Anti-tumor vaccine and its application

Abstract

The present invention relates to the field of medical materials technology, and in particular to an anti-tumor vaccine and its applications. The anti-tumor vaccine comprises anti-tumor nanoparticles comprising a nucleic acid encoding a plasma membrane vesicle-associated protein (PLVAP), a cationic lipid, and a non-cationic lipid. Research has shown that an anti-tumor vaccine based on an antigen molecule specifically expressed on tumor vascular endothelial cells (PLVAP) can effectively activate the immunosuppressive microenvironment of tumor patients, inducing the generation of a large number of cytotoxic T cells. These T cells are capable of targeting and killing tumor vascular endothelial cells expressing the PLVAP protein, effectively destroying tumor blood vessels. This vaccine holds significant value in the field of anti-tumor biomaterials.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and in particular to an anti-tumor vaccine and application thereof. Background Art

[0002] Anti-tumor vaccines, due to their ability to induce potent anti-tumor responses with low systemic toxicity, are an effective alternative or supplement to traditional cancer therapies and have been a hot topic in cancer research in recent years. However, since the first cancer vaccine, Provenge, was used to treat prostate cancer, no new therapeutic cancer vaccine products have been approved for marketing. Currently, the market for vaccines that can be used for cancer treatment is virtually blank.

[0003] The development of new anti-tumor vaccines with good clinical therapeutic effects is not only expected to reduce the burden of tumor treatment, but also has significant investment value. However, due to the presence of an immunosuppressive tumor microenvironment, patients' clinical response rates to tumor vaccines and more generally to immunotherapy are generally low. Tumor blood vessels are crucial for maintaining tumor growth and metastasis, and targeted killing of tumor blood vessels can lead to rapid necrosis of highly vascular tumors. In addition, compared with tumor cells, the antigen expression of tumor vascular endothelial cells is more stable and less prone to immune mutations. However, no specific antigen molecules on the tumor vascular endothelium have been reported. Therefore, exploring new antigen components specifically expressed on the surface of tumor vascular endothelium and developing new tumor vascular targeted vaccines based on this have good clinical application potential. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the present invention provides an anti-tumor vaccine and application thereof.

[0005] Tumor-associated antigens are a class of antigenic molecules that are highly expressed on tumor cells. There are many types of tumor-associated antigens, and their expression levels in tumor cells are generally higher than in normal cells. Although some studies have applied tumor-associated antigens to anti-tumor treatment, most tumor-associated antigens are actually not suitable as immunogenic substances for use in the preparation of anti-tumor vaccines. The reason is that tumor-associated antigens are also expressed in normal tissues, but the expression level is lower than that in tumor cells. In this case, some tumor-associated antigens have an increased risk of inducing autoimmune toxicity and may be affected by central or peripheral tolerance, resulting in poor clinical efficacy. In addition, not all tumor-associated antigens are sufficiently immunogenic to stimulate an effective immune response. Some tumor-associated antigens may not effectively activate the immune system due to immune tolerance or other mechanisms.

[0006] After a large number of research experiments, the present invention found that plasma membrane vesicle-associated protein (PLVAP) can be used as an immunogenic substance for anti-tumor vaccines. By preparing it into the form of liposome nanoparticles, it can effectively trigger an immune response in the body, obtain a large number of killer T cells that have significant killing power against tumor cells, and have a significant inhibitory effect on tumors.

[0007] In a first aspect, the present invention provides an anti-tumor nanoparticle comprising: a nucleic acid encoding a plasma membrane vesicle-associated protein, a cationic lipid, and a non-cationic lipid.

[0008] Furthermore, the cationic lipid is SM102 and / or Lipid5; and the molar ratio of the cationic liposome to the non-cationic lipid is 1:(1~2).

[0009] Furthermore, the non-cationic lipids include: one or more of cholesterol, phospholipids or PEG lipids;

[0010] Preferably, the non-cationic lipid is composed of cholesterol, phospholipid and PEG lipid in a molar ratio of 1: (3-5): (0.1-0.3).

[0011] Furthermore, the plasma membrane vesicle-associated protein comprises the amino acid sequence shown in SEQ ID NO.1.

