CN113577255A

CN113577255A - Tumor nano vaccine, preparation method and application thereof

Info

Publication number: CN113577255A
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: 2021-11-02
Anticipated expiration: 2040-04-30
Also published as: CN113577255B

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 immunologic adjuvant conjugate and tumor-associated antigen/antigen peptide, and the acid-sensitive polymer immunologic adjuvant conjugate is used for immunizingThe adjuvant conjugate has a structure as shown in formula 1 below. The tumor nano vaccine can selectively target lymph nodes, efficiently deliver tumor neoantigens and immunologic adjuvants to dendritic cells, activate the immune effect of antigen-specific T cells, 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 particularly relates to a nano vaccine for co-delivering tumor-associated antigens and an immunologic adjuvant, a preparation method thereof and application thereof in tumor immunotherapy.

Background

In recent years, immunotherapy makes a major breakthrough in clinical treatment of malignant tumors, remarkably improves the life cycle and the quality of life of tumor patients, and brings new hopes for the tumor patients. Tumor-associated antigens, represented by neoantigen (neoantigen), activate anti-tumor immune responses of tumor patients and are expected to be used for personalized immunotherapy. Tumor immunotherapy based on tumor-associated antigens has therefore received widespread attention. The activation of the anti-tumor immune effect of the body requires antigen to be taken up by antigen presenting cells, especially Dendritic Cells (DCs), and the antigen is presented to the original T cells after processing, so as to activate the T cells, thereby generating cytotoxic T cells and realizing the immune elimination of the tumor cells. DCs are key immune cells in the activation of anti-tumor immune processes. However, tumor neoantigens belong to polypeptide molecules, which have poor serum stability and are not easily targeted to lymph nodes. Meanwhile, solid tumors have an immunosuppressive microenvironment, and the antigen recognition and presentation functions of DCs are seriously inhibited.

The immunologic adjuvant can regulate the function of immune cells of an organism and play an auxiliary role in tumor immunotherapy. Among them, Toll-like receptor (TLR) agonists can promote cross presentation of antigens by DCs to activate tumor-specific T lymphocytes. Meanwhile, interferon gene Stimulator (STING) agonists can effectively enhance the cross presentation of DCs to antigens by stimulating DCs to secrete type I interferons. In addition, STING agonists, TLR agonists and the like can promote the secretion of DCs to other inflammatory cytokines, improve the scavenging ability of cytotoxic T cells to tumor cells, and thus improve the tumor immunotherapy effect. However, the small molecular immunologic adjuvant has poor metabolism in vivo, low bioavailability and lacks lymph node and tumor targeting characteristics, thereby restricting the clinical transformation application of the small molecular immunologic adjuvant.

Disclosure of Invention

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

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

tumor-associated antigen/antigen peptide, wherein the tumor-associated antigen/antigen peptide refers to immunogenic protein/protein fragment peptide or derived peptide generated by tumor-associated gene variation, and immunogenic protein/protein fragment peptide or derived peptide related to oncogenic virus gene;

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

wherein, in the formula 1,

R₁selected from the group consisting of N, N-diethylamino, N-dibutylamino, N-diisopropylamino, pentamethyleneamino, hexamethyleneamino;

R₂is a group derived from an immunological adjuvant;

R₃is C_nH_2n+1N is an integer of 3 to 12;

R₄is selected from

Or

Wherein Ligand is a derivatized dendritic cell targeting Ligand;

linker is R₂A 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 of 100 and 140;

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

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

In a particular embodiment, R₁Selected from N, N-dibutylamino, N-diisopropylamino or hexamethyleneamino; r₃Is C_nH_2n+1N is 12; x is 113; y is 40, 42, 50; z is 0, 3 or 5.

In a specific embodiment, the Ligand has a structure represented by formula 2 below:

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

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

In a specific embodiment, the immunoadjuvant R is₂Is selected from the group consisting ofAny one of the following groups:

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

in a specific embodiment, the tumor associated antigen/antigen peptide includes but is not limited to ovalbumin antigen peptide fragments with immunogenicity and human papilloma virus antigen peptides. The ovalbumin antigen peptide segment with immunogenicity can have different amino acid sequences. In specific embodiments, the egg white protein 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 SIIGFEKL or a salt thereof (e.g., trifluoroacetate).

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

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

dissolving the acid-sensitive polymer immunoadjuvant conjugate and the alkylated hexanediol diacrylate derivative in an organic solvent, adding a solution containing tumor-associated antigen/antigen peptide 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 alkanated hexanediol diacrylate.

