A breast cancer mRNA vaccine and its preparation method

Abstract

The present invention provides a breast cancer mRNA vaccine and a preparation method thereof, which comprises lipid nanoparticles and genetic material mRNA. The genetic material is mRNA related to breast cancer-associated antigens HER2 / neu, MUC-1 and / or CEA. The lipid nanoparticles include ionizable cations, stearoyl phosphatidylcholine and polyethylene glycol. The molar ratio of the ionizable cations, stearoyl phosphatidylcholine and polyethylene glycol is 1:(0.1-10):(1-100). The ratio of the nitrogen content in the ionizable cations to the phosphorus content of the mRNA is 1:(0.1-10). At the same time, the present invention also discloses a preparation method of the breast cancer mRNA vaccine. The reasonable and optimized ratio of the ionizable cations in the lipid nanoparticles designed by the present invention realizes the efficient delivery of mRNA encoding breast cancer-associated antigens, so as to achieve the goals of improving the stability, effectiveness and safety of the breast cancer mRNA vaccine. The developed breast cancer mRNA vaccine can not only prevent the occurrence and recurrence of breast cancer, but also achieve the goal of improving its treatment effect through immunotherapy with this vaccine.

Description

Technical Field

[0001] The present invention belongs to the technical field of mRNA vaccines, and particularly relates to a breast cancer mRNA vaccine and a preparation method thereof. Background Art

[0002] Currently, the main treatment methods for breast cancer are surgery, radiotherapy, and chemotherapy. However, these treatment methods all have deficiencies such as large surgical wounds, strong toxic and side effects, incomplete resection, damage to the immune system, and tumor recurrence / metastasis. Moreover, patients also have to endure great pain during the treatment process. Therefore, developing a non-invasive, highly specific, low-toxicity, and highly effective treatment method is a key problem that urgently needs to be solved in the field of breast cancer treatment. In recent years, with the continuous update of tumor treatment methods, tumor mRNA vaccines introduce mRNA containing the coding sequence of tumor-associated antigen proteins into the human body, directly translate it to form the corresponding antigen proteins, thereby inducing the body to produce specific immune responses to achieve the role of preventing and treating tumors. However, there is currently no report on breast cancer mRNA vaccines.

[0003] There are two bottleneck problems in the development of breast cancer mRNA vaccines. One is the selection of tumor-associated antigens. Breast cancer has high heterogeneity, so it is easy for breast cancer cells to escape immunity against a single antigen. The other is the mRNA delivery system. mRNA delivery has always been a key bottleneck hindering the development of mRNA vaccines. Naked mRNA is easily degraded by extracellular RNases, and even if it enters the cell, it is likely to aggregate in lysosomes and cannot reach the place where it plays a role. The mRNA delivery system largely determines the efficacy, stability, and storage conditions of mRNA vaccines. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a multivalent breast cancer mRNA vaccine, its preparation method, and a preparation method of an mRNA delivery system in view of the above-mentioned deficiencies of the prior art. In the lipid nanoparticles designed by this method, the ionizable cations are rationally and optimally proportioned to achieve efficient delivery of mRNA encoding breast cancer-related antigens, so as to improve the stability, effectiveness, and safety of breast cancer mRNA vaccines. The developed breast cancer mRNA vaccine can not only prevent the occurrence and recurrence of breast cancer, but also achieve the goal of improving its treatment effect through immunotherapy with this vaccine.

[0005] To solve the above technical problem, the technical solution adopted by the present invention is: A breast cancer mRNA vaccine, characterized in that it comprises lipid nanoparticles and genetic material mRNA, and the genetic material is mRNA of two or more antigens among breast cancer-related antigens HER2 / neu, MUC-1, and CEA.

[0006] Preferably, the lipid nanoparticles comprise an ionizable cation, stearoyl phosphatidylcholine, and polyethylene glycol; the molar ratio of the ionizable cation, stearoyl phosphatidylcholine, and polyethylene glycol is 1:(0.1-10):(1-100).

[0007] The ionizable cation is one or more of N6-(5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)valeryl)lysine, (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)valeryl)glutamic acid, (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)valeryl)glycine, and (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)valeryl)alanine.

[0008] Preferably, the ratio of the nitrogen content in the ionizable cation to the phosphorus content of the mRNA is 1:(0.1-10).

