CN113577255B

CN113577255B - Tumor nano vaccine, preparation method and application thereof

Info

Publication number: CN113577255B
Application number: CN202010361793.5A
Authority: CN
Inventors: 于海军; 祝奇文; 高晶; 侯博; 李天亮
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2020-04-30
Filing date: 2020-04-30
Publication date: 2023-09-08
Anticipated expiration: 2040-04-30
Also published as: CN113577255A

Abstract

The invention relates to a tumor nano vaccine, a preparation method and application thereof. The tumor nano vaccine is a composite nano micelle formed by an acid sensitive polymer immune adjuvant conjugate and a tumor-associated antigen/antigen peptide, wherein the acid sensitive polymer immune adjuvant conjugate has a structure shown in the following formula 1. The tumor nano vaccine can selectively target lymph nodes and efficiently deliver tumor neoantigens and immune adjuvants to dendritic cells, activate antigen-specific T cell immune effects and efficiently inhibit tumor growth and metastasis.

Description

Tumor nano vaccine, preparation method and application thereof

Technical Field

The invention belongs to the field of biological pharmacy, and in particular relates to a nano vaccine for co-delivering tumor-associated antigens and immune adjuvants, a preparation method thereof and application thereof in tumor immunotherapy.

Background

In recent years, immunotherapy has made a major breakthrough in clinical treatment of malignant tumors, significantly improves the survival time and quality of life of tumor patients, and brings new hopes for tumor patients. Tumor-associated antigens, represented by tumor neoantigens (neoantigens), can activate the anti-tumor immune response of tumor patients, and are expected to be used in personalized immunotherapy. Tumor immunotherapy based on tumor-associated antigens has therefore received a great deal of attention. The antitumor immune effect of the activated organism requires antigen uptake by antigen presenting cells, especially Dendritic Cells (DCs), which are processed and presented to the naive T cells to effect T cell activation, thereby generating cytotoxic T cells to effect immune clearance of tumor cells. DCs are key immune cells in the activation of anti-tumor immunity. However, tumor neoantigens belong to polypeptide molecules, which have poor serum stability and are not easily targeted to lymph nodes. Meanwhile, the solid tumor has an immunosuppressive microenvironment, and seriously inhibits the antigen recognition and presentation functions of DCs.

The immunoadjuvant can regulate the immune cell function of the organism and plays an auxiliary role in tumor immunotherapy. Among other things, toll-like receptor (TLR) agonists can promote cross-presentation of DCs to antigens to activate tumor-specific T lymphocytes. Meanwhile, interferon gene stimulators (stimulator of interferon genes, STING) agonists are effective in enhancing antigen cross presentation by DCs by stimulating the secretion of type I interferons by DCs. In addition, STING agonists, TLR agonists and the like can promote secretion of other inflammatory cytokines by DCs, improve the clearance of cytotoxic T cells from tumor cells, and thus improve the tumor immunotherapeutic effect. However, the micro-molecular immune adjuvant has poor in-vivo metabolism, low bioavailability and lack of lymph node and tumor targeting characteristics, and restricts the clinical transformation application.

Disclosure of Invention

Aiming at the key technical bottleneck how to realize the efficient delivery of tumor-associated antigens and small-molecule immunoadjuvants, the invention aims to provide a nano vaccine which can be used for co-delivering tumor neoantigens and immunoadjuvants, a preparation method thereof and application thereof in anti-tumor immunotherapy.

To achieve the above object, in one aspect, the present invention provides a tumor nano vaccine which is one or more complex nano-micelles composed of an acid-sensitive polymer immunoadjuvant conjugate and a tumor-associated antigen or antigen peptide,

wherein the tumor-associated antigen/antigen peptide refers to a protein/protein fragment peptide or derivative peptide with immunogenicity generated by tumor-associated gene mutation, and a protein/protein fragment peptide or derivative peptide with immunogenicity related to tumorigenic virus genes;

the acid-sensitive polymeric immunoadjuvant conjugate has a structure as shown in formula 1 below:

wherein, in the formula 1,

R ₁ selected from the group consisting of N, N-diethylamino, N-dibutylamino, N-diisopropylamino, pentamethyleneamino, and hexamethyleneamino;