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

In a specific embodiment, the tumor associated antigen/antigenic peptide containing solution is formulated using the solvents DMSO and/or water.

In still another aspect, the present invention also provides a use of the tumor nano-vaccine in the preparation of a medicament for treating malignant tumors, including but not limited to: breast cancer, cervical cancer, liver cancer, stomach cancer, pancreatic cancer, ovarian cancer, colon cancer or prostate cancer.

Advantageous effects

The tumor nano vaccine can selectively target lymph nodes, efficiently deliver tumor-associated antigens including tumor neoantigens and immunologic adjuvants to DCs, activate antigen-specific T cell immune effects, and efficiently resist tumor growth and metastasis. The acid-sensitive polymer in the nano vaccine enables the nano vaccine to have acid sensitivity, protonation can be carried out in lysosomes under the acidic condition, so that the lysosome escape function is realized, and the degradation of lysozymes to antigenic peptides is avoided. The co-delivered small molecule immune adjuvant can enhance the antigen processing and presenting performance of DCs and effectively activate the immune effect of cytotoxic T cells.

Drawings

FIG. 1: mPEG prepared in preparation example 2 of the present invention₁₁₃-b-P(DPA₄₂-co-R848₄) Nuclear magnetic spectrum of (1).

FIG. 2: Mannose-PEG prepared in preparation example 3 of the present invention₁₁₃-b-PDPA₄₀Nuclear magnetic spectrum of (1).

FIG. 3: mPEG prepared in preparation example 4 of the present invention₁₁₃-b-P(DPA₄₂-co-FAA₃) Nuclear magnetic spectrum of (1).

FIG. 4: the nano vaccines PDPR @ OVA and Man-PDPR @ OVA prepared in preparation example 5 of the present invention have a) a dynamic light scattering particle size distribution diagram and b) a scanning electron micrograph (the scale in the figure is 100 nm).

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

FIG. 6: a) OVA standard curve; b) the relationship between the drug loading of OVA in the mannose-modified R848-OVA nano vaccine and the content of PDPE in the nano vaccine (PDPE is C14 alkanised hexanediol diacrylate).

FIG. 7: the tumor nano vaccine of the invention has a growth inhibition curve for B16-OVA mouse transplanted tumor.

Detailed Description

The invention is illustrated by the following examples, without limiting the scope of protection of the invention thereto.

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

methoxy-terminated polyethylene glycol amine 5000, polyethylene glycol diamine 5000, ethyl 2- (diisopropylamino) methacrylate, rasimot (R848), mitoxantone (FAA), 4-cyano-4- [ [ (dodecylthio) thionemethyl ] thio ] pentanoic acid were purchased from sigma aldrich (china). 4-Isothiolanic acid phenyl-alpha-D-mannoside was purchased from Bailingwei science and technology Co. The remaining reagents and solvents were purchased from the national pharmaceutical group (Shanghai) Chemicals, Inc., unless otherwise specified.

The ovalbumin antigenic peptide SIINFEKL (hereinafter referred to as "OVA antigenic peptide") was purchased from national peptide organisms. B16-OVA melanoma cells were purchased from Shanghai cell Bank of Chinese academy of sciences, and cell culture was purchased from Gibco using DMEM medium and fetal bovine serum.

Balb/C white mice and C57BL/6 black mice, 4-5 weeks old, were purchased from Shanghai Si Laike laboratory animals, Inc., and tumor-bearing mouse models of B16-OVA-bearing tumors were constructed by inoculating the right back of the mice with B16-OVA melanoma cancer cells cultured in vitro. The procedure of the whole animal experiment strictly followed the relevant regulations of the Shanghai pharmaceutical research institute animal Care and use Committee.

Sample data were determined by the following instrument: nuclear magnetic resonance hydrogen spectrum (¹H-NMR) using a Varian-MERCURY Plus-400 NMR spectrometer with TMS as internal standard and chemical shift in ppm^-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 microscope photograph was taken with a Tecnai G2F 20S-TWIN type transmission electron microscope.