[0009] Preferably, the preparation method of the ionizable cation is as follows:

[0010] Step 1: 5-Aminovaleric acid and 6-bromo-1,1,1,2,2,3,4,4-monofluorohexane are added to tetrahydrofuran in a molar ratio of 1:2, and then Cs 2 CO 3 is added. The reaction is carried out at 40 °C for 24 h. After the reaction is completed, THF is removed by rotary evaporation under reduced pressure to obtain 5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)valeric acid;

[0011] Step 2: The 5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)valeric acid obtained in Step 1 and an amino acid are added to the solvent tetrahydrofuran in a molar ratio of 1:1, and EDCI and HOBt are added simultaneously. The reaction is carried out for 24 h. After the reaction is completed, the solvent is removed by rotary evaporation under reduced pressure to obtain the ionizable cation;

[0012] When the amino acid in Step 2 is lysine, the ionizable cation is N6-(5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)valeryl)lysine;

[0013] When the amino acid in Step 2 is glutamic acid, the ionizable cation is (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)valeryl)glutamic acid;

[0014] When the amino acid in Step 2 is glycine, the ionizable cation is (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)valeryl)glycine;

[0015] When the amino acid in Step 2 is alanine, the ionizable cation is (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)alanine.

[0016] In addition, the present invention also provides a method for preparing a breast cancer mRNA vaccine, and the method is as follows:

[0017] Dissolve the lipid nanoparticles in 2 μL of ethanol to obtain a mixed solution A; dissolve the genetic material mRNA in 10 μL of citric acid-sodium citrate buffer with a pH of 4 to obtain a mixed solution B, and then add the mixed solution A to the mixed solution B and mix well to obtain the breast cancer mRNA vaccine.

[0018] The present invention has the following advantages compared with the prior art:

[0019] 1. By selecting breast cancer antigens HER2 / neu, MUC-1 and CEA, and rationally designing and optimizing the ratio of the ionizable cation in the lipid nanoparticles, the present invention realizes the efficient delivery of mRNA encoding breast cancer-related antigens (such as HER2 / neu, MUC-1 and CEA) to achieve the goals of improving the stability, effectiveness and safety of the breast cancer mRNA vaccine, and develops a breast cancer mRNA vaccine. This breast cancer mRNA vaccine can not only prevent the occurrence and recurrence of breast cancer, but also achieve the goal of improving its treatment effect through immunotherapy generated by this vaccine.

[0020] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Description of the Drawings

[0021] Figure 1 It is the molecular structure diagram of N6-(5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)lysine disclosed by the present invention.

[0022] Figure 2 It is the molecular structure diagram of (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)glutamic acid disclosed by the present invention.

[0023] Figure 3 It is the molecular structure diagram of (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)glycine disclosed by the present invention.

[0024] Figure 4 It is the molecular structure diagram of (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)alanine disclosed by the present invention.

[0025] Figure 5This is a bioluminescence experimental result graph of the nitrogen content of different ionizable cations of the present invention and the phosphorus content of luciferase mRNA linked to breast cancer antigen HER2 / neu mRNA.

[0026] Figure 6 This is a bioluminescence experimental result graph of the nitrogen content of different ionizable cations of the present invention and the phosphorus content of luciferase mRNA linked to breast cancer antigen MUC-1 mRNA.

[0027] Figure 7 This is a bioluminescence experimental result graph of the nitrogen content of different ionizable cations of the present invention and the phosphorus content of luciferase mRNA linked to breast cancer antigen CEA mRNA. Detailed implementation manners

[0028] Example 1

[0029] This example discloses a lipid nanoparticle, which includes an ionizable cation, stearoyl phosphatidylcholine and polyethylene glycol; the molar ratio of the ionizable cation to stearoyl phosphatidylcholine is 1:(0.1 - 10); the molar ratio of the ionizable cation to polyethylene glycol is 1:(1 - 100).

[0030] The ionizable cation is N6-(5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)lysine.

[0031] In this example, the preparation method of the ionizable cation is as follows:

[0032] Step 1: 5-aminopentanoic acid and 6-bromo-1,1,1,2,2,3,4,4-monofluorohexane are added to tetrahydrofuran in a molar ratio of 1:2, and Cs 2 CO 3 is added, and the reaction is carried out at 40°C for 24 h. After the reaction is completed, THF is removed by rotary evaporation under reduced pressure to obtain 5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoic acid;

[0033] Step 2: The 5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoic acid obtained in Step 1 and an amino acid are added to the solvent tetrahydrofuran in a molar amount ratio of 1:1, and EDCI and HOBt are added simultaneously and the reaction is carried out for 24 h. After the reaction is completed, the solvent is removed by rotary evaporation under reduced pressure to obtain the ionizable cation;

[0034] When the amino acid in Step 2 is lysine, the ionizable cation is N6-(5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)lysine.