R ₂ is a group derived from an immunoadjuvant;

R ₃ is C _n H _2n+1 N is an integer from 3 to 12;

R ₄ selected from the group consisting ofWherein ligands are derivatized dendritic cell targeting ligands;

linker is R ₂ A linking chain with a carbonyl group, the linking chain forming a p, pi-conjugated linkage with the carbonyl group;

x is an integer from 10 to 145, preferably an integer from 100 to 140;

y is an integer from 20 to 60, preferably an integer from 40 to 50;

z is an integer from 0 to 60, preferably an integer from 1 to 10.

In a specific embodiment, R ₁ Selected from N, N-dibutylamino, N-diisopropylamino or hexamethyleneamino; r is R ₃ Is C _n H _2n+1 N is 12; x is 113; y is 40, 42, 50; z is 0, 3 or 5.

In a specific embodiment, the bond has a structure represented by the following formula 2:

in specific embodiments, the Linker is selected from any one of the following structures:

in particular embodiments, the immunoadjuvant comprises: any one selected from STING agonist and TLR agonist.

In a specific embodiment, the immunoadjuvant R ₂ Any one of the following groups:

in particular embodiments, the acid-sensitive polymeric immunoadjuvant conjugate is selected from one or more of the following:

in specific embodiments, the tumor-associated antigens/antigenic peptides include, but are not limited to, ovalbumin antigenic peptide fragments, human papilloma virus antigenic peptides that are immunogenic. The immunogenic ovalbumin antigen peptide fragments may have different amino acid sequences. In specific embodiments, the ovalbumin antigen peptide fragment comprises: an ovalbumin polypeptide having an amino acid sequence of SIINFEKL or a salt thereof (e.g., trifluoroacetate), and an ovalbumin G4 peptide having an amino acid sequence of siifekl or a salt thereof (e.g., trifluoroacetate).

In specific embodiments, the tumor-associated antigen/antigen peptide loading mass ratio is 1 to 90wt% relative to the weight of the composite nanomicelle.

In another aspect, the invention provides a method for preparing the tumor nano vaccine, which comprises the following steps:

dissolving the acid sensitive polymer immune adjuvant conjugate and the alkylated hexanediol diacrylate derivative in an organic solvent, adding a solution containing tumor-associated antigens/antigen peptides under ultrasonic treatment, and then carrying out ultrafiltration or dialysis to obtain the tumor nano vaccine.

In a specific embodiment, the alkylated hexanediol diacrylate derivative is a C14 alkylated hexanediol diacrylate.

In a specific embodiment, the organic solvent comprises: at least one of tetrahydrofuran, methanol, ethanol, N-dimethylformamide, N-dimethylacetamide and dimethylsulfoxide.

In specific embodiments, the tumor associated antigen/antigen peptide-containing solution is formulated using solvent DMSO and/or water.

In yet another aspect, the present invention also provides a use of the tumor nanovaccine in the manufacture of a medicament for treating malignant tumors, including but not limited to: breast cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, ovarian cancer, colon cancer or prostate cancer.

Advantageous effects

The tumor nano vaccine provided by the invention can selectively target lymph nodes, can efficiently deliver tumor-related antigens including tumor neoantigens and immune adjuvants to DCs, activates antigen-specific T cell immune effects, and efficiently resists tumor growth and metastasis. The acid-sensitive polymer in the nano vaccine has acid sensitivity, and can be protonated under the acidic condition in a lysosome so as to realize the escape function of the lysosome and avoid the degradation of the antigen peptide by the lysosome. Co-delivery of small molecule immunoadjuvants can enhance antigen processing and presentation properties of DCs, effectively activating cytotoxic T cell immune effects.

Drawings

Fig. 1: mPEG prepared in preparation example 2 of the present invention ₁₁₃ -b-P(DPA ₄₂ -co-R848 ₃ ) Nuclear magnetic spectrum of (2).