[0012] SEQ ID NO.1:

[0013] MGLAMEHGGSYARAGGSSRGCWYYLRYFFLFVSLIQFLIILGLVLFMVYGNVHVSTESNLQATERRAEGLYSQLLGLTASQSNLTKELNFTTRAKDAIMQMWLNARRDLD RINASFRQCQGDRVIYTNNQRYMAAIILSEKQCRDQFKDMNKSCDALLFMLNQKVKTLEVEIAKEKTICTKDKESVLLNKRVAEEQLVECVKTRELQHQERQLAKEQLQKV QALCLPLDKDKFEMDLRNLWRDSIIPRSLDNLGYNLYHPLGSELASIRRACDHMPSLMSSKVEELARSLRADIERVARENSDLQRQKLEAQQGLRASQEAKQKVEKEAQAR EAKLQAECSRQTQLALEEKAVLRKERDNLAKELEEKKREAEQLRMELAIRNSALDTCIKTKSQPMMPVSRPMGPVPNPQPIDPASLEEFKRKILESQRPPAGIPVAPSSG.

[0014] Furthermore, the nucleic acid encoding the plasma membrane vesicle-associated protein is mRNA or DNA, and preferably includes the nucleotide sequence shown in SEQ ID NO.2.

[0015] SEQ ID NO.2:

[0016]

[0017] Furthermore, it also includes: a nucleic acid encoding MUC1.

[0018] In a second aspect, the present invention provides an anti-tumor vaccine, comprising: the anti-tumor nanoparticles.

[0019] In a third aspect, the present invention provides use of the anti-tumor nanoparticles in the preparation of drugs for preventing or treating cancer.

[0020] The present invention further provides the use of the anti-tumor nanoparticles or the anti-tumor vaccine in preventing or treating cancer.

[0021] Furthermore, the prevention or treatment of cancer includes: reducing tumor volume, or improving survival rate.

[0022] The present invention has the following beneficial effects:

[0023] This study discovered that an antigen molecule (plasma membrane vesicle-associated protein) specifically expressed on tumor vascular endothelial cells, when used as an immunogenic component in a vaccine, can effectively stimulate the body's anti-tumor immune response and specifically destroy tumor blood vessels. Furthermore, this tumor vaccine can induce the production of memory T cells, leading to long-term and effective destruction of tumor blood vessels. This destruction of tumor vessels not only blocks the supply of nutrients and oxygen to tumor tissue but also relieves the immunosuppression caused by tumor vascular endothelial cells, transforming "cold" tumors that are poorly responsive to immunotherapy into "hot" tumors that are responsive to immunotherapy, thereby significantly improving patient response and efficacy of tumor vaccines.

[0024] The tumor vaccine provided by the present invention can effectively reduce tumor volume and improve survival rate, and has important value in the field of anti-tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 This is the expression and identification result of specific antigen molecules on the surface of tumor vascular endothelial cells provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0027] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0028] Unless otherwise specified, the experimental methods involved in the following examples are all conventional methods in the art. For example, reference can be made to experimental manuals in the art, or the conditions recommended by the manufacturer's instructions.

[0029] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources, for example:

[0030] Example 1

[0031] The key to this project is to identify specific antigen molecules on the surface of tumor vascular endothelial cells and to find safe and effective tumor vascular endothelial antigen proteins. The previous single-cell sequencing results of this invention revealed that compared with normal tissue vessels, a variety of solid tumor tissues with rich blood supply highly expressed vascular endothelial specific antigen molecules, plasma membrane vesicle-associated protein PLVAP, and their expression was verified by flow cytometry ( Figure 1 ).

[0032] The amino acid sequence of the plasma membrane vesicle associated protein PLVAP is shown in SEQ ID NO.1, and the nucleotide sequence encoding the plasma membrane vesicle associated protein PLVAP is shown in SEQ ID NO.2. mRNA is obtained by in vitro transcription as follows:

[0033] First, PLVAP-expressing Escherichia coli was constructed, cultured, and amplified. Plasmid DNA was then extracted and purified. A linear DNA template was prepared. T7-based in vitro transcription was then performed. Nucleoside modification (N1-Methylpseudo-UTP), enzymatic capping (Cap1), and template-based tailing with a 110 nt polyA tail were then performed. Finally, the template was digested with DNase I and the mRNA was purified using magnetic beads.