[0035] In this embodiment, the ionizable cation can also be (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)valeryl)glutamic acid, (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)valeryl)glycine, or (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)valeryl)alanine.

[0036] In the preparation method of the ionizable cation, when the amino acid in step 2 is glutamic acid, the ionizable cation is (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)valeryl)glutamic acid; when the amino acid in step 2 is glycine, the ionizable cation is (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)valeryl)glycine; when the amino acid in step 2 is alanine, the ionizable cation is (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)valeryl)alanine.

[0037] In this embodiment, the molar ratio of the ionizable cation to stearoyl phosphatidylcholine can be any one of 1:0.1, 1:1, 1:3, 1:6, 1:10; the molar ratio of the ionizable cation to polyethylene glycol can be any one of 1:1, 1:10, 1:30, 1:50, 1:80, 1:100. Preferably, the molar ratio of the ionizable cation, stearoyl phosphatidylcholine, and polyethylene glycol is 1:1:50.

[0038] Example 2

[0039] This embodiment discloses a bioactive substance delivery system. In the bioactive substance delivery system, the lipid nanoparticles disclosed in the example are used as a carrier. Specifically, the bioactive substance delivery system is a breast cancer mRNA vaccine, and the breast cancer mRNA vaccine contains the lipid nanoparticles and the genetic material mRNA disclosed in Example 1, and the genetic material is the mRNA of two or more antigens related to breast cancer antigens HER2 / neu, MUC-1, and CEA.

[0040] The preparation method of the breast cancer mRNA vaccine is as follows:

[0041] Dissolve the lipid nanoparticles in 2 μL of ethanol to obtain a mixed solution A; dissolve the genetic material mRNA in 10 μL of a buffer solution with a pH of 4 to obtain a mixed solution B. The ratio of the nitrogen content in the ionizable cation in the lipid nanoparticles to the phosphorus content of the mRNA is 1:(0.1 - 10). Subsequently, add the mixed solution A to the mixed solution B and mix well to obtain the breast cancer mRNA vaccine.

[0042] Bioluminescence assay:

[0043] (1) Dissolve N6-(5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)lysine, stearoyl phosphatidylcholine, and polyethylene glycol with a molar ratio of 1:1:50 in 2 μL of ethanol to obtain mixed solution A. Dissolve breast cancer antigen HER2 / neu mRNA linked to luciferase mRNA in 10 μL of a buffer with a pH of 4 to obtain mixed solution B. Subsequently, add mixed solution A to mixed solution B to obtain mixed solution C. Six groups of experiments are set up for mixed solution C. In the six groups of experiments, the ratio of the nitrogen content in N6-(5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)lysine to the phosphorus content in breast cancer antigen HER2 / neu mRNA is 1:(0.5, 1, 2, 4, 8, 16) respectively. The molar ratio of breast cancer antigen HER2 / neu mRNA to luciferase mRNA in breast cancer antigen HER2 / neu mRNA linked to luciferase mRNA is 1:1. The six groups of mixed solution C are respectively added to 293T cells and cultured for 24 h, and detected by an enzyme-linked immunosorbent assay (ELISA) to obtain the graph as shown in Figure 5 shown in the figure, Figure 5 where KA1 in Table 6 represents N6-(5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)lysine. It can be seen from Figure 5 that: Breast cancer antigen HER2 / neu mRNA can be successfully expressed, proving the reliability of this breast cancer mRNA vaccine.

[0044] (2) Dissolve N6-(5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)lysine, stearoyl phosphatidylcholine, and polyethylene glycol with a molar ratio of 1:0.1:80 in 2 μL of ethanol to obtain mixed solution A. Dissolve breast cancer antigen HER2 / neu mRNA, MUC-1 mRNA linked to luciferase mRNA in 10 μL of a buffer with a pH of 4 to obtain mixed solution B. Subsequently, add mixed solution A to mixed solution B to obtain mixed solution C. Six groups of experiments are set up for mixed solution C. In the six groups of experiments, the ratio of the nitrogen content in N6-(5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)lysine to the phosphorus content in breast cancer antigen MUC-1 mRNA in MUC-1 mRNA linked to luciferase mRNA is 1:(0.5, 1, 2, 4, 8, 16) respectively. The molar ratio of breast cancer antigen HER2 / neu mRNA, MUC-1 mRNA, and luciferase mRNA is 1:1:1. The six groups of mixed solution C are respectively added to 293T cells and cultured for 24 h, and detected by an enzyme-linked immunosorbent assay (ELISA) to obtain the graph as shown in Figure 6 shown in the figure, and KA1 in Table 6 represents N6-(5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)lysine. It can be seen from Figure 6It can be seen that the breast cancer antigens HER2 / neu mRNA and MUC-1mRNA can be successfully expressed, proving the reliability of the breast cancer mRNA vaccine.

