Use of immunological agents in promoting cell immunogenic death

Abstract

The application discloses application of an immunological preparation in promoting cell immunogenic death and belongs to the technical field of cell immunity. The immunological preparation comprises a liposome delivery system; the liposome delivery system comprises ionizable phospholipids, double decyl dimethyl ammonium bromide, cholesterol and dimyristyl glycerol-polyethylene glycol 2000; and the molar proportion of the ionizable phospholipids in the liposome delivery system is 10-69%. Unlike traditional strategies for inducing cell immunogenic death, the application starts from the liposome, and the outstanding effects of the immunological preparation in inducing cell immunogenic death and promoting immune activation are verified through cell and animal experiments.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cellular immunity, and particularly relates to application of an immunological preparation in promoting immunogenic death of cells. BACKGROUND

[0002] Cell death is an important step for maintaining normal operation of a living body, which can be roughly divided into two categories of apoptosis and necrosis. Apoptosis refers to cell autonomous and orderly death controlled by a series of genes for maintaining stability of the body, which basically does not affect surrounding cells. Necrosis is active or passive immunogenic death of cells affected by external factors such as bacterial and viral infection, which is manifested as cell membrane damage, leakage of cell contents such as organelles and DNA fragments, and causes inflammation of surrounding tissues. Active induction of immunogenic death of target cells can be used for tumor immunotherapy on the one hand, and as a vaccine adjuvant on the other hand. Therefore, development of a new and safe strategy for immunogenic death of cells has important significance for cancer treatment and vaccine design. SUMMARY

[0003] To solve the above technical problems, the application provides application of an immunological preparation in promoting immunogenic death of cells from the perspective of liposomes, and outstanding effects of the immunological preparation in inducing immunogenic death of cells and promoting immune activation are verified through cell and animal experiments.

[0004] To achieve the above application purposes, the application provides the following technical solutions.

[0005] In one aspect, the application provides application of an immunological preparation in promoting immunogenic death of cells.

[0006] The immunological preparation comprises a liposome delivery system.

[0007] The liposome delivery system comprises ionizable phospholipids, double-decanediyl dimethyl ammonium bromide, cholesterol and dimyristyl glycerol-polyethylene glycol 2000.

[0008] The ionizable phospholipids have a molar ratio of 10-69% in the liposome delivery system.

[0009] Optionally, the ionizable phospholipids, double-decanediyl dimethyl ammonium bromide, cholesterol and dimyristyl glycerol-polyethylene glycol 2000 have a molar ratio of:

[0010] 13-90:0-77:40:0.4.

[0011] Optionally, the ionizable phospholipids have the following structural formula:

[0012]

[0013] Optionally, the immunological preparation further comprises a cyclic dinucleotide.

[0014] Optionally, the cyclic dinucleotide is encapsulated in the liposome delivery system.

[0015] Optionally, the dose of the cyclic dinucleotide in the immunological preparation is 0.25-1 mg / kg.

[0016] Optionally, the cyclic dinucleotide has the following structural formula:

[0017]

[0018] Optionally, the mass ratio of the cholesterol to the cyclic dinucleotide is 0.5-5.

[0019] Optionally, the cells are immune cells and / or tumor cells.

[0020] Optionally, the immune cells include macrophages RAW-ISG and dendritic cells DC2.4.

[0021] The tumor cells include melanoma B16F10.

[0022] Optionally, in the application process, the immunological preparation induces cell immunogenic death by activating the programmed necrosis pathway.

[0023] Optionally, the manifestations of the cell immunogenic death include cell content release, cell membrane damage and lymph node inflammation.

[0024] Compared with the prior art, the present application has the following beneficial effects:

[0025] (1) Unlike the traditional strategy of inducing cell immunogenic death, the present application designs a liposome delivery system based on ionizable phospholipid IP9 from the perspective of liposomes to load and deliver the immunostimulant cyclic dinucleotide, and constructs a new immunological preparation STINGsome.

[0026] (2) The application selects cyclic dinucleotide CDG as a representative of stimulants to construct an immune preparation, and studies the effect of different IP9 proportions in inducing cell immunogenic death. Among them, the proportion of IP9 is divided into 46% (named STINGsome60, abbreviated as STS60) and 23% (named STINGsome30, abbreviated as STS30). The corresponding empty vectors (without CDG) are named S60 and S30. Based on the experiments of immune cells (macrophage RAW-ISG and dendritic cell DC2.4) and tumor cells (melanoma B16F10), it is proved that STINGsome can induce high proportion of cell death and content release, and the higher the proportion of IP9, the better the mortality.

