DC vaccine as well as preparation method and application thereof

Abstract

The invention provides a DC vaccine as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The invention provides a preparation method of a DC vaccine. The preparation method comprises the following steps: obtaining bone marrow-derived dendritic cells; synthesizing mRNA (messenger Ribonucleic Acid) for coding the tumor-associated The method comprises the following steps: delivering mRNA into bone marrow-derived dendritic cells by adopting a polymer carrier delivery method, and inducing the bone marrow-derived dendritic cells to be mature by virtue of an adjuvant, so as to prepare the DC vaccine. The preparation method of the DC vaccine provided by the invention is simple, and the prepared DC vaccine has the characteristics of strong specificity, remarkable curative effect and the like, realizes prevention and treatment of tumors, and has wide application prospects in the field of tumor immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a DC vaccine and a preparation method and application thereof. Background Art

[0002] Tumor immunotherapy uses the human immune system to identify and attack cancer cells, maintain the tumor-immune cycle, and strengthen the body's recognition and elimination of tumor cells, thereby achieving cancer treatment. This treatment method includes cancer vaccines, immune checkpoint inhibitors, and cell immunotherapy, among which cell immunotherapy, as a treatment that activates autoimmune function, has attracted much attention due to its broad spectrum, significant effects, fewer side effects, and prevention of recurrence and metastasis. It cultivates and transforms immune cells in the body that can recognize and attack cancer cells, especially antigen presenting cells (APCs), and re-infuses them into the body, effectively and specifically activating immune cells (such as T cells or NK cells) to fight cancer cells [see C. Liu, Q. Shi, X. Huang, S. Koo, N. Kong, W. Tao, Nature Reviews Cancer. 2023, 23, 526-543, N. Gong, M. Alameh, R. El-Mayta, L. Xue, D. Weissman, MJ Mitchell, Nature Reviews Drug Discovery. 2024, 23, 607-625]. Dendritic cells (DCs) are considered the most potent antigen-presenting cells (APCs) that initiate antigen-specific T cell activation in adaptive immunity.

[0003] Given the central role of DCs vaccines in controlling immune responses, they have been widely studied for cancer immunotherapy, and some of them have even been approved by the FDA for clinical use, such as Sipuleucel-T, which is one of the first cancer immunotherapies approved by the FDA for prostate cancer. At present, DC cell vaccines have been used in clinical studies for the treatment of various tumors worldwide, but the actual clinical effect is difficult to meet expectations [see M. Saxena, S. Balan, V. Roudko, N. Bhardwaj, Nature Biomedical Engineering, 2018, 2, 341-346]. This is mainly due to the fact that in vitro transcribed (IVT) mRNA usually enters cells through the endolysosomal system, but on the one hand, endosomal localization allows IVT mRNA to interact with Toll-like receptors (such as TLR 3, TLR7) on the endosomal membrane, leading to nonspecific immune activation in cells, inhibiting mRNA translation and protein expression. On the other hand, the delivery vector affects the interaction of mRNA with pattern recognition receptors (PRRs) through unknown pathways; and the immunogenicity of the vector itself can also lead to enhanced innate immune activation, further inhibiting the RNA translation process. Endosomal escape and the release of IVT mRNA pose a major obstacle to RNA translation. The antigen types, loading strategies and vaccine delivery methods used in the trial still need to be improved, and tumor-induced immunosuppression further limits the effectiveness of DC vaccines [see KH Loomis, JL Kirschman, S. Bhosle, RV Bellamkonda, PJ Santangelo, Journal of Materials Chemistry B, 2016, 4, 1619-1632, Q. He, H. Gao, D. Tan, H. Zhang, J. Wang, Acta Pharmaceutica Sinica B, 2022, 12, 2969-2989].

[0004] Therefore, how to safely and efficiently deliver mRNA to DCs and achieve efficient expression of specific antigens without activating the innate immune pathway, and at the same time enhance antigen presentation and promote DC cell maturation after reaching a certain antigen accumulation, and stimulate T cell activation and proliferation has become the core issue in improving the efficacy of DC vaccines.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The first purpose of the present invention is to provide a method for preparing a DC vaccine, wherein the prepared DC vaccine has significantly improved antigen expression, and its antigen presentation ability and its ability to induce tumor-specific cytotoxic T cells in vitro are also significantly enhanced, so as to solve the above-mentioned technical problems.