Fig. 2: mannose-PEG prepared in preparation example 3 of the present invention ₁₁₃ -b-PDPA ₄₀ Nuclear magnetic spectrum of (2).

Fig. 3: mPEG prepared in preparation example 4 of the present invention ₁₁₃ -b-P(DPA ₄₂ -co-FAA ₃ ) Nuclear magnetic spectrum of (2).

Fig. 4: the nanovaccines PDPR@OVA and Man-PDPR@OVA prepared in preparation example 5 of the present invention were a) dynamic light scattering particle size distribution profile and b) scanning electron microscopy (scale of 100nm in the figure).

Fig. 5: the structure of the nano micelle prepared by the invention is schematically shown.

Fig. 6: a) An OVA standard curve; b) Relationship between the drug loading of OVA in mannose modified R848-OVA nanovaccine and the amount of PDPE in nanovaccine (PDPE is C14 alkylated hexanediol diacrylate).

Fig. 7: growth inhibition curve of the tumor nano vaccine of the invention on B16-OVA mouse transplanted tumor.

Detailed Description

The invention is illustrated by the following examples, but the scope of the invention is not limited thereto.

The reagents and apparatus used in the following examples are as follows:

methoxy-terminated polyethylene glycol amine 5000, polyethylene glycol diamine 5000, ethyl 2- (diisopropylamino) methacrylate, raschimot (R848), mi Tuola ketone (FAA), 4-cyano-4- [ [ (dodecylthio) thiomethyl ] thio ] pentanoic acid were all purchased from sigma aldrich (china). 4-Isothioate phenyl-alpha-D-mannoside was purchased from carboline technologies Co. The remaining reagents and solvents were purchased from the national drug group (Shanghai) chemical reagent Co., ltd unless otherwise specified.

Ovalbumin antigen peptide SIINFEKL (hereinafter referred to as "OVA antigen peptide") was purchased from a national peptide organism. B16-OVA melanoma cells were purchased from Shanghai cell Bank, and DMEM medium and fetal bovine serum for cell culture were purchased from Gibco corporation.

Balb/C white mice and C57BL/6 black mice of 4-5 weeks old were purchased from Shanghai Laek laboratory animal Limited and tumor-bearing mouse models were constructed with B16-OVA tumors by inoculating B16-OVA melanoma cancer cells cultured in vitro on the right back of the mice. The operation of the whole animal experiment strictly follows the relevant regulations of the animal care and use committee of Shanghai pharmaceutical research institute.

Sample data were determined by the following instrument: nuclear magnetic resonance hydrogen spectrum [ ] ¹ H-NMR) with a Varian-MERCURY Plus-400 Nuclear magnetic resonance apparatus with TMS as internal standard in ppm chemical Displacement ^-1 ；

The hydrodynamic particle size and surface potential of the cationic micelle were measured by a MALVERN NANO SIZER type particle size analyzer, and a transmission electron micrograph was obtained by a Tecnai G2F 20S-TWIN type transmission electron microscope.

In the present invention, unless otherwise specified, the equipment and test methods used are those conventional in the art.

Preparation example 1: PEGylated Raft initiator mPEG ₁₁₃ Synthesis of CTA

4-cyano-4- [ [ (dodecylthio) thioketomethyl]Thio group]Valeric acid (CTA) (19.3 mg,0.048 mmol) was dissolved in 5mL of N, N-dimethylformamide, to which 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) (27.5 mg,0.144 mmol), 1-Hydroxybenzotriazole (HOBT) (19.4 mg,0.144 mmol), triethylamine (TEA) (16.16 mg,0.160 mmol) was added and reacted at room temperature for 1.5h. Subsequently, mPEG is added thereto ₁₁₃ -NH ₂ (200.8 mg,0.040 mmol) and then reacted at room temperature for 24 hours, the reaction liquid is dialyzed with ethanol and deionized water, the cut-off molecular weight of a dialysis bag is 3500D,182mg of pale yellow powdery solid is obtained after freeze-drying, and the yield is 84.3%. ¹ H NMR(400MHz,CDCl ₃ )δ3.84–3.80(m,3H),3.69–3.62(m,456H),3.56(dd,J＝9.9,5.1Hz,6H),3.47(dd,J＝9.2,4.7Hz,6H),3.39(s,3H),3.35–3.30(m,2H),2.51(d,J＝3.9Hz,3H),1.69(dt,J＝15.0,7.5Hz,2H),1.45–1.36(m,4H),1.35–1.28(m,8H),1.26(s,12H),0.88(t,J＝6.8Hz,3H).

