WO2021169484A1 - Nanovaccine and preparation method therefor - Google Patents

Abstract

A multi-component vector-free integrated nanovaccine constructed from an unsaturated fatty acid and containing an antigen peptide, an immunologic adjuvant, and a small molecule drug, and a method therefor. The nanovaccine contains an antigen, an immunologic adjuvant, and an unsaturated fatty acid, the antigen is covalently coupled to the unsaturated fatty acid by means of a -SH group, and the immunologic adjuvant is coupled to the unsaturated fatty acid by means of a S-S bond. The antigen peptide and the immunologic adjuvant which are tumor-targeting are modified with the unsaturated fatty acid, self-assembly is enabled in an aqueous solution by taking the unsaturated fatty acid as a hydrophobic core, and the small molecule drug having high hydrophobicity can be loaded in the hydrophobic core. The nanovaccine can prolong the residence time of the antigen peptide in a lymph gland, enhance endocytosis of DC cells for the antigen peptide, and improve the anti-tumor immune effect.

Description

Nano vaccine and preparation method thereof Technical field

The present invention relates to the field of medical biotechnology, in particular to the technical field of vaccines; in particular, it relates to a multi-component carrier-free integrated nano vaccine containing antigen peptides, immune adjuvants and small molecule drugs established by using unsaturated fatty acids.

Background technique

With the intensification of factors such as population aging, the incidence and mortality of cancer in my country continue to rise, and cancer has become the leading cause of death in my country, seriously endangering the health of Chinese people. In recent years, anti-tumor immunotherapy has developed rapidly, especially immune checkpoint inhibitor therapies (for example: PD-1 inhibitors and CTLA-4 inhibitors) have achieved significant clinical effects. The main principle of immune checkpoint inhibitor therapy is to block the immunosuppressive complex formed between tumor cells and effector T cells (CTL) through antibodies or small molecule compounds (such as PD-1/PD-L1, CTLA-4/ B7 complex), and then relieve the immunosuppressive signal in CTL, and restart CTL's attack on tumor cells.

However, immune checkpoint inhibitor therapy also has certain limitations. First, drug responsiveness varies greatly among different tumor types. One of the main reasons for this difference is that tumors with low mutation load will produce less tumor-specific antigen (TSA), which weakens the efficiency of spontaneous recognition of tumor cells by immune cells and hinders immune checkpoint inhibitor therapy. Widely used, it also reminds us that we need to cooperate with better tumor-specific antigen presentation methods to assist immune checkpoint inhibitor therapy. Secondly, in some patients, the side effects (immune-related side effects) of immune checkpoint inhibitor therapy are more obvious, including skin itching, rash, vitiligo, diarrhea, even liver damage and heart failure, which are mainly immune cells Caused by a non-specific attack on non-tumor cells. Therefore, in the process of anti-tumor immunotherapy, the number of highly active and tumor-targeting CTLs is the key to improving anti-tumor efficacy and reducing side effects.

The activation of CTL mainly relies on antigen-presenting cells (APC), especially dendritic cells (DC) with cross-presentation effects, which can present the endocytosed TSA on the cell surface through MHCI ( Cross-presentation), which specifically activates CTL to kill tumors. Moreover, while DC presents the antigen peptide-MHC molecular complex, it can highly express costimulating factors, directly provide dual signals to activate the initial CD8+ T cells, make them express and secrete IL-2, and promote their own proliferation and differentiation. For CTL.

However, the uptake rate of free peptides by DC cells is not high, and the circulation period of free peptides in the body is short, which greatly limits the effect of anti-tumor vaccines. To solve this problem, anti-tumor vaccines are often used with immune adjuvants (such as CpG-ODN, Montanide, R848) to further stimulate DC and CTL cells, but immune adjuvants can also cause some side effects, such as muscle atrophy at the injection site. , Flu-like reaction, headache. Therefore, the optimization and innovation of immune adjuvant administration methods are also a research hotspot and difficulty in the field of anti-tumor immunity. The current immune adjuvant nanomedicine is mainly in the form of a single-drug adjuvant packaged by a nanocarrier. It does not form a connection with the tumor-specific antigen peptide. It cannot be guaranteed that the same DC can receive the immune adjuvant and tumor-specific antigen peptide at the same time, which will reduce The efficacy of adjuvant/tumor-specific antigen peptide combination therapy also increases immune-related toxic and side effects. In order to solve the above-mentioned problems, the present invention comes from this.