[0027] (3) The inhibitor experiment of the application shows that STINGsome may induce the cell programmed necrosis pathway (necroptosis). After subcutaneous injection of mice, the transcriptome analysis of the draining lymph nodes shows that S60 and S30 also have certain immune stimulating activity. Based on this, the immunogenic cell death system induced by STINGsome can provide a new breakthrough point for the development of cancer treatment and vaccine adjuvant. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Survival rate analysis of DC2.4, RAW-ISG, B16F10 treated by different STS60 and STS30 of the application (Note: *** represents p<0.001);

[0029] Figure 2 Proportion analysis of LDH released by DC2.4, RAW-ISG, B16F10 induced by STS60 and STS30 of the application (Note: *** represents p<0.001);

[0030] Figure 3 Proportion analysis of cells positive for Annexin V-mCherry (cell membrane staining) / SYTOX Green of DC2.4, RAW-ISG, B16F10 treated by STS60 and STS30 of the application (Note: ** represents p<0.01; *** represents p<0.001);

[0031] Figure 4 STS60 treated cells of the application can be colored by Annexin V-mCherry (cell membrane staining) / SYTOX Green (nucleus staining), which proves the destruction of cell integrity (Note: A represents CLSM analysis diagram of DC2.4 cells; B represents enlarged diagram of single DC2.4 cell; C represents CLSM analysis diagram of B16F10 cells; D represents enlarged diagram of single B16F10 cell);

[0032] Figure 5 The difference in the immune activation characteristics of the draining lymph nodes stimulated by STS60 and STS30 after subcutaneous injection at the base of the mouse tail for the present application;

[0033] Figure 6 The pretreatment of the programmed necrosis inhibitors Nec-1 and Nec-2 of the present application can significantly alleviate the death of DC2.4 cells and the release of LDH caused by STS60 (Note: *** represents p<0.001). DETAILED DESCRIPTION

[0034] The present application will be further described below in conjunction with specific examples. The following description is only a few embodiments of the present application, and does not limit the present application in any form. Although the present application is disclosed as follows with preferred embodiments, it is not intended to limit the present application. Any skilled person in the art can make some changes or modifications to the disclosed technical content without departing from the scope of the technical solution of the present application, and such equivalent embodiments are equivalent to the equivalent embodiments, which are within the scope of the technical solution.

[0035] Unless otherwise specified, the raw materials in the examples of the present application are purchased through commercial channels and are directly used without any special treatment.

[0036] Unless otherwise specified, the analysis methods in the examples all use the conventional settings of instruments or equipment and conventional analysis methods.

[0037] Example 1

[0038] Preparation of STINGsome immunological agent:

[0039] (1) The synthesis route of ionizable phospholipid IP9 is as follows:

[0040]

[0041] P2 synthesis:

[0042] Take n-nonyl alcohol 2g (P1) and triethylamine 8.42g and add them to a round-bottom flask containing 60mL of anhydrous tetrahydrofuran (THF); take 12.7g of phosphorus oxychloride (POCl3) and dissolve it in 60mL of THF, and cool it to -30℃; add P1 dropwise to the POCl3 solution, stir the reaction at 0℃ for 1h after the dropwise addition is completed, and concentrate the reaction crude product P2 by rotary evaporator after the reaction is completed;

[0043] P3 synthesis:

[0044] Take n-bromooctane (P3a) 3.8g, ethanolamine (P3b) 30g, diisopropylethylamine (DIEA) 20g and potassium iodide (KI) 1.03g dissolved in 300mL ethanol, heated to 90℃, and react for 15h. After the reaction is completed, the reaction product is concentrated by a rotary evaporator, and column chromatography is used for separation and purification to obtain the product P3;

[0045] Synthesis of IP9:

[0046] Take P2 2.56g dissolved in 20mL THF, and cool to 0℃; take P3 2g and triethylamine 0.708g dissolved in 20mL THF and dropwise added to the P2 solution, and stir for 4h. After the reaction is completed, the reaction product is concentrated by a rotary evaporator, and high performance liquid chromatography (C 18 column) is used for separation and purification to obtain the target product IP9.

[0047] The cyclic dinucleotide CDG used in the application has the following structural formula:

[0048]

[0049] The compound is synthesized based on the route of the previous patent CN111592570B of the present inventor.

[0050] (2) Preparation of STINGsome:

[0051] First, the ethanol solutions of the four compounds IP9, DDAB (dodecyl dimethyl ammonium bromide), cholesterol and DMG-PEG2000 (dimyristyl glycerol-polyethylene glycol 2000) are mixed in a molar ratio of 60:30:40:0.4 (STS60) and 30:60:40:0.4 (STS30) respectively. Then, according to the mass ratio of cholesterol:CDG=2.36, CDG is added to the mixed solution of STS60 and STS30 respectively, mixed uniformly, and then diluted 4 times with phosphate buffered saline PBS to complete the preparation of STS60 and STS30.

[0052] (3) Preparation of empty vectors S60, S30:

[0053] Except that no CDG is added, the preparation steps of S60 and S30 are the same as those of STS60 and STS30 respectively.