Preparation example 2: raximote polymer conjugate mPEG ₁₁₃ -b-P(DPA ₄₂ -co-R848 ₃ ) Is synthesized by (a)

Compound 1 (6.0 g,0.04 mol), TEA (8.08 g,0.08 mol) was dissolved in 10mL of methylene chloride, and a solution of 10mL of methacryloyl chloride (2.08 g,0.08 mol) in methylene chloride was slowly added dropwise thereto under an ice water bath, and the mixture was reacted at room temperature for 12 hours after the addition. The reaction mixture was washed with water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and separated by column to give 3.8g of Compound 2 in 87.2% yield. ¹ H NMR(400MHz,CDCl ₃ )δ6.15(s,1H),5.64–5.55(m,1H),4.35–4.30(m,2H),3.79–3.75(m,2H),3.75–3.71(m,2H),3.69(s,4H),3.64–3.60(m,2H),1.96(s,3H).

Compound 2 (0.20 g,0.91 mmol), N, N-Diisopropylethylamine (DIEA) (354 mg,2.75 mmol) was dissolved in 10mL of tetrahydrofuran, and di (p-nitrophenyl) carbonate (NPC) (418 mg,1.37 mmol) was added thereto under an ice-water bath, and reacted at room temperature for 12 hours. After the reaction was completed, the reaction mixture was washed with water, saturated ammonium chloride solution, extracted with methylene chloride, the organic phases were combined, dried over anhydrous sodium sulfate, and then subjected to column separation to obtain 285mg of compound 3 in a yield of 81.8%. ¹ HNMR(400MHz,CDCl ₃ )δ8.29(d,J＝9.2Hz,2H),7.40(d,J＝9.2Hz,2H),6.14(s,1H),5.59(s,1H),4.47–4.43(m,2H),4.35–4.31(m,2H),3.85–3.81(m,2H),3.80–3.76(m,2H),3.72(s,4H),1.96(s,3H).

Compound 3 (80 mg,0.21 mmol), raximote (55 mg,0.17 mmol) was dissolved in 2mL of N, N-Dimethylformamide (DMF), and 1-Hydroxybenzotriazole (HOBT) (35.1 mg,0.26 mmol) was added thereto and reacted at room temperature for 12 hours. After the reaction, the solvent was removed by spin-drying, the reaction mixture was washed with water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and then 72mg of Compound 4 was isolated in 77.7% yield. ¹ H NMR(400MHz,CDCl ₃ )δ8.47(s,1H),8.14(d,J＝8.2Hz,2H),7.58(dd,J＝11.3,4.1Hz,1H),7.49–7.43(m,1H),6.12(s,1H),5.59–5.53(m,1H),4.90(s,2H),4.78(s,2H),4.46–4.40(m,2H),4.30–4.24(m,2H),3.84–3.79(m,2H),3.77(dd,J＝5.5,4.2Hz,2H),3.71(s,4H),3.64(q,J＝7.0Hz,2H),3.35(s,1H),1.93(s,3H),1.33(s,6H),1.25(t,J＝7.0Hz,3H).