Summary of the invention

The purpose of the present invention is to improve the anti-tumor vaccine immune adjuvant in the prior art to reduce the curative effect and existing side effects of the adjuvant/tumor-specific antigen peptide, and to provide a chemical modification of the antigen peptide and immune adjuvant by using unsaturated fatty acid. Carrier-free self-assembly technology loads antigen peptides, immune adjuvants and hydrophobic small molecule drugs in the same nanoparticle tumor vaccine to improve the immune effect of tumor antigen polypeptide vaccine.

In order to achieve the above objective, the present invention is achieved through the following technical solutions: a nano-vaccine comprising: an antigen, an immune adjuvant and an unsaturated fatty acid, characterized in that the antigen is coupled to the unsaturated fatty acid through a covalent bond, and the The immune adjuvant is coupled to the same type or different types of unsaturated fatty acids through covalent bonds.

Preferably, the antigen is selected from the form of a peptide, protein, glycoprotein, glycopeptide, proteoglycan, or a combination thereof without Cys.

Preferably, a Cys-(b) sequence or Cys-(a)-(b) sequence is added to the N-terminus of the antigen without Cys without cysteine, wherein (a) is a hydrophilic group and (b) is Cathepsin enzyme cleavage site group. This (a) sequence is used to balance the hydrophilicity of the entire antigen peptide. If the subsequent antigen peptide has poor hydrophilicity, it is necessary to increase the (a) hydrophilic group. If the antigen peptide itself has good hydrophilicity, it can be There is only one amino acid or even none.

Preferably, said (a) is a peptide fragment without Cys, including but not limited to a single amino acid, dipeptide or polypeptide; more preferably, said (a) is selected from -Ser- group,- Gly-group, dipeptide -Ser-Ser- or -Ser-Gly-.

The b is an amino acid sequence selected from the restriction site of any one of cathepsin A--Z, and the cathepsin AZ is known from the prior art from A, B, C, D, E, F. ..... Cathepsins of X, Y, Z. More preferably, (b) is an amino acid sequence of any one of cathepsin B, F, H, K, L, S, V, B, C, H, X. In a specific technical scheme of the present invention, (b) is the enzyme cleavage site of cathepsin S, namely -Val-Val-Arg-tripeptide, VVR is conducive to the endocytosis of the nanoparticle by DC cells, and the lysosome The cleavage under the action of cathepsin S releases the model antigen.

Preferably, the immune adjuvant is selected from adjuvants containing -OH, more preferably, is selected from compounds that can activate the antigen presentation ability of DC cells, including but not limited to: TLR signaling pathway agonists, STING signaling pathway agonists, And a compound containing an -OH group inside the molecular structure; more preferably, the immune adjuvant includes but not limited to one or more of R848, CpG, and Imiquimod.

In a specific technical scheme of the present invention, the antigen is modified at the N-terminus of the Cys-(b) sequence or Cys-(a)-(b) sequence and then is covalently coupled through the -SH of the Cys residue of cysteine. Saturated fatty acids, the immune adjuvant is coupled to unsaturated fatty acids of the same type or different types of unsaturated fatty acids through SS bonds. More preferably, the -OH of the immune adjuvant is coupled to the unsaturated fatty acid through an S-S bond.

Preferably, the unsaturated fatty acid is selected from: monounsaturated fatty acid or polyunsaturated fat, and the monounsaturated fatty acid is selected from one or more of oleic acid, erucic acid, palmitoleic acid, and trans oleic acid ; Polyunsaturated fat, selected from one or more of linoleic acid, linolenic acid, arachidonic acid, docosahexaenoic acid DHA.