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

Preparation example 1: PEGylated Raft initiator mPEG₁₁₃Synthesis of-CTA

Reacting 4-cyano-4- [ [ (dodecylthio) thione methyl]Sulfur based radicals]Pentanoic acid (CTA) (19.3mg, 0.048mmol) was dissolved in 5mL of N, N-dimethylformamide, and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) (27.5mg,0.144mmol), 1-Hydroxybenzotriazole (HOBT) (19.4mg,0.144mmol), Triethylamine (TEA) (16.16mg,0.160mmol) were added thereto and reacted at room temperature for 1.5 h. Subsequently, mPEG was added thereto₁₁₃-NH₂(200.8mg, 0.040mmol), reacting at room temperature for 24h, dialyzing the reaction solution with ethanol and deionized water, carrying out dialysis bag with molecular weight cut-off of 3500D, and freeze-drying to obtain 182mg of pale yellow powdery solid with yield of 84.3%.¹H NMR(400MHz,CDCl₃)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: racemate polymer conjugates mPEG₁₁₃-b-P(DPA₄₂-co-R848₃) Synthesis of (2)

Compound 1(6.0g,0.04mol), TEA (8.08g,0.08mol) were dissolved in 10mL of dichloromethane, and 10mL of a solution of methacryloyl chloride (2.08g,0.08mol) in dichloromethane was slowly added dropwise thereto under an ice-water bath, and after completion of the dropwise addition, the reaction was carried out at room temperature for 12 hours. The reaction mixture was washed with water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and subjected to column separation to obtain 3.8g of compound 2, which was 87.2% in yield.¹H NMR(400MHz,CDCl₃)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(0.20g,0.91mmol), N, N-Diisopropylethylamine (DIEA) (354mg,2.75mmol) was dissolved in 10mL of tetrahydrofuran, and di (p-nitrophenyl) carbonate (NPC) (418mg,1.37mmol) was added thereto under ice-water bath and reacted at room temperature for 12 hours. After the reaction, the reaction solution was washed with water and then with a saturated ammonium chloride solution, extracted with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, and then subjected to column separation to obtain 285mg of compound 3, with a yield of 81.8%.¹H NMR(400MHz,CDCl₃)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(80mg, 0.21mmol), Rasimotent (55mg, 0.17mmol) was dissolved in 2mL of N, N-Dimethylformamide (DMF), and 1-Hydroxybenzotriazole (HOBT) (35.1mg,0.26mmol) was added thereto and reacted at room temperature for 12 hours. After the reaction, the solvent was removed by rotation, the reaction solution was washed with water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and then subjected to column separation to obtain 72mg of compound 4, which was 77.7% in yield.¹H NMR(400MHz,CDCl₃)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(65mg,0.12mmol), mPEG₁₁₃CTA (89.5mg, 16.6. mu. mol),2- (diisopropylamino) methacrylic acid (DPA) (177mg,0.83mmol) and Azobisisobutyronitrile (AIBN) (0.27mg, 1.7. mu. mol) were dissolved in 1mL anhydrous Dioxane (Dioxane), oxygen was removed from the system by three freeze-thaw cycles, and then reacted at 70 ℃ for 24 hours. After the reaction is finished, dialyzing the reaction solution by using ethanol and deionized water, freeze-drying, and obtaining 210mg of light yellow white solid mPEG with the cut-off molecular weight of 3500D by using a dialysis bag₁₁₃-b-P(DPA₄₂-co-R848₃) The yield was 75.8%.¹The H-NMR is shown in FIG. 1.

Preparation example 3:mannose-derived polymer Mannose-PEG₁₁₃-b-PDPA₄₀Synthesis of (2)

Reacting 4-cyano-4- [ [ (dodecylthio) thione methyl]Sulfur based radicals]Valeric acid (CTA) (19.7mg,0.048mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) (28.1mg,0.144mmol), 1-hydroxybenzotriazole (19.8mg,0.144mmol), triethylamine (16.2mg,0.160mmol) were dissolved in 5mL of N, N-dimethylformamide solution and reacted at room temperature for 2h, and NH was added thereto₂-PEG₁₁₃-NH₂(201.2mg,0.040mmol), and reacted at room temperature for 24 h. 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, 182mg of light yellow white solid powder NH₂-PEG₁₁₃CTA, yield 84.5%.