[0007] The second object of the present invention is to provide a DC vaccine.

[0008] The third object of the present invention is to provide the use of the above DC vaccine in preventing tumors.

[0009] In order to achieve the above objectives, the following technical solutions are proposed:

[0010] In a first aspect, the present invention provides a method for preparing a DC vaccine, comprising the following steps:

[0011] a. Obtain bone marrow-derived dendritic cells (BMDC);

[0012] b. Synthesize mRNA encoding tumor-associated antigens;

[0013] c. using a polymer carrier delivery method to deliver the mRNA in step b to the bone marrow-derived dendritic cells in step a, and then inducing the maturation of the bone marrow-derived dendritic cells with an adjuvant to prepare a DC vaccine;

[0014] The polymer carrier has a structure shown in Formula 1:

[0015]

[0016] wherein x is selected from 10 to 1000, and y is selected from 1 to 200;

[0017] R1 is selected from -NH(CH2) m R or -O(CH2) m R;

[0018] The R is selected from -NR a R b , a substituted or unsubstituted 5- to 6-membered nitrogen-containing heterocyclic group, a substituted or unsubstituted 5- to 6-membered nitrogen-containing heteroaromatic ring group or -N + (R c )3X - ;

[0019] The R c is selected from C1-C4 alkyl, and X is selected from halogen;

[0020] The R a , R b Independently selected from -H, -(CH2) m NH2, substituted or unsubstituted C1~C 10 One or more of the alkyl groups of

[0021] The m is selected from an integer between 2 and 5;

[0022] The R2 is selected from -NH(CH2)n R d or -OCH2R e ;

[0023] The n is selected from an integer between 0 and 15;

[0024] The R d Selected from substituted or unsubstituted C1~C 16 Alkyl, substituted or unsubstituted C6~C 14 aryl, substituted or unsubstituted C3-C8 heteroaryl, -CH(COOCH3)(CH2) p One or more of NHC(NH)NH2;

[0025] The R' is selected from substituted or unsubstituted C6~C 14 The aromatic group;

[0026] The p is selected from an integer between 1 and 6;

[0027] The R e Selected from substituted or unsubstituted C1~C 16 The alkyl group;

[0028] The R3 is selected from -(CH2) L R f ;

[0029] The L is selected from an integer between 0 and 15;

[0030] The R f Selected from substituted or unsubstituted C1~C 16 substituted or unsubstituted 5- to 8-membered cycloalkyl, substituted or unsubstituted 5- to 6-membered heteroaromatic ring group, substituted or unsubstituted C6-C 14 Aryl, -COR c , -C(R g )3, -(O(CH2) m )3OH or more;

[0031] Among them, R c is selected from C1-C4 alkyl, q1 is selected from 40-120, R g Selected from substituted or unsubstituted C6~C 14 The aromatic group.

[0032] As a further technical solution, the method for preparing bone marrow-derived dendritic cells comprises: isolating single bone marrow cells from the bone marrow, and then culturing them in a culture medium containing GM-CSF and IL-4 to prepare bone marrow-derived dendritic cells.

[0033] As a further technical solution, the concentration of GM-CSF is 8 to 12 ng / mL;

[0034] The concentration of IL-4 is 4-6 ng / mL.

[0035] As a further technical solution, the culture temperature is 37°C;

[0036] The culture time is 5 to 7 days.

[0037] As a further technical solution, the mRNA encoding tumor-associated antigens includes GFP mRNA (mGFP), OVA mRNA (mOVA), AFP mRNA (mAFP), EGFR mRNA (mEGFR) and MUC-1 mRNA (mMUC-1).

[0038] As a further technical solution, the adjuvant includes lipopolysaccharide (LPS), monophosphoryl lipid A (MPL), resiquimod (R848), 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP), imiquimod (R837) and Plasmodium falciparum merozoite surface antigen 2 (MSA-2).