Preferably, the nano-tumor vaccine also contains a small molecule drug with strong hydrophobicity. The small molecule drugs with strong hydrophobicity are selected from one or more of small molecule drugs that regulate DC cell signaling pathways; including but not limited to: STAT3 inhibitor stattic, Artesunate artesunate, and C188-9.

In some embodiments, the antigen is coupled to the unsaturated fatty acid I through a covalent bond, and the immune adjuvant is coupled to the unsaturated fatty acid I of the same kind through a covalent bond. In some embodiments, the antigen is coupled to the unsaturated fatty acid I through a covalent bond, and the immune adjuvant is coupled to the unsaturated fatty acid II of the same type through a covalent bond.

In some embodiments, when the antigen is an antigen peptide sequence containing Cys, the antigen peptide sequence should be replaced so that it does not contain Cys.

The present invention also provides a method for preparing the above-mentioned nano-vaccine, which includes the following steps:

(1) Prepare the antigen,

(2) N-(2-aminoethyl)maleimide and unsaturated fatty acid are subjected to condensation reaction to obtain intermediate product I, and then intermediate product I and the antigen are covalently coupled to the antigen by Michael addition reaction unsaturated fatty acid:

(3) Intermediate product II, -unsaturated fatty acid -SS-OH is obtained by esterification reaction of 2,2'-dithiodiethanol and unsaturated fatty acid, and -unsaturated fatty acid -SS-OH is combined with p-nitrochloroformic acid Intermediate product III is obtained by phenyl ester reaction, and the final product is obtained by transesterification reaction of intermediate product III and immune adjuvant: the immune adjuvant is covalently coupled to unsaturated fatty acid;

(4) Dissolve the unsaturated fatty acid-coupled antigen obtained in step (2) and the unsaturated fatty acid-coupled immune adjuvant obtained in step (3) together in an organic solvent, and mix them thoroughly;

(5) The mixture obtained in step (4) is subjected to ultrasonic treatment, and then it is added dropwise to ultrapure water for injection to obtain a self-assembled nano-vaccine.

Preferably, the antigen is in the form of a peptide, protein, glycoprotein, glycopeptide, proteoglycan, or a combination thereof without Cys.

Preferably, a Cys-(b) sequence or Cys-(a)-(b) sequence is added to the N-terminus of the antigen without Cys without cysteine, wherein (a) is a hydrophilic group and (b) is Cathepsin enzyme cleavage site group.

Preferably, the unsaturated fatty acid in step (2) and the unsaturated fatty acid in step (3) are the same type or different types.

Preferably, in step (4), a small molecule drug with strong hydrophobicity is also added to the organic solvent. In step (4), the organic solvent is selected from DMSO, ethanol, and acetone.

In some specific embodiments, the volume of ultrapure water for injection in step (5) is at least 9 times that of the mixture.

In some embodiments, a small molecule drug with strong hydrophobicity refers to a small molecule that is hardly 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, B cell antigens are addictive substances. In some embodiments, the B cell antigen is a toxin. In some embodiments, T cell antigens are degenerative disease antigens, infectious disease antigens, cancer antigens, allergic disease antigens, autoimmune disease antigens, allogeneic antigens, xenoantigens, allergens, addictive Material or metabolic disease enzymes or enzyme products. In some embodiments, the T cell antigen is a universal T cell antigen.

In some embodiments, the nanovaccine is formed by self-assembly. Self-assembly refers to the process of forming a nano-vaccine and/or a carrier by using components that can adapt itself in a foreseeable manner, predictably and repeatedly, to form a nano-vaccine and/or a vaccine carrier. In some embodiments, a nano-vaccine is formed by using polar or amphoteric biomaterials (which are themselves set to each other to form nanomaterials with predictable size, composition, and position of the components). According to the present invention, the polar biological material can be combined with immunomodulatory reagents, immunostimulatory reagents and/or target reagents, so that when the nanovaccine is self-assembled, there is a repeatable positioning and density of the reagents on the nanovaccine/in the nanovaccine style.