[0054] Example 2

[0055] Analysis of STS60, STS30 promoting immune cell and tumor cell death:

[0056] Macrophage RAW-ISG, dendritic DC2.4 and melanoma B16F10 cells were used. Cells were plated at 1-4x104 / well in 96-well plates after culture completion and incubated overnight. CDG was dosed at 0.5, 1, 3, 5 μg / mL. Incubation was terminated after dosing according to cell phenotype change and cell viability was analyzed by CCK-8 and ATP cell viability (as shown in Figure 1 The results showed that the growth inhibition of STS60, STS30 on the three cells increased significantly with the increase of concentration. And compared with STS30, STS60 had higher cytotoxicity (as shown in Figure 1

[0057] Example 3

[0058] STS60, STS30 promote immune cells and tumor cells content release analysis:

[0059] Macrophage RAW-ISG, dendritic DC2.4 and melanoma B16F10 cells were used. Cells were plated at 1-4x104 / well in 96-well plates after culture completion and incubated overnight. CDG was dosed at 5 μg / mL. Incubation was terminated after dosing according to cell phenotype change and cell content release ratio was analyzed by lactate dehydrogenase (LDH) release (Promega) (as shown in Figure 2 The results showed that STS60, STS30 could cause the release of the contents of the three cells. And compared with STS30, STS60 had higher LDH release efficiency (as shown in Figure 2 In addition, empty vectors S60, S30 could also cause LDH release, and S60 had higher LDH release efficiency (as shown in Figure 2

[0060] Example 4

[0061] STS60, STS30 affect immune cells and tumor cells membrane integrity analysis:

[0062] Macrophage RAW-ISG, dendritic DC2.4 and melanoma B16F10 cells were used. Cells were plated at 1-4x104 / well in 96-well plates after culture completion and incubated overnight. CDG was dosed at 5 μg / mL. Incubation was terminated after dosing according to cell phenotype change and the incubated cells were stained using Annexin V-mCherry (stains cell membrane) / SYTOX Green (stains cell nucleus) apoptosis detection kit (Biological) to analyze the proportion of double positive cells (as shown in Figure 3 ​​The experimental results show that STS60 and STS30 can cause integrity destruction of the three kinds of cells, and further cause successful dyeing of the dye. And, compared with STS30, STS60 can cause higher proportion of cell membrane destruction (such as Figure 3 The laser confocal microscope imaging results also prove that the increase of cell membrane permeability causes the cell nucleus and cell membrane to be dyed by SYTOX Green and Annexin V, respectively (such as Figure 4

[0063] Example 5

[0064] In vivo lymph node activation evaluation of S60 and S30:

[0065] PBS, S60 and S30 were subcutaneously injected into the tail root of BALB / c mice. Four hours after injection, the proximal inguinal lymph nodes were collected and rapidly frozen with liquid nitrogen, and transcriptome sequencing was performed. The results show that S60 and S30 have certain immune stimulating activity, and can significantly up-regulate the levels of immune genes such as IL-1β, CCL12 and CCL7 in the lymph nodes (such as Figure 5

[0066] Example 6

[0067] STS60 induces cell immunogenic death through the necroptosis pathway:

[0068] Dendritic cells DC2.4 were used. After cell culture was completed, 1-4 million cells / well were plated into a 96-well plate and cultured overnight. After pre-treatment with necroptosis inhibitors (Nec-1, Nec-2), STS60 was added for incubation. The CDG administration concentration was 5 μg / mL. After administration, the incubation was terminated according to the cell phenotype change, and the survival of the cells was analyzed by CCK-8 and lactate dehydrogenase (LDH) release (Promega) (such as Figure 6 The experimental results show that the necroptosis inhibitors can alleviate the cell death caused by STS60 and inhibit the release of LDH, proving that STINGsomes can cause cell immunogenic death by inducing the necroptosis pathway.

[0069] The above is only a few embodiments of the present application, and does not limit the present application in any form. Although the above preferred embodiments are disclosed, they are not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above disclosed technical contents without departing from the scope of the technical solutions of the present application, and such equivalent embodiments are also equivalent to the equivalent embodiments, and all belong to the scope of the technical solutions.​​

Claims

1. Application of immunomodulatory agents in the preparation of drugs for treating melanoma; The immunomodulatory agent includes a liposome delivery system; The liposome delivery system contains ionizable phospholipids, bis(decyl)dimethylammonium bromide, cholesterol, and dimyristic glycerol-polyethylene glycol 2000; The ionizable phospholipids constitute 23% or 46% of the molar percentage in the liposome delivery system. The ionizable phospholipid has the following structural formula: ； The immunomodulator also contains cyclic dinucleotides; The cyclic dinucleotide is encapsulated in the liposome delivery system; The cyclic dinucleotide has the following structural formula: 。 2. The application according to claim 1, characterized in that, The molar ratio of the ionizable phospholipid, didecyl dimethyl ammonium bromide, cholesterol, and dimyristoyl glycerol-polyethylene glycol 2000 is as follows: 13~90:0~77:40:0.4。 3. The application according to claim 1, characterized in that, The dosage of cyclic dinucleotide in the immunomodulator is 0.25~1 mg / kg.

4. The application according to claim 1, characterized in that, The mass ratio of cholesterol to cyclic dinucleotide is 0.5 to 5.

5. The application according to claim 1, characterized in that, The immunomodulator induces immunogenic cell death by activating the programmed necrosis pathway.

6. The application according to claim 5, characterized in that, The manifestations of immunogenic cell death include the release of cell contents, cell membrane damage, and lymph node inflammation.

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