[0039] As a further technical solution, in step c, bone marrow-derived dendritic cells are treated with a polymer carrier loaded with mRNA encoding tumor-associated antigens for 4 to 8 hours, and then an adjuvant is added to continue stimulating for 24 to 30 hours to prepare a DC vaccine.

[0040] As a further technical solution, the final concentration of the mRNA encoding the tumor-associated antigen is 0.8 to 1.2 μg / mL;

[0041] The final concentration of the polymer carrier is 1.25-10 μg / mL;

[0042] The final concentration of the adjuvant is 0.1-10 μg / mL.

[0043] In a second aspect, the present invention provides a DC vaccine prepared by the above-mentioned preparation method.

[0044] In a third aspect, the present invention provides the use of the above DC vaccine in preventing tumors.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The preparation method of the DC vaccine provided by the present invention is simple. The mRNA encoding tumor-associated antigens is delivered to BMDCs through a low-immunogenic polymer carrier in a way that bypasses the endosome to avoid disturbance and non-specific activation of BMDCs, thereby achieving efficient intracellular translation of exogenous RNA and obtaining a large amount of encoded antigens. Further stimulation by adjuvants significantly enhances the BMDC antigen presentation level and the expression of costimulatory molecules CD80 and CD86, promotes the maturation of BMDCs, and effectively activates CD8 + T cells regulate the body's immune microenvironment and achieve effective tumor immunotherapy. The DC vaccine prepared by this preparation method has the characteristics of strong specificity and significant efficacy, and achieves the prevention and treatment of tumors, and has broad application prospects in the field of tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0048] Figure 1 Agarose gel electrophoresis evaluation results of carrier-encapsulated OVA mRNA nanoparticles in Example 3;

[0049] Figure 2 The evaluation results of the vector / GFP mRNA nanoparticle cell transfection effect in Example 4 (top) and the bright field image of cells after incubation with vector / GFP mRNA in vitro (bottom);

[0050] Figure 3 The presentation effect diagram of BMDC in Example 5;

[0051] Figure 4 Activation effect diagram of BMDC in Example 5;

[0052] Figure 5 Activation effect diagram of BMDC in Example 6;

[0053] Figure 6 The presentation effect diagram of BMDC in Example 9;

[0054] Figure 7 The presentation effect diagram of BMDC in Example 11;

[0055] Figure 8 Example 31 CD8 + T cell proliferation evaluation results diagram;

[0056] Fig. 9The results of DC vaccine in Example 32 preventing melanoma in mice;

[0057] Fig.10 The results of DC vaccine treatment of mouse melanoma in Example 33 are shown in FIG. DETAILED DESCRIPTION

[0058] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0059] In a first aspect, the present invention provides a method for preparing a DC vaccine, comprising the following steps:

[0060] a. Obtain bone marrow-derived dendritic cells;

[0061] b. Synthesize mRNA encoding tumor-associated antigens;

[0062] c. using a polymer carrier delivery method to deliver the mRNA in step b to the bone marrow-derived dendritic cells in step a, and then inducing the maturation of the bone marrow-derived dendritic cells with an adjuvant to prepare a DC vaccine;

[0063] The polymer carrier has a structure shown in Formula 1:

[0064]

[0065] wherein x is selected from 10 to 1000, and y is selected from 1 to 200;

[0066] R1 is selected from -NH(CH2) m R or -O(CH2) m R;

[0067] The R is selected from -NR a R b , a substituted or unsubstituted 5- to 6-membered nitrogen-containing heterocyclic group, a substituted or unsubstituted 5- to 6-membered nitrogen-containing heteroaromatic ring group or -N + (R c )3X - ;

[0068] The R c is selected from C1-C4 alkyl, and X is selected from halogen;

[0069] The R a , R b Independently selected from -H, -(CH2) mNH2, substituted or unsubstituted C1~C 10 One or more of the alkyl groups of

[0070] The m is selected from an integer between 2 and 5;

[0071] The R2 is selected from -NH(CH2) n R d or -OCH2R e ;

[0072] The n is selected from an integer between 0 and 15;

[0073] The R d Selected from substituted or unsubstituted C1~C 16 Alkyl, substituted or unsubstituted C6~C 14 aryl, substituted or unsubstituted C3-C8 heteroaryl, -CH(COOCH3)(CH2) p One or more of NHC(NH)NH2;