In some embodiments, the antigen is covalently coupled to unsaturated fatty acids, the immune adjuvant is covalently coupled to unsaturated fatty acids and hydrophobic small molecule drugs to be dissolved in an organic solvent, and then ultrasonically mixed in pure water, and finally self-assembled into Nano vaccine; the molar ratio of the three is about 1-1.2:1-1.2:0.7-1.5. In some embodiments, the molar ratio of the antigen covalently coupled to unsaturated fatty acid, the immunoadjuvant covalently coupled to unsaturated fatty acid and the hydrophobic small molecule drug is about 1:1:1.

In some embodiments, the nano-vaccine is a microparticle, nanoparticle, or picomi particle. In some embodiments, the microparticles, nanoparticles, or picomi particles are self-assembled.

In some embodiments, the nanovaccine of the composition provided herein has an average geometric diameter of 500 nm or less. In some embodiments, the nano vaccine has an average geometric diameter above 50 nm but below 500 nm. In some embodiments, the average geometric diameter of the nanovaccine population is about 75nm, 100nm, 125nm, 150nm, 175nm, 200nm, 225nm, 250nm, 275nm, 300nm, 325nm, 350nm, 375nm, 400nm, 425nm, 450nm, or 475nm. In some embodiments, the average geometric diameter is 100-400 nm, 100-300 nm, 100-250 nm, or 100-200 nm. In some embodiments, the average geometric diameter is 60-400 nm, 60-350 nm, 60-300 nm, 60-250 nm, or 60-200 nm. In some embodiments, the average geometric diameter is 75-250 nm. In some embodiments, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the nanovaccine population has a diameter of 500 nM or less. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the nanovaccine population in the population of nanovaccine has 100nm or more but 250nm or more. diameter of.

Studies have shown that DC cells have a high phagocytic capacity for nanoparticles of 20-200nm scale, and nanotechnology can help improve the effect of anti-tumor vaccines. The present invention chemically modifies antigen peptides and immune activation adjuvants (take R848 as an example), covalently couples unsaturated fatty acids (take DHA as an example) safe to the human body, and uses unsaturated fatty acids to spontaneously form a hydrophobic core in an aqueous solution. With the characteristics of forming the central structure of the nanoparticle, and at the same time encapsulating the extremely hydrophobic small molecule inhibitor (take the STAT3 inhibitor stattic as an example), it is possible to prepare a non-antigenic peptide-immune activation adjuvant-small molecule inhibitor at the same time. The carrier self-assembles nanoparticles. The nano-vaccine also contains cathepsin S (not limited to S-type) cleavage module and glutathion (GSH) reaction module to ensure that the nanoparticle can be used in cathepsin and ester Under the triple action of enzymes and GSH, free antigen peptides, immune adjuvants and small molecule drugs are released, ultimately achieving the purpose of enhancing the anti-tumor immune effect.

The nano-vaccine of the present invention can significantly increase the residence time of antigen peptides in lymph nodes and increase the endocytosis of antigen peptides by DC cells. After the nanoparticles are endocytosed, under the action of intracellular cathepsin S and intracellular high-concentration GSH , Release antigen peptides, immune adjuvants and small molecule drugs to improve immune response.

Description of the drawings

Figure 1 is an example model of the nano-vaccine of the present invention.

Figure 2 shows the dynamic light scattering method to determine the particle size of the nano vaccine particles.

Figure 3 is a graph of the surface potential of the nano vaccine particle measured by the Zeta potentiometer.

Figure 4 shows the surface morphology of nano-vaccine particles detected by transmission electron microscopy.

Figure 5 is a comparison diagram of fluorescence imaging detection of lymph nodes after injection of the fluorescently modified nano-vaccine in mice.

Figure 6 shows the proportion of FITC-positive cells to CD11c-positive DC cells analyzed by flow cytometry.

Figure 7A is a comparison chart of physiological detection of mouse melanoma in situ tumor model (CD8+T cell immunohistochemical results), 7B is a statistical chart of tumor volume in each group of mouse melanoma in situ tumor model; 7C is mouse peripheral blood The proportion of CD69+ cells in CD8+ T cells, this indicator can reflect the degree of activation of CD8+ T cells.

