccine and preparation method thereof - Google Patents
Nano vaccine and preparation method thereof Download PDFInfo
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- CN111249453B CN111249453B CN202010119256.XA CN202010119256A CN111249453B CN 111249453 B CN111249453 B CN 111249453B CN 202010119256 A CN202010119256 A CN 202010119256A CN 111249453 B CN111249453 B CN 111249453B
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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Abstract
The invention relates to a multi-component carrier-free integrated nano vaccine which is established by using unsaturated fatty acid and contains antigen peptide, immunologic adjuvant and micromolecule medicine and a method thereof. The nano vaccine comprises an antigen, an immunologic adjuvant and unsaturated fatty acid, wherein the antigen is covalently coupled with the unsaturated fatty acid through a-SH group, and the immunologic adjuvant is coupled with the unsaturated fatty acid through an S-S bond. The invention utilizes unsaturated fatty acid to modify tumor-targeted antigen peptide and immunologic adjuvant, takes the unsaturated fatty acid as a hydrophobic core in aqueous solution to carry out self-assembly, and small molecular drugs with strong hydrophobicity can be loaded in the hydrophobic core. The nano vaccine can improve the residence time of the antigen peptide in lymph nodes, increase the endocytosis of DC cells to the antigen peptide and improve the anti-tumor immunity effect.
Description
Technical Field
The invention relates to the technical field of medical biology, in particular to the technical field of vaccines; in particular to a multi-component carrier-free integrated nano vaccine which is established by utilizing unsaturated fatty acid and contains antigen peptide, immunologic adjuvant and small molecular medicine.
Background
With the aggravation of factors such as aging of population and the like, the morbidity and mortality of cancer in China continuously rise, and the cancer becomes a main cause of death of people in China and seriously harms the health of people in China. In recent years, anti-tumor immunotherapy has been rapidly developed, and particularly, immune checkpoint inhibitor therapy (e.g., PD-1 inhibitor and CTLA-4 inhibitor) has clinically significant efficacy. The main principle of immune checkpoint inhibitor therapy is to block the immunosuppressive complex (e.g. PD-1/PD-L1, CTLA-4/B7 complex) formed between tumor cells and effector T Cells (CTLs) by antibodies or small molecule compounds, thereby releasing the immunosuppressive signal in the CTLs and restarting the CTL attack on the tumor cells.
However, immune checkpoint inhibitor therapy also has certain limitations. First, drug responsiveness varies widely between different tumor types. One of the main reasons for this difference is that low mutation-loaded tumors produce less tumor-specific antigens (TSA), which in turn weaken the efficiency of spontaneous immune cell recognition of tumor cells, preventing the widespread use of immune checkpoint inhibitor therapy, and also suggesting a need to coordinate better tumor-specific antigen presentation methods to assist immune checkpoint inhibitor therapy. Secondly, in some patients, side effects (immune-related toxic side effects) of immune checkpoint inhibitor therapy are more pronounced, including skin itching, rash, vitiligo, diarrhea, even liver damage and heart failure, which are mainly caused by non-specific attack of immune cells on non-tumor cells. Therefore, in the course of anti-tumor immunotherapy, the number of high-activity tumor-targeting CTLs is the key to improve the anti-tumor efficacy and reduce the side effects.
The activation of CTLs depends mainly on Antigen Presenting Cells (APCs), particularly cross-Presenting Dendritic Cells (DCs), which can present endocytosed TSA on the Cell surface via MHCI (cross-presentation), specifically activating CTLs for tumor killing. Moreover, DCs can express a high level of costimulatory molecules (costimulating factor) while presenting antigen peptide-MHC molecule complexes, and directly provide dual signals to activate naive CD8+ T cells to express and secrete IL-2, thereby promoting autologous proliferation and differentiation into CTLs.
However, the uptake rate of the free peptide fragment by the DC cell is not high, and the in vivo circulation period of the free peptide fragment is short, so that the effect of the anti-tumor vaccine is greatly limited. To solve this problem, anti-tumor vaccines are often used with immune adjuvants (e.g., CpG-ODN, Montanide, R848) to further stimulate DC and CTL cells, but immune adjuvants also cause side effects such as muscular atrophy at the injection site, influenza-like reactions, headache. Therefore, the optimization and innovation of the administration mode of the immune adjuvant are also a research hotspot and difficulty in the field of anti-tumor immunity. The existing immunologic adjuvant nano-drug is mainly in the form that a nano-carrier wraps an adjuvant single-drug, is not connected with tumor specific antigen peptide, and cannot ensure that the same DC can simultaneously receive the immunologic adjuvant and the tumor specific antigen peptide, so that the curative effect of adjuvant/tumor specific antigen peptide combination therapy can be reduced, and the immune-related toxic and side effects are increased. The present invention has been made to solve the above problems.