[0074] The R' is selected from substituted or unsubstituted C6~C 14 The aromatic group;

[0075] The p is selected from an integer between 1 and 6;

[0076] The R e Selected from substituted or unsubstituted C1~C 16 The alkyl group;

[0077] The R3 is selected from -(CH2) L R f ;

[0078] The L is selected from an integer between 0 and 15;

[0079] The R f Selected from substituted or unsubstituted C1~C 16 substituted or unsubstituted 5- to 8-membered cycloalkyl, substituted or unsubstituted 5- to 6-membered heteroaromatic ring group, substituted or unsubstituted C6-C 14 Aryl, -COR c , -C(R g )3, -(O(CH2) m )3OH or more;

[0080] Among them, R c is selected from C1-C4 alkyl, q1 is selected from 40-120, R g Selected from substituted or unsubstituted C6~C 14 The aromatic group.

[0081] It should be noted that the polymer carrier described in the present invention is the polymer in patent 202410220208.8, for example, it can be the polymer of Example 1 in patent 202410220208.8. The inventors have found that after the carrier is loaded with mRNA, it enters the cytoplasm through disulfide exchange, bypasses the endosomal pathway, avoids mRNA and endosomal membrane Toll-like receptor (TLR) recognition and activation of cell-related pathways leading to mRNA degradation, and rapidly degrades and releases mRNA to the cytoplasm under the response of intracellular GSH.

[0082] The preparation method of the DC vaccine provided by the present invention is simple. The mRNA encoding tumor-associated antigens is delivered to BMDCs through a low-immunogenic polymer carrier in a way that bypasses the endosome to avoid disturbance and non-specific activation of BMDCs, thereby achieving efficient intracellular translation of exogenous RNA and obtaining a large amount of encoded antigens. Further stimulation by adjuvants significantly enhances the BMDC antigen presentation level and the expression of costimulatory molecules CD80 and CD86, promotes the maturation of BMDCs, and effectively activates CD8 + T cells regulate the body's immune microenvironment and achieve effective tumor immunotherapy. The method provided by the present invention to promote the efficacy of DC vaccines through a strategy of first transfection and then activation provides a new idea for tumor prevention and treatment.

[0083] In some optional embodiments, the method for preparing bone marrow-derived dendritic cells comprises: isolating single bone marrow cells from the bone marrow, and then culturing the single bone marrow cells in a culture medium containing GM-CSF and IL-4 to prepare bone marrow-derived dendritic cells.

[0084] In some optional embodiments, the concentration of GM-CSF may be, for example, but not limited to, 10 ng / mL;

[0085] The concentration of IL-4 may be, for example, but not limited to, 5 ng / mL.

[0086] In some optional embodiments, the culture temperature may be, for example, but not limited to, 37°C;

[0087] The culture period may be, for example, but not limited to, 5 days, 6 days or 7 days.

[0088] In some optional embodiments, the mRNA encoding tumor-associated antigens includes but is not limited to GFP mRNA, OVA mRNA, AFP mRNA, EGFR mRNA and MUC-1 mRNA.

[0089] In some optional embodiments, the adjuvant includes but is not limited to lipopolysaccharide (LPS), monophosphoryl lipid A (MPL), resiquimod (R848), 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP), imiquimod (R837) and Plasmodium falciparum merozoite surface antigen 2 (MSA-2).

[0090] The delayed addition of the above adjuvants can activate BMDCs without affecting mRNA translation, promote the expression of co-stimulatory signals and enhance antigen presentation, and enhance the efficacy of DC vaccines.

[0091] In some optional embodiments, in step c, bone marrow-derived dendritic cells are treated with a polymer carrier loaded with mRNA encoding a tumor-associated antigen for 4 to 8 hours, and then an adjuvant is added to continue stimulating for 24 to 30 hours to prepare a DC vaccine.

[0092] Treating immature BMDCs with mRNA-loaded vectors for 4 to 8 hours in advance helps achieve sufficient cellular internalization and mRNA translation. The low immunogenicity of the vector and the uptake that bypasses the endosomal pathway can avoid the interaction between mRNA and pattern recognition receptors (PRRs) on the endosomal membrane, preventing the restriction of mRNA translation caused by nonspecific activation of BMDCs, thereby achieving massive protein expression.