The obtained NH₂-PEG₁₁₃CTA (100mg,0.019mmol),2- (diisopropylamino) methacrylic acid (DPA) (237.3mg,1.114mmol) and Azobisisobutyronitrile (AIBN) (0.31mg,0.0019mmol) were dissolved in 1mL anhydrous dioxane, oxygen was removed from the system by three freeze-thaw cycles, and then reacted at 70 ℃ for 24 h. After the reaction is finished, the reaction solution is dialyzed by ethanol and deionized water and then is freeze-dried, and the molecular weight cut-off of the dialysis bag is 3500D. Obtained NH₂-PEG₁₁₃-b-DPA₄₀Dissolved in 3ml of N, N-dimethylformamide, and thereto were added compound 5(6.3mg,0.02mmol) and N, N-Diisopropylethylamine (DIEA) (2.6mg,0.02mmol), followed by reaction 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 a light yellow solid obtained after freeze-drying is Mannose-PEG₁₁₃-b-DPA₄₀。¹The H-NMR is shown in FIG. 2.

Preparation example 4: FAA-derivatized polymer mPEG₁₁₃-b-P(DPA₄₂-co-FAA₃) Synthesis of (2)

Compound 6(192.5mg,1.55mmol), Triethylamine (TEA) (303.5mg,3.00mmol) was dissolved in 10mL of anhydrous dichloromethane and 2mL of methacryloyl chloride (104.5mg,1.00mmol) 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, column separation was performed to obtain 289mg of Compound 7 in 97.0% yield.

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

Compound 8(260.2mg,0.296mmol), mPEG₁₁₃CTA (100.4mg,0.019mmol),2- (diisopropylamino) methacrylic acid (DPA) (237.3mg,1.114mmol) and Azobisisobutyronitrile (AIBN) (0.31mg,0.0019mmol) were dissolved in 1mL anhydrous dioxane, oxygen was removed from the system by three freeze-thaw cycles, and then reacted at 70 ℃ for 24 h. After the reaction is finished, the reaction solution is dialyzed by ethanol and deionized water and then is freeze-dried, and the cut-off molecular weight of a dialysis bag is 3500D, thus obtaining 295mg of pale yellowish white solid mPEG₁₁₃-b-P(DPA₄₀-co-FAA₁₀) Yield 84.3%.¹H-NMR is shown in FIG. 3.

Preparation example 5: preparation of nano-vaccine

The polymer mPEG prepared in preparation example 2 was dispersed in pure water to prepare an OVA aqueous solution having a concentration of 1mg/mL₁₁₃-b-P(DPA₄₂-co-R848₄) And C14 alkanated hexanediol diacrylate (PDPE) was dissolved in tetrahydrofuran and gradually added to the OVA antigen peptide aqueous solution under ultrasonic conditions. And then, carrying out ultrafiltration and centrifugation for 1h to remove free peptide and tetrahydrofuran (the molecular weight cut-off of an ultrafiltration centrifugal tube is 50000D) in the system, thus obtaining the nano vaccine PDPR @ OVA.

Dispersing OVA antigen peptide DMSO solution in pure water to prepare OVA aqueous solution with concentration1 mg/mL. The polymers prepared in preparation examples 2 and 3 were mPEG₁₁₃-b-P(DPA₄₂-co-R848₄),Mannose-PEG₁₁₃-b-DPA₄₀And C14 alkanated hexanediol diacrylate (PDPE) was dissolved in tetrahydrofuran and gradually added to the OVA antigen peptide aqueous solution under ultrasonic conditions. And then, carrying out ultrafiltration and centrifugation for 1h to remove free peptide and tetrahydrofuran (the molecular weight cut-off of an ultrafiltration centrifugal tube is 50000D) in the system, thus obtaining the nano vaccine Man-PDPR @ OVA. The dynamic light scattering instrument is used for respectively measuring the hydrodynamic radius of the micelle, and meanwhile, the shape of the micelle is determined by a transmission electron microscope photo, and the test result is shown in figure 4.

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

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

Test example 1: determination of OVA content in nano vaccine

Free OVA antigen peptides were dissolved in methanol solutions to give solutions of 4,8,16,32,64, 128. mu.g/mL, and the absorption area at 280nm was measured by HPLC to plot a standard curve, as shown in FIG. 6 a. The nano-micelle PDPR @ OVA prepared in preparation example 5 was lyophilized, and 1mg thereof was weighed, dissolved in 2mL of methanol, and insoluble matter was removed through a 0.22 μm filter, and then the content of OVA antigen peptide was measured under the same chromatographic conditions, and the test results are shown in FIG. 6 b.