Compound 4 (65 mg,0.12 mmol), mPEG ₁₁₃ CTA (89.5 mg, 16.6. Mu. Mol), 2- (diisopropylamino) methacrylic acid (DPA) (177 mg,0.83 mmol) and Azobisisobutyronitrile (AIBN) (0.27 mg, 1.7. Mu. Mol) were dissolved in 1mL of anhydrous Dioxane (Dioxane), and oxygen was removed from the system by three freeze thawing cycles, followed by reaction at 70℃for 24 hours. After the reaction is finished, the reaction solution is dialyzed by ethanol and deionized water and then freeze-dried, the cut-off molecular weight of a dialysis bag is 3500D, and 210mg of pale yellow white solid mPEG is obtained ₁₁₃ -b-P(DPA ₄₂ -co-R848 ₃ ) The yield was 75.8%. ¹ H-NMR is shown in FIG. 1.

Preparation example 3: mannose-derivatized polymer Mannose-PEG ₁₁₃ -b-PDPA ₄₀ Is synthesized by (a)

4-cyano-4- [ [ (dodecylthio) thioketomethyl]Thio group]Valeric acid (CTA) (19.7 mg,0.048 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) (28.1 mg,0.144 mmol), 1-hydroxybenzotriazole (19.8 mg,0.144 mmol), triethylamine (16.2 mg,0.160 mmol) were dissolved in 5mL of N, N-dimethylformamide solution and after 2h reaction at room temperature, NH was added thereto ₂ -PEG ₁₁₃ -NH ₂ (201.2 mg,0.040 mmol) was reacted at room temperature for 24 hours. After the reaction is finished, the reaction solution is dialyzed by ethanol and deionized water, the cut-off molecular weight of a dialysis bag is 2500D, 182mg of pale yellow white solid powder NH ₂ -PEG ₁₁₃ CTA, yield 84.5%.

The NH obtained ₂ -PEG ₁₁₃ CTA (100 mg,0.019 mmol), 2- (diisopropylamino) methacrylic acid (DPA) (237.3 mg,1.114 mmol) and Azobisisobutyronitrile (AIBN) (0.31 mg,0.0019 mmol) were dissolved in 1mL anhydrous di-waterIn the oxygen hexacycle, oxygen in the system was removed by three freeze-thawing cycles, followed by a reaction at 70℃for 24 hours. After the reaction is finished, the reaction solution is dialyzed by ethanol and deionized water and then freeze-dried, and the cut-off molecular weight of a dialysis bag is 3500D. The NH obtained ₂ -PEG ₁₁₃ -b-DPA ₄₀ Dissolved in 3ml of N, N-dimethylformamide, to which was added compound 5 (6.3 mg,0.02 mmol) and N, N-Diisopropylethylamine (DIEA) (2.6 mg,0.02 mmol) and reacted at room temperature for 24 hours. After the reaction is finished, the reaction solution is dialyzed by ethanol and deionized water (the cut-off molecular weight of a dialysis bag is 3500D), and the pale yellow solid obtained after freeze drying is Mannose-PEG ₁₁₃ -b-DPA ₄₀ 。 ¹ H-NMR is shown in FIG. 2.

Preparation example 4: FAA-derived Polymer mPEG ₁₁₃ -b-P(DPA ₄₂ -co-FAA ₃ ) Is synthesized by (a)

Compound 6 (192.5 mg,1.55 mmol), triethylamine (TEA) (303.5 mg,3.00 mmol) was dissolved in 10mL of anhydrous dichloromethane, and a solution of 2mL of methacryloyl chloride (104.5 mg,1.00 mmol) in dichloromethane was slowly added dropwise thereto under an ice water bath. After the addition, the reaction was carried out at room temperature for 12 hours. After the reaction solution was spin-dried, 289mg of compound 7 was obtained by column separation in 97.0% yield.

The obtained compound 7 (288.3 mg,1.50 mmol) was dissolved in anhydrous dichloromethane, to which FAA (282.5 mg,1.00 mmol), 2- (7-benzotriazol-oxide) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) (1.140 g,3.0 mmol) and Triethylamine (TEA) (303.5 mg,3.00 mmol) were added and reacted at room temperature for 12 hours. After the reaction, the reaction mixture was washed with water, extracted with dichloromethane, dried over anhydrous sodium sulfate and spin-dried, and the resultant was separated on a column to give 270mg of Compound 8 in 59.4% yield.