Disclosure of Invention
The invention aims to improve the curative effect and side effects of the anti-tumor vaccine immunoadjuvant in the prior art for reducing adjuvant/tumor specific antigen peptide, and provides a tumor vaccine which utilizes unsaturated fatty acid to chemically modify the antigen peptide and the immunoadjuvant and utilizes a carrier-free self-assembly technology to load the antigen peptide, the immunoadjuvant and hydrophobic small molecular drugs on the same nanoparticle, thereby improving the immune effect of the tumor antigen polypeptide vaccine.
In order to achieve the purpose, the invention is realized by the following technical scheme: a nano-vaccine, comprising: the antigen, the immune adjuvant and the unsaturated fatty acid are characterized in that the antigen is coupled with the unsaturated fatty acid through a covalent bond, and the immune adjuvant is coupled with the same or different unsaturated fatty acid through a covalent bond.
Preferably, the antigen is selected from the form of a peptide, protein, glycoprotein, glycopeptide, proteoglycan, or combination thereof, that does not contain cysteine Cys.
Preferably, the N-terminal of the antigen without cysteine Cys is added with Cys- (b) sequence or Cys- (a) - (b) sequence, wherein, (a) is hydrophilic group, and (b) is enzyme cutting site group of cathepsin. The sequence (a) is used to balance the hydrophilicity of the whole antigen peptide, if the hydrophilicity of the following antigen peptide is poor, the hydrophilic group (a) needs to be added, and if the hydrophilicity of the antigen peptide is good, only one amino acid or even none can be added.
Preferably, said (a) is a peptide fragment without cysteine Cys, including but not limited to a single amino acid, a dipeptide or a polypeptide; more preferably, said (a) is selected from the group consisting of-Ser-group, -Gly-group, dipeptide-Ser-Ser-or-Ser-Gly-.
B is the amino acid sequence of an enzyme cleavage site selected from any one of cathepsins A-Z, known in the art as cathepsin from A, B, C, D, E, F … … X, Y, Z. More preferably, (b) is any one of the enzyme cutting site amino acid sequences selected from cathepsin B, F, H, K, L, S, V, B, C, H, X. In a specific technical scheme of the invention, the (b) is an enzyme cutting site of cathepsin S, namely-Val-Val-Arg-tripeptide, and VVR is favorable for breaking and releasing the model antigen under the action of the cathepsin S in lysosomes after the nanoparticles are endocytosed by DC cells.
Preferably, the immunoadjuvant is selected from-OH containing adjuvants, more preferably from compounds that activate the antigen presenting ability of DC cells, including but not limited to: a TLR signaling pathway agonist, a STING signaling pathway agonist, and a compound comprising an-OH group within the molecular structure; more preferably, the immunological adjuvant includes, but is not limited to, one or more of R848, CpG, Imiquimod.
In a specific technical scheme of the invention, the antigen is covalently coupled with unsaturated fatty acid through-SH of cysteine Cys residue after the N terminal modifies Cys- (b) sequence or Cys- (a) - (b) sequence, and the immunoadjuvant is coupled with unsaturated fatty acid of the same kind or different kinds through S-S bond. More preferably, the-OH of the immunoadjuvant is coupled to the unsaturated fatty acid via an S-S bond.
Preferably, the unsaturated fatty acid is selected from: monounsaturated fatty acids or polyunsaturated fats, said monounsaturated fatty acids selected from one or more of oleic acid, erucic acid, palmitoleic acid, trans-oleic acid; polyunsaturated fat selected from one or more of linoleic acid, linolenic acid, arachidonic acid, and DHA.
Preferably, the nano tumor vaccine also contains a small molecule drug with strong hydrophobicity. The small molecule drug with strong hydrophobicity is selected from one or more small molecule drugs for regulating a DC intracellular signal pathway; including but not limited to: STATIC, artemisinite Artesunate, C188-9, STATIC, STATUS 3 inhibitors.