Test results show that the content of C14 chain alkylated hexanediol diacrylate (PDPE) in the nanomicelle PDPR @ OVA has a remarkable influence on the antigen peptide loading capacity of nanoparticles, and the antigen peptide loading capacity of the nanoparticles is gradually increased up to about 70% along with the increase of the content of C14 chain alkylated hexanediol diacrylate (PDPE).

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

After inoculating B16-OVA cells to the right back of C57BL/6 mice, when the tumor volume reaches 50mm³The mice are randomly divided into 5 groups at the left and right, each group comprises 6 mice, and the groups are respectively PBS; OVA; PDPR; PDPR @ OVA; Man-PDPR @ OVA was administered 1 time every seven days, twice every three days, and tumor volumes were recorded every three days, with the results shown in fig. 7. In FIG. 7, OVA group was free antigenic peptide and administered at a dose of 2.5 mg/kg; the PDPR group is a nano micelle group formed by a Racemate polymer, and the administration dosage is equivalent to 1mg/kg of Racemate; the PDPR @ OVA group is a nano vaccine simultaneously loaded with Rasimotent and antigen peptide, and the administration dosage is equivalent to 1mg/kg of Rasimotent and 2.5mg/kg of OVA; the Man-PDPR @ OVA group is mannose-functionalized Racemide and antigen peptide loaded nano vaccine, the administration dose is equivalent to 1mg/kg of Racemide and 2.5mg/kg of OVA, except that the R848 group is intraperitoneal administration, the administration modes of other groups are foot pad subcutaneous injection.

Test results show that the nano vaccine loaded with the Racemate and the antigenic peptide can effectively inhibit the growth of B16-OVA tumors, and the Toll-like receptor agonist can remarkably promote the recognition of the immune system of an organism on the antigenic peptide and realize the growth inhibition of B16-OVA transplanted tumors. After functionalization by mannose, the growth inhibition effect of the nanoparticles on B16-OVA transplanted tumors can be effectively enhanced.

Sequence listing

<110> Shanghai pharmaceutical research institute of Chinese 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 immunoadjuvant conjugates and tumor-associated antigens/antigen peptides,

wherein, the tumor-associated antigen/antigen peptide refers to immunogenic protein/protein fragment peptide or derived peptide generated by tumor-associated gene variation, and immunogenic protein/protein fragment peptide or derived peptide related to oncogenic virus gene,

wherein, in the formula 1,

R₂is a group derived from an immunological adjuvant;

R₃is C_nH_2n+1N is an integer of 3 to 12;

R₄is selected from

Or

Wherein Ligand is a derivatized dendritic cell targeting Ligand;

linker is R₂A linking chain to a carbonyl group, the linking chain forming a p, pi-conjugated linkage with an adjacent carbonyl group;

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

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

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

2. The tumor nano-vaccine of claim 1, wherein,

R₁selected from N, N-dibutylamino, N-diisopropylamino or hexamethyleneamino;

R₃is C_nH_2n+1N is 12;

x is 113; y is 40, 42 or 50; z is 0, 3 or 5.

3. The tumor nano-vaccine of claim 1, wherein,

the Ligand has a structure represented by the following formula 2:

4. the tumor nano-vaccine of claim 1, wherein,

the Linker is selected from any one of the following structures:

5. the tumor nano-vaccine of claim 1, wherein,

the immunologic adjuvant packageComprises the following steps: any one selected from the group consisting of STING agonists and TLR agonists, preferably, said R₂Any one selected from the following groups:

6. the tumor nano-vaccine of claim 1, wherein,

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

7. the tumor nano-vaccine of claim 1, wherein,

the tumor-associated antigen/antigen peptide comprises an ovalbumin antigen peptide segment with immunogenicity and a human papilloma virus antigen peptide, and preferably, the ovalbumin antigen peptide segment comprises: an ovalbumin polypeptide with an amino acid sequence of SIINFEKL or a salt thereof, such as trifluoroacetate of the ovalbumin polypeptide, and an ovalbumin G4 peptide with an amino acid sequence of SIIGFEKL or a salt thereof, such as trifluoroacetate of the ovalbumin G4 peptide.

8. The tumor nano-vaccine of claim 1, wherein,

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

9. A method for preparing a tumor nano-vaccine according to any one of claims 1 to 8, comprising the steps of:

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

10. Use of the tumor nano-vaccine of any one of claims 1 to 8 in the manufacture of a medicament for treating a malignant tumor comprising: breast cancer, cervical cancer, liver cancer, stomach cancer, pancreatic cancer, ovarian cancer, colon cancer or prostate cancer.