[0045] (III) N6-(5-(bis(3,3,4,4,5,6,6-heptafluoropentyl)amino)valeryl)lysine, stearoyl phosphatidylcholine and polyethylene glycol with a molar ratio of 1:3:100 are dissolved in 2 μL of ethanol to obtain a mixed solution A. The breast cancer antigens HER2 / neu, MUC-1 and CEA mRNA ligated with luciferase mRNA are dissolved in 10 μL of a buffer solution with a pH of 4 to obtain a mixed solution B. Subsequently, the mixed solution A is added to the mixed solution B to obtain a mixed solution C. Six groups of experiments are set up for the mixed solution C. In the six groups of experiments, the ratio of the nitrogen content in N6-(5-(bis(3,3,4,4,5,6,6-heptafluoropentyl)amino)valeryl)lysine to the phosphorus content in the breast cancer antigen CEA mRNA is 1:(0.5, 1, 2, 4, 8, 16) respectively, and the molar ratio of the breast cancer antigens HER2 / neu mRNA, MUC-1mRNA, CEA mRNA and luciferase mRNA is 1:1:1:1. The six groups of mixed solution C are respectively added to 293T cells and cultured for 24 h, and detected by an enzyme-linked immunosorbent assay (ELISA) to obtain the figure as Figure 7 shown, Figure 7 where KA1 represents N6-(5-(bis(3,3,4,4,5,6,6-heptafluoropentyl)amino)valeryl)lysine. It can be seen from Figure 7 that HER2 / neu mRNA, MUC-1mRNA and CEA mRNA can be successfully expressed, proving the reliability of the breast cancer mRNA vaccine.

[0046] The above are only the preferred embodiments of the present invention and do not impose any limitations on the present invention. Any simple modifications, changes and equivalent changes made to the above embodiments according to the technical essence of the invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A breast cancer mRNA vaccine, characterized in that: The method comprises lipid nanoparticles and gene material mRNA, wherein the gene material is mRNA of two or more antigens among breast cancer-related antigens HER2 / neu, MUC-1 and CEA; The lipid nanoparticles are prepared from ionizable cations, stearoyl phosphatidylcholine and polyethylene glycol; the molar ratio of the ionizable cations, stearoyl phosphatidylcholine and polyethylene glycol is 1:(0.1-10):(1-100); The ionizable cation is one or more of N6-(5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)lysine, (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)glutamate, (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)glycine and (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)alanine; The ratio of the nitrogen content in ionizable cations to the phosphorus content in the genetic material mRNA is 1:(1-10).

2. A breast cancer mRNA vaccine according to claim 1, characterized in that: The preparation method of the ionizable cation is: Step 1, 5-aminopentanoic acid and 6-bromo-1,1,1,2,2,3,4,4-monofluorohexane are added to tetrahydrofuran in a molar ratio of 1:2, and then Cs2CO3 is added, and the reaction is carried out at 40°C for 24 hours. After the reaction is completed, THF is removed by rotary evaporation under reduced pressure to obtain 5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoic acid; Step 2: Add tetrahydrofuran solvent according to the molar ratio of 1:1 to 5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoic acid and amino acid obtained in step 1, and add EDCI HOBt to react for 24 hours. After the reaction is completed, remove the solvent by vacuum rotary evaporation to obtain an ionizable cation; When the amino acid in step 2 is lysine, the ionizable cation is N6-(5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)lysine; When the amino acid in step 2 is glutamic acid, the ionizable cation is (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)glutamic acid; When the amino acid in step 2 is glycine, the ionizable cation is (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)glycine; When the amino acid in step 2 is alanine, the ionizable cation is (5-(bis(3,3,4,4,5,6,6-monofluorohexyl)amino)pentanoyl)alanine.

3. A method for preparing a breast cancer mRNA vaccine as claimed in claim 1 or 2, characterized in that: The method is: The lipid nanoparticles were dissolved in 2 uL of ethanol to obtain a mixed solution A; the genetic material mRNA was dissolved in 10 uL of a citric acid-sodium citrate buffer solution with a pH of 4 to obtain a mixed solution B, and then the mixed solution A was added to the mixed solution B and mixed to obtain a breast cancer mRNA vaccine.

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