In some embodiments, the antigen is coupled to the unsaturated fatty acid I by a covalent bond and the immunoadjuvant is coupled to the homologous unsaturated fatty acid I by a covalent bond. In some embodiments, the antigen is coupled to unsaturated fatty acid I by a covalent bond and the immunoadjuvant is coupled to the cognate unsaturated fatty acid II by a covalent bond.
In some embodiments, where the antigen is an antigenic peptide sequence containing cysteine Cys, the antigenic peptide sequence should be selected for replacement so that it does not contain Cys.
The invention also provides a preparation method of the nano vaccine, which comprises the following steps:
(1) preparing an antigen, and preparing the antigen,
(2) carrying out condensation reaction on N- (2-aminoethyl) maleimide and unsaturated fatty acid to obtain an intermediate product I, and carrying out Michael addition reaction on the intermediate product I and the antigen to obtain the antigen covalent coupling unsaturated fatty acid:
(3) carrying out esterification reaction on 2, 2' -dithiodiethanol and unsaturated fatty acid to obtain an intermediate product II, carrying out esterification reaction on-unsaturated fatty acid-S-S-OH, carrying out reaction on-unsaturated fatty acid-S-OH and p-nitro phenyl chloroformate to obtain an intermediate product III, and carrying out ester exchange reaction on the intermediate product III and an immunologic adjuvant to obtain a final product: the immunologic adjuvant is coupled with unsaturated fatty acid through covalent bond;
(4) dissolving the antigen of the coupling unsaturated fatty acid obtained in the step (2) and the immune adjuvant of the coupling unsaturated fatty acid obtained in the step (3) into an organic solvent, and fully and uniformly mixing;
(5) and (4) carrying out ultrasonic treatment on the mixture obtained in the step (4), and then dropwise adding the mixture into ultrapure water for injection to obtain the self-assembled nano vaccine.
Preferably, the antigen is in the form of a peptide, protein, glycoprotein, glycopeptide, proteoglycan, or combination thereof, that does not contain cysteine Cys.
Preferably, the N-terminal of the antigen without cysteine Cys is added with Cys- (b) sequence or Cys- (a) - (b) sequence, wherein, (a) is hydrophilic group, and (b) is enzyme cutting site group of cathepsin.
Preferably, the unsaturated fatty acid in step (2) and the unsaturated fatty acid in step (3) are of the same kind or different kinds.
Preferably, in the step (4), a small molecule drug with strong hydrophobicity is also added into the organic solvent. In the step (4), the organic solvent is selected from DMSO, ethanol and acetone.
In some embodiments, the volume of the ultrapure water for injection in step (5) is at least 9 times or more the volume of the mixture.
In some embodiments, a small molecule drug with strong hydrophobicity refers to a small molecule that is poorly soluble in water, has a solubility of less than 0.01g/100g solvent, and is soluble in organic solvents (greater than 1g/100g solvent).
In some embodiments, the antigen is a B cell antigen or a T cell antigen. In some embodiments, the B cell antigen is a weakly immunogenic antigen. In some embodiments, the B cell antigen is a small molecule. In some embodiments, the B cell antigen is a carbohydrate. In some embodiments, the B cell antigen is an addictive substance. In some embodiments, the B cell antigen is a toxin. In some embodiments, the T cell antigen is a degenerative disease antigen, an infectious disease antigen, a cancer antigen, an allergic disease antigen, an autoimmune disease antigen, a alloantigen, a xenoantigen, an allergen, an addictive substance, or a metabolic disease enzyme or enzyme product. In some embodiments, the T cell antigen is a universal T cell antigen.
In some embodiments, the nano-vaccine is formed by self-assembly. Self-assembly refers to the process of forming a nano-vaccine and/or vector using components that can adapt themselves to predictably and reproducibly form a nano-vaccine and/or vaccine vector in a predictable manner. In some embodiments, the nano-vaccine is formed by using polar or amphoteric biomaterials (which themselves are set to form nanomaterials of predictable size, composition, and composition location with respect to one another). According to the present invention, immunomodulatory, immunostimulatory, and/or targeting agents may be bound to the polar biomaterial such that when the nano-vaccine self-assembles, there is a reproducible pattern of localization and density of the agents on/within the nano-vaccine.