Fig. 8A is a comparison diagram of observation and measurement between the mouse melanoma lung metastasis model control and the experimental group; 8B is a statistical analysis diagram of observation and measurement between the mouse melanoma lung metastasis model control and the experimental group.

Fig. 9A is a statistical diagram of tumor volume in the anti-melanoma experiment of the combination of nano-vaccine and PD-1 antibody; 9B is a statistical diagram of the survival rate of mice in the anti-melanoma experiment of the combination of nano-vaccine and PD-1 antibody.

Detailed ways

In order to further understand the present invention, the preferred solutions of the present invention will be described below in conjunction with specific examples, but it should be understood that these descriptions are only to further illustrate the features and advantages of the present invention, rather than limiting the claims of the present invention.

The above solution will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are used to illustrate the present invention and not to limit the scope of the present invention. The implementation conditions used in the examples can be further adjusted according to the conditions of specific manufacturers, and implementation conditions not specified are usually conditions in routine experiments.

Example 1

A method for preparing a nano-vaccine includes the following steps:

1. Synthesis of peptide-maleimide-docosahexaenoic acid/peptide-mal-DHA

1.1 Prepare the antigen peptide for use, the N-terminal of the antigen is connected to the -Cys-(a)-(b) sequence, (a) is the hydrophilic group, (b) is the cathepsin enzyme cleavage site group;

1.2 N-(2-aminoethyl)maleimide and docosahexaenoic acid undergo condensation reaction to obtain intermediate product I;

2,2'-Dithiodiethanol and docosahexaenoic acid are dissolved in dichloromethane, the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and the base is React at 43°C for 24 hours under the condition of N,N-diisopropylethylamine, spin off the solvent, separate and extract, and pass through a silica gel column to obtain the intermediate product I (DHA-mal);

1.3 Intermediate product I and polypeptide are through Michael addition reaction to obtain the final product peptide-mal-DHA:

The intermediate product I and the polypeptide (containing a sulfhydryl group) are dissolved in dimethyl sulfoxide, stirred at room temperature, and the reaction process is detected by high performance liquid phase. After the reaction is completed, it is lyophilized to a solid powder to obtain the final product peptide-mal-DHA.

2. Synthesis of Resimot-Disulfide-Docosahexaenoic Acid/R848-ss-DHA

2.1 2,2’-Dithiodiethanol and docosahexaenoic acid are esterified to obtain intermediate product II:

2,2'-Dithiodiethanol and docosahexaenoic acid are dissolved in dichloromethane, the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and the base is React at 43°C for 24 hours under the condition of 4-dimethylaminopyridine, spin off the solvent, separate and extract, and pass through a silica gel column to obtain intermediate product II (DHA-ss-OH);

2.2 Intermediate product II is reacted with phenyl p-nitrochloroformate to obtain intermediate product III:

Intermediate product II and phenyl p-nitrochloroformate are dissolved in dichloromethane, N,N-diisopropylethylamine is used as a base, reacted at room temperature for 8 hours, the solvent is spin-dried, separated and extracted, and then passed through a silica gel column to obtain Intermediate product III;

2.3 Intermediate product III reacts with Resimod R848 to obtain the final product R848-ss-DHA through transesterification reaction:

Intermediate product III and Resimod R848 were dissolved in dichloromethane, N,N-diisopropylethylamine as base, reacted at 43°C for 24 hours, spin-dried the solvent, separated and extracted, and passed through a silica gel column to obtain the final product The product R848-ss-DHA.

3. Nanoparticle assembly:

Peptide-mal-DHA, R848-DHA and hydrophobic small molecule drug stattic are dissolved in organic solvent DMSO (ethanol, acetone, etc.), and the molar ratio of the three is about 1:1:1. Place 900ul ultrapure water for injection in an ultrasonic cleaner, and slowly drop the above mixed solution into pure water while performing ultrasonic. After fully mixing, a carrier-free self-assembled nano vaccine is obtained, as shown in the schematic diagram in Figure 1.