Compound 8 (260.2 mg, 0.298 mmol), mPEG ₁₁₃ CTA (100.4 mg,0.019 mmol), 2- (diisopropylamino) methacrylic acid (DPA) (237.3 mg,1.114 mmol) and Azobisisobutyronitrile (AIBN) (0.31 mg,0.0019 mmol) were dissolved in 1mL anhydrous dioxane, and oxygen was removed from the system by three freeze thawing cycles, followed by reaction at 70℃for 24 hours. ReactionAfter the completion, the reaction solution is dialyzed by ethanol and deionized water and then freeze-dried, the cut-off molecular weight of a dialysis bag is 3500D, thus 295mg of pale yellow white solid mPEG is obtained ₁₁₃ -b-P(DPA ₄₀ -co-FAA ₁₀ ) The yield was 84.3%. ¹ H-NMR is shown in FIG. 3.

Preparation example 5: preparation of nanovaccine

The polymer mPEG prepared in preparation example 2 was prepared by dispersing an OVA antigen peptide DMSO solution in pure water to prepare an OVA aqueous solution at a concentration of 1mg/mL ₁₁₃ -b-P(DPA ₄₂ -co-R848 ₃ ) And C14 alkylated hexanediol diacrylate (PDPE) was dissolved in tetrahydrofuran and gradually added to the OVA antigenic peptide aqueous solution under ultrasound. And then, ultrafiltering and centrifuging for 1h, and removing free peptide and tetrahydrofuran (the molecular weight cut-off of an ultrafilter centrifuge tube is 50000D) in the system, thus obtaining the nano vaccine PDPR@OVA.

The OVA antigen peptide DMSO solution was dispersed in pure water to prepare an OVA aqueous solution at a concentration of 1mg/mL. Polymer mPEG prepared in preparation examples 2 and 3 ₁₁₃ -b-P(DPA ₄₂ -co-R848 ₃ ),Mannose-PEG ₁₁₃ -b-DPA ₄₀ And C14 alkylated hexanediol diacrylate (PDPE) was dissolved in tetrahydrofuran and gradually added to the OVA antigenic peptide aqueous solution under ultrasound. And then, ultrafiltering and centrifuging for 1h, and removing free peptide and tetrahydrofuran (the molecular weight cut-off of an ultrafiltration centrifuge tube is 50000D) in the system, thus obtaining the nano vaccine Man-PDPR@OVA. The hydrodynamic radius of the micelle was measured by a dynamic light scattering instrument, and the morphology was determined by a transmission electron micrograph, and the test results are shown in fig. 4.

As shown in figure 4, the average hydration particle sizes of the two formed nano vaccines are 46.28nm and 43.30nm respectively, and the average particle size shown by a transmission electron microscope photo is close to 70nm, which indicates that the system can form nano particles with good physical morphology, and mannosylation modification has no obvious influence on the particle size of nano micelles.

The schematic structure of the prepared nano micelle is shown in fig. 5.

Test example 1: determination of OVA content in nanovaccine

The free OVA antigen peptide was dissolved in methanol solution to prepare 4,8,16,32,64,128. Mu.g/mL solutions, and the absorption area thereof was measured at 280nm wavelength by high performance liquid chromatography, and a standard curve was drawn as shown in FIG. 6 a. The nano micelle PDPR@OVA prepared in preparation example 5 was lyophilized, 1mg was weighed, dissolved in 2mL of methanol, and insoluble matters were removed through a 0.22 μm filter membrane, and the content of the OVA antigen peptide was measured under the same chromatographic conditions, and the test result is shown in FIG. 6 b.

The test results show that the content of the C14 chain alkylated hexanediol diacrylate (PDPE) in the nano micelle PDPR@OVA has a significant effect on the loading of the antigen peptide of the nano particles, and the loading capacity of the nano particles on the antigen peptide is gradually increased as the content of the C14 chain alkylated hexanediol diacrylate (PDPE) is increased, up to about 70%.