In some specific embodiments, the antigen covalent coupling unsaturated fatty acid, the immunologic adjuvant covalent coupling unsaturated fatty acid and the hydrophobic small molecule drug are dissolved in an organic solvent, then are ultrasonically and uniformly mixed in pure water, and finally are self-assembled into the nano vaccine; the molar ratio of the three is about 1-1.2: 1-1.2: 0.7 to 1.5. In some embodiments, the molar ratio of antigen covalently coupled unsaturated fatty acid, immunoadjuvant covalently coupled unsaturated fatty acid, and hydrophobic small molecule drug is about 1: 1: 1.
in some embodiments, the nano-vaccine is a microparticle, nanoparticle, or picoparticle. In some embodiments, the microparticles, nanoparticles, or picoparticles are self-assembled.
In some embodiments, the nano-vaccines of the compositions provided herein have an average geometric diameter of 500nm or less. In some embodiments, the nano-vaccine has an average geometric diameter of 50nm or more but 500nm or less. In some embodiments, the average geometric diameter of the population of nano-vaccines is about 75nm, 100nm, 125nm, 150nm, 175nm, 200nm, 225nm, 250nm, 275nm, 300nm, 325nm, 350nm, 375nm, 400nm, 425nm, 450nm, or 475 nm. In some embodiments, the average geometric diameter is 100-. In some embodiments, the average geometric diameter is 60-400nm, 60-350nm, 60-300nm, 60-250nm, or 60-200 nm. In some embodiments, the average geometric diameter is 75 to 250 nm. In some embodiments, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the nano-vaccines in the population of nano-vaccines have a diameter of 500nM or less. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the population of nano-vaccines have a diameter above 100nm but below 250 nm.
Researches show that the DC cells have higher phagocytosis capacity on nanoparticles with the size of 20-200nm, and the nanotechnology is favorable for improving the effect of the anti-tumor vaccine. According to the invention, the antigen peptide and the immune activation adjuvant (taking R848 as an example) are chemically modified, unsaturated fatty acid (taking DHA as an example) safe to a human body is covalently coupled, the characteristic that the unsaturated fatty acid spontaneously forms a hydrophobic core in an aqueous solution is utilized to form the central structure of the nanoparticle, and meanwhile, the small molecule inhibitor (taking STATIC 3 inhibitor as an example) with extremely strong hydrophobicity is wrapped, so that the carrier-free self-assembly nanoparticle containing the antigen peptide, the immune activation adjuvant and the small molecule inhibitor can be prepared. The nano vaccine also contains a cathepsin S (which can be not limited to S type) shearing module and a Glutathione (GSH) reaction module, so that after entering a DC lysosome, the nano particles can release free antigen peptide, immunologic adjuvant and small molecular drugs under triple actions of cathepsin, esterase and GSH, and the aim of enhancing the anti-tumor immune effect is finally achieved.
The nano vaccine can obviously improve the residence time of the antigen peptide in lymph nodes, improve the endocytosis of DC cells to the antigen peptide, release the antigen peptide, immunologic adjuvant and micromolecule medicine under the action of intracellular cathepsin S and intracellular high-concentration GSH after the nanoparticles are endocytosed, and improve the immunoreaction.
Drawings
FIG. 1 is a model of one embodiment of the nano-vaccine of the present invention.
Fig. 2 is a graph showing the measurement of the particle size of the nano vaccine particles by a dynamic light scattering method.
FIG. 3 is the surface potential diagram of the nano vaccine particles measured by Zeta potentiometer.
FIG. 4 is a transmission electron microscope image of the surface topography of the nano vaccine particles.
FIG. 5 is a comparison graph of lymph node fluorescence imaging detection after the fluorescence-modified nano vaccine is injected into mice.
FIG. 6 is a flow cytometer analyzing the proportion of FITC positive cells to CD11c positive DC cells.
FIG. 7A is a comparison graph of physiological detection of mouse melanoma tumor in situ model (CD8+ T cell immunohistochemistry result graph), and FIG. 7B is a statistical graph of tumor volumes of various groups of mouse melanoma tumor in situ model; 7C is the proportion of CD69+ cells in peripheral blood of the mice to CD8+ T cells, and the index can reflect the activation degree of CD8+ T cells.