Example 2 Preparation method of nano vaccine

(A) The linker sequence CSSVVR was added to the front end of the model antigen ovalbumin (OVA, amino acid sequence SIINFEKL) 257-264 sequence, which was synthesized by Zhejiang Ontop Biotechnology Co., Ltd.;

N-(2-aminoethyl)maleimide and linolenic acid are subjected to condensation reaction to obtain intermediate product I, and then intermediate product I and the antigen are subjected to Michael addition reaction to obtain OVA covalently coupled to linolenic acid: The cysteine on C is used to connect the highly hydrophobic DHA, so that the modified peptide has a hydrophobic form at one end and a hydrophilic form at the other end, which is conducive to the formation of self-assembled nanoparticles; the VVR sequence is the cleavage site of cathepsin S When the nano-vaccine particles are endocytosed by DC cells, they are broken under the action of cathepsin S in the lysosome to release the model antigen OVA.

(B) The active hydroxyl group of the immune adjuvant R848 is coupled with linolenic acid through the SS bond, which is conducive to the formation of self-assembled nanoparticles with linolenic acid as the hydrophobic core of R848-DHA and OVA-CSSVVR-linolenic acid. After the cell, under the action of the high concentration of GSH in the cell, it will break and release the active drug.

Intermediate product II, -linolenic acid-SS-OH is obtained by esterification reaction of 2,2'-dithiodiethanol and unsaturated fatty acid, and intermediate product II is obtained by reaction of -linolenic acid-SS-OH with phenyl p-nitrochloroformate. Product III, intermediate product III and immune adjuvant through transesterification reaction to obtain the final product: R848 is covalently coupled to linolenic acid;

(C) Nanoparticle assembly: Dissolve 200nM OVA-CSSVVR-DHA, 200nM R848-DHA and 200nM hydrophobic small molecule drug stattic in 100μL DMSO (organic solvents such as ethanol and acetone can also be used). Place 900ul ultrapure water for injection in an ultrasonic cleaner, and slowly drop the above mixed solution into pure water while performing ultrasonic. After thorough mixing, 1 mL of carrier-free self-assembled OVA nano vaccine is obtained.

Nanoparticle characterization:

The particle size of the nano vaccine particles measured by the dynamic light scattering method is 105.5±1.758 nm, as shown in Figure 2.

The Zeta potentiometer measured the surface potential of the nano vaccine particles to be 40.83±1.528mV, as shown in Figure 3.

The surface morphology of the nano-vaccine particles was observed by transmission electron microscope, as shown in Figure 4.

Animal experiment:

(1) Nano vaccines can extend the residence time of antigen peptides in lymph nodes: the K amino acid residue side chain in the CSSVVR-OVA sequence is modified with fluorescein FITC, and DHA modification and nano vaccine assembly are performed according to the aforementioned steps. The mice were injected subcutaneously at the base of the tail of the nano-vaccine (100 μL/mouse) prepared as described above, with 12 rats in each group. At 4h, 24h, 48h after injection, 4 mice in each group were sacrificed, the inguinal reflux lymph nodes were taken out, and fluorescent imaging was performed with a small animal imager, as shown in Figure 5; the experimental results showed that: compared with the same dose of free drug components , The nano-vaccine modified by DHA and nano-assembly can reside in the reflux lymph nodes in a larger amount and longer, which is conducive to initiating a stronger anti-tumor immune response.

(2) Nano vaccines can improve the endocytosis efficiency of DC cells to antigen peptides: the K amino acid residue side chain of the CSSVVR-OVA sequence is modified with fluorescein FITC, and the DHA modification and nano vaccine assembly are carried out according to the aforementioned steps. The mice were injected subcutaneously at the base of the tail of the nano vaccine (100 μL/mouse) prepared as described above, with 3 rats in each group. After the injection of the mice, the inguinal reflux lymph nodes were taken out, the DC cells were labeled with PE fluorescently labeled CD11c antibody, and the proportion of FITC-positive cells to CD11c-positive DC cells was analyzed by flow cytometry, as shown in Figure 6.