Test example 2: tumor proliferation inhibition of B16-OVA transplanted tumor-bearing mice by nano vaccine

After inoculating B16-OVA cells on the right back of C57BL/6 mice, the tumor volume reached 50mm ³ When the mice are left and right, the mice are randomly divided into 5 groups of 6 mice each, and the groups are PBS respectively; OVA; PDPR; PDPR@OVA; man-PDPR@OVA was administered 1 time every seven days, twice, tumor volumes were recorded every three days, and the test results are shown in FIG. 7. In FIG. 7, OVA group was free antigenic peptide administered at a dose of 2.5mg/kg; the PDPR group is a nano micelle group formed by the Raximote polymer, and the administration dosage is equivalent to 1mg/kg of Raximote; the PDPR@OVA group is a nano vaccine loaded with the raschimod and the antigen peptide simultaneously, and the administration dosage is equivalent to 1mg/kg of the raschimod and 2.5mg/kg of the OVA; the Man-PDPR@OVA group is mannose functionalized nano vaccine loaded with the raschimod and the antigen peptide, the administration dose is equivalent to 1mg/kg of the raschimod, the OVA is 2.5mg/kg, and other groups except the R848 group are intraperitoneal administration, and the other groups are subcutaneous injections of the foot pads.

The test result shows that the nano vaccine loaded with the raschimot and the antigen peptide can effectively inhibit the growth of B16-OVA tumor, which shows that the Toll-like receptor agonist can obviously promote the recognition of the antigen peptide by the immune system of the organism and realize the growth inhibition of B16-OVA transplanted tumor. After the functionalization of mannose, the growth inhibition effect of the nano particles on B16-OVA grafted tumor can be effectively enhanced.

Sequence listing

<110> Shanghai pharmaceutical institute of China academy of sciences

<120> tumor nano vaccine, preparation method and application thereof

<130> DI20-0473-XC03

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> ovalbumin polypeptide

<400> 1

Ser Ile Ile Asn Phe Glu Lys Leu

1 5

<210> 2

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> ovalbumin G4 peptide

<400> 2

Ser Ile Ile Gly Phe Glu Lys Leu

1 5

Claims

1. A tumor nano vaccine is a composite nano micelle formed by one or more acid sensitive polymer immune adjuvant conjugates and tumor related antigens/antigen peptides,

wherein the tumor-associated antigen/antigen peptide refers to a protein/protein fragment peptide or derivative peptide with immunogenicity generated by tumor-associated gene mutation, and a protein/protein fragment peptide or derivative peptide with immunogenicity related to tumorigenic virus gene,

the acid-sensitive polymeric immunoadjuvant conjugate is selected from one or more of the following:

and the tumor-associated antigen/antigenic peptide is an ovalbumin antigenic peptide fragment having immunogenicity, and the tumor is melanoma.

2. The tumor nanovaccine of claim 1, wherein,

the ovalbumin antigen peptide fragment comprises: the amino acid sequence is SIINFEKL ovalbumin polypeptide or its salt.

3. The tumor nanovaccine of claim 1, wherein,

the ovalbumin antigen peptide fragment comprises: the amino acid sequence is SIIGFEKL ovalbumin G4 peptide or its salt.

4. The tumor nanovaccine according to claim 2 or 3, wherein,

the salt is trifluoroacetate.

5. The tumor nanovaccine of claim 1, wherein,

the loading mass ratio of the tumor-associated antigen/antigen peptide is 1-90wt% relative to the composite nano-micelle.

6. A method of preparing a tumor nanovaccine as claimed in any one of claims 1 to 5, comprising the steps of:

dissolving the acid sensitive polymer immunoadjuvant conjugate and the alkylated hexanediol diacrylate derivative according to claim 1 in an organic solvent, adding a solution containing tumor associated antigens/antigen peptides under ultrasonic treatment, and then performing ultrafiltration or dialysis to obtain the tumor nano vaccine.

7. Use of a tumor nanovaccine according to any one of claims 1 to 5 in the manufacture of a medicament for the treatment of a malignancy, which is melanoma.