[0093] In some optional embodiments, the final concentration of the mRNA encoding the tumor-associated antigen may be, for example, but not limited to, 0.8 μg / mL, 1 μg / mL or 1.2 μg / mL;

[0094] The final concentration of the polymer carrier may be, for example, but not limited to, 1.25 μg / mL, 5 μg / mL or 10 μg / mL;

[0095] The final concentration of the adjuvant may be, for example, but not limited to, 0.1 μg / mL, 1 μg / mL or 10 μg / mL.

[0096] In a second aspect, the present invention provides a DC vaccine prepared by the above-mentioned preparation method.

[0097] This DC vaccine has the characteristics of strong specificity and significant efficacy and can be used for the prevention and treatment of tumors. It has broad application prospects in the field of tumor immunotherapy.

[0098] In a third aspect, the present invention provides the use of the above DC vaccine in preventing and treating tumors.

[0099] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0100] Example 1 Extraction and culture of mouse bone marrow-derived dendritic cells

[0101] The bone marrow of C57BL / 6 mice was taken, and single bone marrow cells were washed with sterile PBS at a speed of 350g for 8 minutes. The single cells separated above were resuspended in 10mL 1640 medium containing 10ng / mL GM CSF and 5ng / mL IL 4, and transferred to a culture dish. They were cultured in a cell culture incubator at 37°C with 5% carbon dioxide for 7 days. During this period, the medium was changed according to the cell status to ensure that the concentrations of GM CSF and IL 4 remained unchanged. After 7 days, the semi-adherent cells were resuspended and the cells were washed twice with sterile PBS at a speed of 350g for 8 minutes to obtain immature BMDCs. The collected cells were centrifuged at 1×10 4 mL -1 The cells were seeded in 12-well plates and cultured in a cell culture incubator at 37°C containing 5% carbon dioxide for 24 hours.

[0102] Example 2 In vitro transcription synthesis of mRNA

[0103] Plasmids containing GFP, OVA, AFP, EGFR and MUC-1 were constructed respectively, and the nucleotide sequences were obtained from Gene Bank; the plasmids were linearized by treatment with BspQ1 endonuclease, and the mRNAs of GFP, OVA, AFP, EGFR and MUC-1 were synthesized by in vitro transcription.

[0104] Example 3 Preparation of carrier / mRNA composite nanoparticles

[0105] The polymer carrier P1 in Example 1 of Patent 202410220208.8 is respectively dissolved in a reduced serum medium Opti-MEM with a composite ratio of 2.5 / 1 or 5 / 1 with green fluorescent protein expression mRNA (GFP mRNA), ovalbumin expression mRNA (OVA mRNA), alpha-fetoprotein expression mRNA (AFP mRNA), transmembrane mucin expression mRNA (MUC1 mRNA), and epidermal growth factor receptor protein expression mRNA (EGFR mRNA). TM A homogenous solution was formed in which the final concentration of mRNA was 1 μg / mL. After vortexing for 30 seconds, the mixture was incubated at room temperature for 15 minutes to obtain P1 / mRNA nanoparticles. The agarose gel electrophoresis evaluation results of the carrier-encapsulated OVA mRNA nanoparticles are shown in Figure 2. Figure 1 (In the figure, from left to right: RNA ladder (0.2, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0 and 6.0 kb from bottom to top), naked OVA mRNA (1.59 kb), P1 / OVA mRNA).

[0106] Example 4 Evaluation of transfection effect of vector / mRNA composite nanoparticles in BMDC

[0107] The prepared P1 / mGFP nanoparticles were added to a 12-well cell plate at a dosage of 1 μg mRNA / mL, and the FDA-approved lipid nanoparticles LNP and commercial transfection reagent Lipofectamine MessengerMAX were used. TM (Lipo) was used as a positive control for the transfection experiment. BMDC cells were cultured for 24 hours in a cell culture incubator at 37°C with 5% carbon dioxide. The 12-well cell culture plate was taken out of the incubator and the expression of GFP protein was observed using an inverted fluorescence microscope. The results showed that ( Figure 2 ), the vector / mRNA nanoparticles of the present invention showed excellent transfection efficiency in BMDC, and the transfection efficiency was significantly higher than that of the commercial mRNA transfection reagent Lipofectamine MessengerMAX TM .