(3) Melanoma orthotopic tumor model: Inoculate 10^5 B16/F10-OVA melanoma cells expressing OVA antigen in the armpit of the right upper limb of male SPF grade C57B/6 mice at 6 weeks of age (day 0), 5 in each group. On the 4th, 11th, and 18th days, the mice were injected subcutaneously at the base of the tail of the nano vaccine (100 μL/mouse) prepared as described above. The tumor size was recorded with a vernier caliper every two days, and the results are shown in Figures 7A, 7B, and 7C. Experimental results show that the effect of nano-vaccine in inhibiting tumor growth in situ is better than that of free components without nano-assembly. Nano-vaccine can significantly increase the number of CD8+ T cells inside the tumor and increase the degree of activation of T cells in peripheral blood ( Take CD69 positive as the standard).

(4) Melanoma lung metastasis model: In 6-week-old male SPF-grade C57B/6 mice, the nano-vaccine (100μL/mouse) prepared according to the aforementioned method was injected subcutaneously at the base of the tail of the mouse on days 1, 8, and 15. 8 per group. On the 20th day, mice were injected with 10^5 B16/F10-OVA melanoma cells expressing OVA antigen through the tail vein. The mice were sacrificed on the 40th day, the lungs were taken out, and the number of black lesions on the surface of the lungs of the mice was observed and counted. The results are shown in Figures 8A and 8B. The experimental results show that the nano-vaccine can effectively reduce the transfer of melanoma cells to the lungs through the blood circulation.

(5) Anti-tumor experiment of the combination of nano-vaccine and PD-1 antibody: Inoculate 2*10^5 B16/F10-OVA expressing OVA antigen in the armpit of the right upper limb of male SPF-grade C57B/6 mice at 6 weeks of age The melanoma cells (day 0) were injected subcutaneously at the base of the tail of the mouse on the 4th, 11th, and 18th days, respectively, with the nano-vaccine prepared as described above (100 μL/mouse). On days 6, 9, 13, 16, 20, and 23, 100 μg/mouse PD-1 antibody was injected through the tail vein of the mouse. The tumor size was recorded with a vernier caliper every two days. The results are shown in Figure 9A and 9B. The experimental results show that both nano-vaccine and anti-PD-1 antibody exhibit significant anti-tumor effects. The combination of the two can produce stronger anti-tumor effects, inhibit tumor growth and prolong mice The tumor-bearing survival time.

The present invention uses unsaturated fatty acids to modify tumor-targeted antigen peptides and immune adjuvants, and uses unsaturated fatty acids as the hydrophobic core to self-assemble in an aqueous solution, and the hydrophobic core can also be loaded with small-molecule drugs with strong hydrophobicity. The nano vaccine can increase the residence time of the antigen peptide in the lymph node, increase the endocytosis of the antigen peptide by DC cells, and improve the anti-tumor immune effect.

The technical content and technical features of the present invention have been disclosed as above, but those skilled in the art may still make various substitutions and modifications based on the teachings and disclosures of the present invention without departing from the spirit of the present invention. Therefore, the scope of protection of the present invention should not be limited The content disclosed in the embodiments should include various replacements and modifications that do not deviate from the present invention, and are covered by the claims of this patent application.

Claims (16)