FIG. 8A is a graph comparing observed measurements of a control and experimental group of a mouse melanoma lung metastasis model; 8B is a statistical analysis chart of observation measurement of a mouse melanoma lung metastasis model control group and a test group.
FIG. 9A is a statistical plot of tumor volumes for a combination of nano-vaccine and PD-1 antibody for anti-melanoma experiment; and 9B is a statistical chart of the survival rate of the mouse subjected to the anti-melanoma experiment by combining the nano vaccine and the PD-1 antibody.
Detailed Description
For a further understanding of the invention, preferred embodiments of the invention are described below in conjunction with specific examples, but it is to be understood that these descriptions are merely provided to further illustrate features and advantages of the invention, and are not intended to limit the scope of the claims.
The above-described scheme is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes and are not intended to limit the scope of the present invention. The conditions used in the examples may be further adjusted according to the conditions of the particular manufacturer, and the conditions not specified are generally the conditions in routine experiments.
Example 1
A preparation method of a nano vaccine comprises the following steps:
synthesis of polypeptide-maleimide-docosahexaenoic acid/peptide-mal-DHA
1.1 preparing antigen peptide for later use, wherein the N end of the antigen is connected with a-Cys- (a) - (b) sequence, (a) is a hydrophilic group, and (b) is a restriction enzyme cutting site group of cathepsin;
1.2N- (2-aminoethyl) maleimide and docosahexaenoic acid are subjected to condensation reaction to obtain an intermediate product I;
dissolving 2, 2' -dithiodiethanol and docosahexaenoic acid in dichloromethane, reacting at 43 ℃ for 24 hours under the condition that a condensing agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and a base is N, N-diisopropylethylamine, spin-drying the solvent, separating and extracting, and passing through a silica gel column to obtain an intermediate product I (DHA-mal);
1.3 the intermediate product I and the polypeptide are subjected to Michael addition reaction to obtain a final product peptide-mal-DHA:
and dissolving the intermediate product I and the polypeptide (containing sulfydryl) in dimethyl sulfoxide, stirring at room temperature, detecting the reaction process through a high performance liquid chromatography, and freeze-drying to obtain solid powder after the reaction is finished to obtain the final product peptide-mal-DHA.
Synthesis of Rasimotent-disulfide bond-docosahexaenoic acid/R848-ss-DHA
2.12, 2' -dithiodiethanol and docosahexaenoic acid are subjected to esterification reaction to obtain an intermediate product II:
dissolving 2, 2' -dithiodiethanol and docosahexaenoic acid in dichloromethane, reacting at 43 deg.C for 24 hr under the condition that the condensing agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and the alkali is 4-dimethylaminopyridine, spin-drying the solvent, separating, extracting, and passing through silica gel column to obtain intermediate product II (DHA-ss-OH);
2.2 reaction of intermediate II with p-nitrophenylchloroformate to give intermediate III:
dissolving the intermediate product II and p-nitrophenyl chloroformate in dichloromethane, taking N, N-diisopropylethylamine as a base, reacting for 8 hours at room temperature, spin-drying the solvent, separating, extracting, and passing through a silica gel column to obtain an intermediate product III;
2.3 reaction of intermediate III with Rasimoter R848 to obtain the final product R848-ss-DHA by transesterification:
dissolving the intermediate product III and Rasimoter R848 in dichloromethane, taking N, N-diisopropylethylamine as a base, reacting for 24 hours at 43 ℃, spin-drying the solvent, separating, extracting, and passing through a silica gel column to obtain the final product R848-ss-DHA.
Thirdly, assembling nanoparticles:
dissolving peptide-mal-DHA, R848-DHA and hydrophobic small molecule drug stattic in organic solvent DMSO (ethanol, acetone, etc.) at a molar ratio of about 1: 1: 1. 900ul of ultrapure water for injection was placed in an ultrasonic cleaning apparatus, and the mixed solution was slowly dropped into the pure water while performing ultrasonic treatment. After fully mixing, the carrier-free self-assembly nano vaccine is obtained, as shown in the schematic diagram of figure 1.