[0108] Example 5 Evaluation of the activation and presentation effect of carrier / mRNA composite nanoparticles in BMDC

[0109] The prepared P1 / mOVA nanoparticles were added to a 12-well cell plate at a dosage of 1 μg mRNA / mL, and the FDA-approved lipid nanoparticles LNP and commercial transfection reagent Lipofectamine MessengerMAX were used. TM As a positive control for the transfection experiment, BMDC cells were cultured for another 24 hours in a cell culture incubator at 37°C containing 5% carbon dioxide. The 12-well cell culture plate was removed from the incubator, and BMDCs were collected and treated as follows: washed once with PBS, and then BMDCs were stained with antibodies (anti-CD11c antibody, anti-CD80 antibody, anti-CD86 antibody, anti-MHC II antibody, and anti-OVA antibody). Then, the cells were washed twice with PBS. Finally, BMDCs were resuspended in 4% paraformaldehyde (PFA) for FACS analysis. The results showed that ( Figure 3 , Figure 4 ), the vector / mRNA nanoparticles of the present invention showed excellent presentation efficiency in BMDC, and the antigen presentation efficiency was significantly higher than that of the commercial mRNA transfection reagent Lipofectamine MessengerMAX TM However, compared with other groups, it does not stimulate BMDC over-maturation and avoids BMDC cell disorder.

[0110] Example 6 Study on the promotion of BMDC maturation by vectors

[0111] The polymer carrier P1 in Example 1 of Patent 202410220208.8 was incubated in BMDCs for 24 hours with 2.5 or 5 μg / mL, and the BMDCs were collected and treated as follows: washed once with PBS, and then stained with antibodies (anti-CD11c antibody, anti-CD80 antibody, anti-CD86 antibody, and anti-MHC II antibody). Then, the cells were washed twice with PBS. Finally, the BMDCs were resuspended in 4% PFA for FACS analysis. FDA-approved lipid nanoparticles LNP and commercial transfection reagent Lipofectamine MessengerMAX were used TM As a positive control for this transfection experiment. Figure 5 As shown, compared with other groups, vector P1 alone did not lead to obvious phenotypic maturation of BMDCs, achieving the function of non-perturbed delivery.

[0112] Example 7-30 Preparation of DC vaccine

[0113] Experimental group: The prepared P1 / mRNA nanoparticles were added to a 12-well cell plate at a dosage of 1 μg mRNA / mL, and the BMDC cells were cultured for 6 hours in a cell culture incubator at 37°C containing 5% carbon dioxide. The adjuvant was added to the well plate at a determined dosage, and cultured for 24 hours in a cell culture incubator at 37°C containing 5% carbon dioxide. The 12-well cell culture plate was taken out of the incubator, and BMDCs were collected as the DC vaccine (recorded as P1 / mRNA-6h-adjuvant group). According to this experimental method, nanoparticles, adjuvants, and adjuvant dosages were selected according to Table 1 to obtain the DC vaccines of Examples 7 to 30.

[0114] Control group 1 (denoted as P1 / mRNA group): The prepared P1 / mRNA nanoparticles were added to a 12-well cell plate at a dosage of 1 μg mRNA / mL, and the BMDC cells were cultured for 30 hours in a cell culture incubator at 37°C containing 5% carbon dioxide. The 12-well cell culture plate was taken out from the incubator, and the BMDCs were collected as the DC vaccine of the control group 1. According to this experimental method, the control groups 1 of Examples 7 to 30 were obtained respectively.

[0115] Control group 2 (denoted as P1 / mRNA+adjuvant group): The prepared P1 / mRNA nanoparticles were added to a 12-well cell plate at a dosage of 1 μg mRNA / mL, and the adjuvant was added to the well plate at a determined dosage, and the BMDC cells were cultured for 30 hours in a cell culture incubator at 37°C containing 5% carbon dioxide. The 12-well cell culture plate was taken out from the incubator, and the BMDCs were collected as the DC vaccine of the control group 2. According to this experimental method, the control groups 1 of Examples 7 to 30 were obtained respectively.