1. A nano vaccine comprising: an antigen, an immune adjuvant and an unsaturated fatty acid, characterized in that the antigen is coupled to an unsaturated fatty acid through a covalent bond, and the immune adjuvant is coupled to the same type or different types through a covalent bond. Types of unsaturated fatty acids.
2. The nano-vaccine according to claim 1, wherein the antigen is in the form of a peptide, protein, glycoprotein, glycopeptide, proteoglycan, or a combination thereof without Cys.
3. The nano-vaccine according to claim 1, wherein the Cys-(b) sequence or Cys-(a)-(b) sequence is added to the N-terminus of the antigen without cysteine Cys, wherein (a) It is a hydrophilic group, (b) is a cathepsin enzyme cleavage site group.
4. The nano-vaccine according to claim 3, wherein the (a) is a peptide fragment without Cys, and the (a) is selected from the group consisting of -Ser-, -Gly-, Dipeptide Ser-Ser- or -Ser-Gly-.
5. The nano-vaccine according to claim 3, wherein the (b) is an amino acid sequence of an enzyme cleavage site selected from any one of cathepsin A-Z.
6. The nano-vaccine according to claim 1, wherein the antigen is modified at the N-terminal of the Cys-(b) sequence or the Cys-(a)-(b) sequence and passes through the -SH of the Cys residue of cysteine. The unsaturated fatty acid is covalently coupled, and the immune adjuvant is coupled to the unsaturated fatty acid of the same kind or different kinds of unsaturated fatty acids through the SS bond.
7. The nano-vaccine according to claim 1, wherein the immune adjuvant is selected from compounds containing -OH that can activate the antigen presentation ability of DC cells, preferably, the immune adjuvant is selected from TLR signaling pathway agonists , STING signaling pathway agonist, and one or more of the compounds containing -OH group in the molecular structure.
8. The nano-vaccine according to claim 1, wherein the immune adjuvant is selected from one or more of R848, CpG, and imiquimod.
9. The nano-vaccine according to claim 1, wherein the unsaturated fatty acid is selected from monounsaturated fatty acid or polyunsaturated fat, and the monounsaturated fatty acid is selected from oleic acid, erucic acid, palmitoleic acid, One or more of trans oleic acid; polyunsaturated fat, selected from one or more of linoleic acid, linolenic acid, arachidonic acid, and docosahexaenoic acid DHA.
10. The nano-vaccine according to claim 1, wherein the nano-vaccine further contains a small molecule drug with strong hydrophobicity; preferably, the small molecule drug with strong hydrophobicity is selected from STAT3 inhibitor stattic, Artesunate Succinate, C188-9.
11. The preparation method of the nano vaccine according to any one of claims 1-10, comprising the following steps:

    (1) Prepare the antigen,

    (2) N-(2-aminoethyl)maleimide and unsaturated fatty acid are subjected to condensation reaction to obtain intermediate product I, and then intermediate product I and the antigen are covalently coupled to the antigen by Michael addition reaction unsaturated fatty acid:

    (3) Intermediate product II, -unsaturated fatty acid -SS-OH is obtained by esterification reaction of 2,2'-dithiodiethanol and unsaturated fatty acid, and -unsaturated fatty acid -SS-OH is combined with p-nitrochloroformic acid Intermediate product III is obtained by phenyl ester reaction, and the final product is obtained by transesterification reaction of intermediate product III and immune adjuvant: the immune adjuvant is covalently coupled to unsaturated fatty acid;

    (4) Dissolve the unsaturated fatty acid-coupled antigen obtained in step (2), the unsaturated fatty acid-coupled immune adjuvant and hydrophobic small molecules obtained in step (3) together in an organic solvent, and mix them thoroughly;

    (5) The mixture obtained in step (4) is subjected to ultrasonic treatment, and then it is added dropwise to ultrapure water for injection to obtain a self-assembled nano vaccine.
12. The preparation method according to claim 11, wherein in step (1), the antigen is in the form of a peptide, protein, glycoprotein, glycopeptide, proteoglycan or a combination thereof without Cys.
13. The preparation method according to claim 11 or 12, wherein in step (1), a Cys-(b) sequence or Cys-(a)-(b) is added to the N-terminus of the antigen without Cys. ) Sequence, wherein (a) is a hydrophilic group and (b) is a cathepsin enzyme cleavage site group.
14. The preparation method according to claim 11, wherein the unsaturated fatty acid in step (2) and the unsaturated fatty acid in step (3) are of the same type or different types.
15. The preparation method according to claim 11, wherein in step (4), a small molecule drug with strong hydrophobicity is added to the organic solvent.
16. The preparation method according to claim 11, wherein the organic solvent is selected from DMSO, ethanol, and acetone.

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