EXAMPLE 2 preparation of Nanoprotein vaccine
(A) Adding a linker sequence CSSVVR at the front end of a 257-one 264 sequence of a model antigen ovalbumin (OVA, with the amino acid sequence of SIINFEKL), and synthesizing by Zhejiang ang On Tuotuolaisi biotechnology limited;
carrying out condensation reaction on N- (2-aminoethyl) maleimide and linolenic acid to obtain an intermediate product I, and carrying out Michael addition reaction on the intermediate product I and the antigen to obtain OVA covalently coupled linolenic acid: cysteine on the C is used for connecting DHA with strong hydrophobicity, so that the modified peptide segment is in a form that one end is hydrophobic and the other end is hydrophilic, and the formation of self-assembled nanoparticles is facilitated; the VVR sequence is a shearing site of cathepsin S, and after the nano vaccine particles are endocytosed by DC cells, the nano vaccine particles are broken under the action of the cathepsin S in lysosomes to release the pattern antigen OVA.
(B) Linolenic acid is coupled to an active hydroxyl group of the immunologic adjuvant R848 through an S-S bond, which is beneficial to the formation of self-assembled nanoparticles taking the linolenic acid as a hydrophobic core by the R848-DHA and the OVA-CSSVVR-linolenic acid, and the S-S bond can be broken under the action of high-concentration GSH in cells after entering the cells, so that active drugs are released.
Carrying out esterification reaction on 2, 2' -dithiodiethanol and unsaturated fatty acid to obtain an intermediate product II, linolenic acid-S-S-OH, reacting linolenic acid-S-S-OH with p-nitro phenyl chloroformate to obtain an intermediate product III, and carrying out ester exchange reaction on the intermediate product III and an immunologic adjuvant to obtain a final product: r848 covalent bond is coupled with linolenic acid;
(C) assembling nanoparticles: 200nM OVA-CSSVVR-DHA, 200nM R848-DHA and 200nM stattic, hydrophobic small molecule drug are dissolved in 100. mu.L DMSO (or organic solvent such as ethanol and acetone). 900ul of ultrapure water for injection was placed in an ultrasonic cleaning apparatus, and the mixed solution was slowly dropped into the pure water while performing ultrasonic treatment. And (3) fully mixing to obtain 1mL of carrier-free self-assembly OVA nano vaccine.
Characterization of nanoparticles:
the particle size of the nano vaccine particles was 105.5 + -1.758 nm as measured by dynamic light scattering method, as shown in FIG. 2.
The surface potential of the nano vaccine particles measured by a Zeta potentiometer is 40.83 +/-1.528 mV, as shown in figure 3.
The surface morphology of the nano vaccine particles was observed by transmission electron microscopy as shown in fig. 4.
Animal experiments:
(1) the nano-vaccine can prolong the residence time of the antigen peptide in lymph nodes: and modifying fluorescein FITC at the side chain of the K amino acid residue in the CSSVVR-OVA sequence, and performing DHA modification and nano vaccine assembly according to the steps. The nano-vaccine (100. mu.L/mouse) prepared as described above was injected subcutaneously into the tail base of mice, 12 per group. 4 mice were sacrificed at 4h, 24h, 48h post-injection per group, inguinal reflux lymph nodes were removed, and fluorescence imaging was performed with a small animal imager, as shown in fig. 5; the experimental results show that: compared with the free drug component with equal dose, the nano vaccine modified by DHA and nano assembled can be resided in the reflux lymph node in a larger amount for a longer time, which is beneficial to starting stronger anti-tumor immune response.
(2) The nano vaccine can improve the endocytosis efficiency of DC cells to antigen peptide: and modifying fluorescein FITC at the side chain of the K amino acid residue of the CSSVVR-OVA sequence, and performing DHA modification and nano vaccine assembly according to the steps. The nano-vaccine (100. mu.L/mouse) prepared as described above was injected subcutaneously into the tail base of mice, 3 per group. In mice after injection, inguinal reflux lymph nodes were removed, DC cells were labeled with CD11c antibody fluorescently labeled with PE, and the ratio of FITC-positive cells to CD11 c-positive DC cells was analyzed by flow cytometry, as shown in fig. 6.