[0116] The presentation effects of BMDC in Examples 9 and 11 are as follows: Figure 6 and Figure 7 The results of the remaining examples are similar to those of Examples 9 and 11, and the presentation effect of the P1 / mRNA-6h-adjuvant group is better than that of the control group 1 and the control group 2.

[0117] Table 1 Preparation of different DC vaccines

[0118] Example mRNA types Adjuvant type Adjuvant dosage (μg / mL) 7 OVA mRNA LPS 0.1 8 OVA mRNA R848 2.0 9 OVA mRNA 2'3'-cGAMP 1.8 10 OVA mRNA R837 2.0 11 OVA mRNA MSA-2 5.9 12 OVA mRNA MPL 0.1 13 AFP mRNA LPS 0.1 14 AFP mRNA R848 2.0 15 AFP mRNA 2'3'-cGAMP 1.8 16 AFP mRNA R837 2.0 17 AFP mRNA MSA-2 5.9 18 AFP mRNA MPL 0.1 19 EGFR mRNA LPS 0.1 20 EGFR mRNA R848 2.0 21 EGFR mRNA 2'3'-cGAMP 1.8 22 EGFR mRNA R837 2.0 23 EGFR mRNA MSA-2 5.9 24 EGFR mRNA MPL 0.1 25 MUC-1mRNA LPS 0.1 26 MUC-1mRNA R848 2.0 27 MUC-1mRNA 2'3'-cGAMP 1.8 28 MUC-1mRNA R837 2.0 29 MUC-1mRNA MSA-2 5.9 30 MUC-1mRNA MPL 0.1

[0119] Example 31 DC vaccine mediated CD8 + Effects of T cell proliferation and activation

[0120] Initial CD8 + T cell isolation kit isolates naive CD8 from OT1 mice + T cells, sorted cells were stained with CFSE, resuspended in T cell culture medium and counted. 2.5×10 4 The DCs of control group 1 and control group 2 and the DCs of the experimental group obtained by the treatment in Example 7 were added with 2.5×10 5 Initial CD8 + T cells were co-cultured with DCs. Cells were collected on the 4th day of co-culture and CD8 + The proliferation of T cells. Figure 8 As shown, the experimental group P1 / mOVA-6h-LPS can effectively promote the proliferation of T cells and enhance the immune response.

[0121] Example 32 Study on the prevention of melanoma in mice by DC vaccine

[0122] The DC vaccine of Example 7 was injected into the right dorsal flank of female C57BL / 6J mice. The dose of DC vaccine containing 1 million BMDC cells was administered to each mouse once every 3 days for a total of 3 times. 6 B16OVA cells were injected into the right dorsal flank of mice, and the tumor growth of mice in each group was recorded. The mice were killed on the 16th day for analysis.

[0123] Experimental results ( Fig. 9 ) showed that compared with the untreated group and the DC-P1 / mOVA group without adjuvant treatment, the melanoma of mice was significantly reduced after treatment with DC vaccine enhanced by antigen presentation. This indicates that DC vaccine enhanced by antigen presentation can effectively inhibit the growth of melanoma in mice.

[0124] Example 33 Study on the treatment of melanoma in mice with DC vaccine

[0125] 1.0×10 6 B16OVA cells were injected into the right dorsal flank of female C57BL / 6J mice to establish a B16OVA mouse melanoma tumor model. When the tumor volume grew to about 80-120 mm 3 At 14:00, B16OVA tumor-bearing mice were randomly divided into three groups: PBS, DC-P1 / mOVA, and DC-P1 / mOVA-6h-LPS (Example 7). Each mouse was administered a DC vaccine containing 1 million BMDC cells, once every 3 days, for a total of 3 times. The tumor growth of each group of mice was recorded during the administration period, and the mice were killed on the 16th day for analysis.

[0126] Experimental results ( Fig.10 ) showed that, compared with the PBS and DC-P1 / mOVA groups, the melanoma in mice was significantly reduced after treatment with DC vaccine enhanced by antigen presentation. This indicates that DC vaccine enhanced by antigen presentation can effectively inhibit the growth of melanoma in mice.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a DC vaccine, characterized in that: The following steps are involved: a. Obtain bone marrow-derived dendritic cells; b. Synthesize mRNA encoding tumor-associated antigens; c. using a polymer carrier delivery method to deliver the mRNA in step b to the bone marrow-derived dendritic cells in step a, and then inducing the maturation of the bone marrow-derived dendritic cells with an adjuvant to prepare a DC vaccine; The polymer carrier has a structure shown in Formula 1: wherein x is selected from 10 to 1000, and y is selected from 1 to 200; R1 is selected from -NH(CH2) m R or -O(CH2) m R; The R is selected from -NR a R b , a substituted or unsubstituted 5- to 6-membered nitrogen-containing heterocyclic group, a substituted or unsubstituted 5- to 6-membered nitrogen-containing heteroaromatic ring group or -N + (R c )3X - ; The R c is selected from C1-C4 alkyl, and X is selected from halogen; The R a , R b Independently selected from -H, -(CH2) m NH2, substituted or unsubstituted C1~C 10 One or more of the alkyl groups of The m is selected from an integer between 2 and 5; The R2 is selected from -NH(CH2) n R d or -OCH2R e ; The n is selected from an integer between 0 and 15; The R d Selected from substituted or unsubstituted C1~C 16 Alkyl, substituted or unsubstituted C6~C 14 aryl, substituted or unsubstituted C3-C8 heteroaryl, -CH(COOCH3)(CH2) p One or more of NHC(NH)NH2; The R' is selected from substituted or unsubstituted C6~C 14 The aromatic group; The p is selected from an integer between 1 and 6; The R e Selected from substituted or unsubstituted C1~C 16 The alkyl group; The R3 is selected from -(CH2) L R f ; The L is selected from an integer between 0 and 15; The R f Selected from substituted or unsubstituted C1~C 16 substituted or unsubstituted 5- to 8-membered cycloalkyl, substituted or unsubstituted 5- to 6-membered heteroaromatic ring group, substituted or unsubstituted C6-C 14 Aryl, -COR c , -C(R g )3, -(O(CH2) m )3OH or more; Among them, R c is selected from C1-C4 alkyl, q1 is selected from 40-120, R g Selected from substituted or unsubstituted C6~C 14 The aromatic group.

2. The preparation method according to claim 1, characterized in that: The method for preparing bone marrow-derived dendritic cells comprises: isolating single bone marrow cells from bone marrow, and then culturing the single bone marrow cells in a culture medium containing GM-CSF and IL-4 to prepare bone marrow-derived dendritic cells.

3. The preparation method according to claim 2, characterized in that: The concentration of GM-CSF is 8-12 ng / mL; The concentration of IL-4 is 4-6 ng / mL.

4. The preparation method according to claim 2, characterized in that: The culture temperature is 37°C; The culture time is 5 to 7 days.

5. The preparation method according to claim 1, characterized in that: The mRNA encoding tumor-associated antigens includes GFP mRNA, OVA mRNA, AFP mRNA, EGFR mRNA and MUC-1 mRNA.

6. The preparation method according to claim 1, characterized in that: The adjuvants include lipopolysaccharide, monophosphoryl lipid A, resiquimod, 2',3'-cyclic guanosine monophosphate-adenosine monophosphate, imiquimod and Plasmodium falciparum merozoite surface antigen 2.

7. The preparation method according to claim 1, characterized in that: In step c, bone marrow-derived dendritic cells are treated with a polymer carrier loaded with mRNA encoding tumor-associated antigens for 4 to 8 hours, and then an adjuvant is added to continue stimulating for 24 to 30 hours to prepare a DC vaccine.

8. The preparation method according to claim 7, characterized in that: The final concentration of the mRNA encoding the tumor-associated antigen is 0.8 to 1.2 μg / mL; The final concentration of the polymer carrier is 1.25-10 μg / mL; The final concentration of the adjuvant is 0.1-10 μg / mL.

9. A DC vaccine, characterized in that: The preparation method is described in any one of claims 1 to 8.

10. Use of the DC vaccine according to claim 9 in preventing tumors.

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