(3) Melanoma in situ tumor model: the right upper arm of 6-week-old male SPF-grade C57B/6 mice was inoculated with 10^5 OVA antigen-expressing B16/F10-OVA melanoma cells (day 0), 5 per group. The nano-vaccine (100. mu.L/mouse) prepared as described above was injected subcutaneously at the base of the tail of the mouse on days 4, 11, and 18, respectively. Tumor size was recorded every two days with a vernier caliper and the results are shown in fig. 7A, 7B, 7C. The experimental result shows that the effect of the nano vaccine for inhibiting the growth of the in-situ tumor is better than that of the free component without nano assembly, the nano vaccine can obviously improve the number of CD8+ T cells in the tumor and improve the activation degree of the T cells in peripheral blood (taking CD69 positive as a standard).
(4) Melanoma lung metastasis model: male SPF grade C57B/6 mice at 6 weeks of age were injected subcutaneously at the base of the tail of the mice on days 1, 8, 15 with 8 vaccines (100. mu.L/mouse) per group prepared as described above. 10^5 OVA antigen expressing B16/F10-OVA melanoma cells were injected intravenously through the tail of mice on day 20. Mice were sacrificed on day 40, lungs were removed and the number of black lesions on the surface of the mice lungs was counted. The results are shown in fig. 8A and 8B, and the experimental results show that the nano vaccine can effectively reduce the metastasis of melanoma cells to the lung through blood circulation.
(5) Combined anti-tumor experiment of nano vaccine and PD-1 antibody: 2 x 10^5 OVA antigen-expressing B16/F10-OVA melanoma cells were inoculated into the axilla of the right upper limb of a male SPF-grade C57B/6 mouse at 6 weeks of age (day 0), and the mice were injected subcutaneously at the base of the tail with the nano-vaccine prepared as described above (100. mu.L/mouse) on days 4, 11, and 18, respectively. On days 6, 9, 13, 16, 20, 23, 100. mu.g/mouse anti-PD-1 antibody was injected via the tail vein of the mice. Tumor size was recorded every two days with a vernier caliper. The results are shown in fig. 9A and 9B, and experimental results show that the nano vaccine and the anti-PD-1 antibody both have significant anti-tumor effects, and the combination of the nano vaccine and the anti-PD-1 antibody can produce stronger anti-tumor effects, inhibit tumor growth and prolong the tumor-bearing survival time of mice.
The invention utilizes unsaturated fatty acid to modify tumor-targeted antigen peptide and immunologic adjuvant, takes the unsaturated fatty acid as a hydrophobic core in aqueous solution to carry out self-assembly, and small molecular drugs with strong hydrophobicity can be loaded in the hydrophobic core. The nano vaccine can improve the residence time of the antigen peptide in lymph nodes, increase the endocytosis of DC cells to the antigen peptide and improve the anti-tumor immunity effect.
Therefore, the scope of the present invention should not be limited to the disclosure of the embodiments, but includes various alternatives and modifications without departing from the scope of the present invention, which is defined by the claims of the present patent application.
Claims (3)
1. A preparation method of a self-assembled nano vaccine comprises the following steps:
(1) preparing an ovalbumin antigen with an amino acid sequence of-Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu-; the N end of the ovalbumin antigen is added with Cys- (a) - (b) sequences, wherein, (a) is-Ser-Ser-, and (b) is-Val-Val-Arg-;
(2) carrying out condensation reaction on N- (2-aminoethyl) maleimide and DHA to obtain an intermediate product I, and carrying out Michael addition reaction on the intermediate product I and the ovalbumin to obtain ovalbumin covalently coupled DHA;
(3) carrying out esterification reaction on 2, 2' -dithiodiethanol and DHA to obtain an intermediate product II: -DHA-S-S-OH, reacting-DHA-S-S-OH with p-nitrophenyl chloroformate to obtain an intermediate product III, and carrying out transesterification reaction on the intermediate product III and R848 to obtain a final product: r848 is covalently linked with DHA;
(4) dissolving the ovalbumin covalently coupled DHA obtained in the step (2), the R848 covalent bond coupled DHA obtained in the step (3) and the STATtic 3 inhibitor in an organic solvent, and fully and uniformly mixing;
(5) and (4) carrying out ultrasonic treatment on the mixture obtained in the step (4), and then dropwise adding the mixture into ultrapure water for injection to obtain the self-assembled nano vaccine.
2. The method of claim 1, wherein the organic solvent is selected from the group consisting of DMSO, ethanol, and acetone.
3. The self-assembled nano-vaccine prepared by the preparation method of claim 1 or 2, which is used for treating melanoma.
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