[0034] The PLVAP-expressing E. coli can be constructed using methods commonly used in the art, such as constructing a nucleotide sequence encoding PLVAP into an expression vector and then transforming the vector into E. coli.

[0035] This embodiment further provides an anti-tumor vaccine, which is prepared by the following method:

[0036] (1) First, prepare an organic phase containing lipid components: SM-102, DMG-PEG2000, DSPC, and cholesterol are mixed into the organic phase at a molar ratio of 47.5:10:40.7:1.8, and the total mass concentration of all components is 2 mg / mL;

[0037] (2) The mRNA component encoding PLVAP was then dissolved in PBS solution at a concentration of 0.5 mg / mL, and the aqueous phase and organic phase were passed through a microfluidic chip at a speed ratio of 3:1 to obtain LNP nanoparticles carrying PLVAP mRNA; subsequently, the mixture was centrifuged at 3000 g for 20 minutes and washed twice with PBS to remove excess anhydrous ethanol, thereby finally obtaining a tumor vaccine.

[0038] Example 2

[0039] The embodiment of the present invention provides a tumor vaccine, which is the same as that of Example 1, except that in step (2), in addition to PLVAP mRNA, an equal concentration of MUC1 mRNA (mucin, also a tumor antigen, the nucleotide sequence encoding MUC1 is shown in SEQ ID NO. 3) is also used.

[0040]

[0041] Comparative Example 1

[0042] The comparative example of the present invention provides a tumor vaccine, which is the same as that of Example 1, except that the plasma membrane vesicle-associated protein PLVAP is replaced with an equal amount of nucleolin (also expressed on vascular endothelial cells).

[0043] Comparative Example 2

[0044] The comparative example of the present invention provides a tumor vaccine, which is the same as Example 1, except that the plasma membrane vesicle-associated protein PLVAP is replaced with an equal amount of VEGF protein (also expressed on vascular endothelial cells).

[0045] Comparative Example 3

[0046] The comparative example of the present invention provides a tumor vaccine, which is the same as that in Example 1, except that the cationic lipid SM-102 is replaced by an equal amount of modRNA-LNP (MC3).

[0047] Comparative Example 4

[0048] The comparative example of the present invention provides a tumor vaccine, which is the same as that of Example 2, except that the cationic lipid SM-102 is replaced by an equal amount of modRNA-LNP (MC3).

[0049] Experimental Example 1

[0050] The particle size, particle size distribution coefficient (PDI), and zeta potential of the tumor vaccine prepared in Test Examples 1-2 of the present invention were obtained. The particle size and particle size distribution coefficient were measured by dynamic light scattering, and the zeta potential was measured by a surface potential meter. The test results are shown in the following table:

[0051] Table 1 Particle size, particle size distribution index (PDI) and Zeta potential value of tumor vaccine

[0052]

[0053] As can be seen from Table 1, the tumor vaccine prepared by the preparation method of the present invention has uniform particle size and excellent dispersibility.

[0054] Experimental Example 2

[0055] The present invention uses LLC lung cancer-bearing mice to evaluate the tumor vaccines obtained in Examples 1 and 2, specifically comprising the following steps:

[0056] (1) Thirty mice bearing LLC lung cancer were divided into three groups, with 10 mice in each group;

[0057] (2) Inject the tumor vaccine prepared in Examples 1 and 2 in the following manner:

[0058] The blank group received an intramuscular injection of 100 μL of normal saline, while the other two groups received an intramuscular injection of 10 μL of the vaccine described in Examples 1 and 2, containing 10 μg of mRNA. Tumor volumes were measured on days 6, 8, 10, 12, 14, 18, and 20 after tumor inoculation, and the average volume was recorded. Tumor volume was determined by multiplying the length by the square of the width, divided by 2. Tumor volumes greater than 2000 mm were considered to be the most common tumor volume. 3 Death was determined after the mice were taken; the changes in weight and survival period of the mice were observed at the same time. The survival rate was recorded as the number of days when the survival rate of the mice was 50% and 0%; the tumor volume (mm 3 ) test results are shown in Table 2, and the survival rate (d) test results are shown in Table 3.

[0059] Table 2 Statistical results of tumor volume

[0060]

[0061] As can be seen from Table 2, compared with the blank group, the tumor vaccines provided in Examples 1 and 2 of the present invention can significantly slow down the growth rate of tumors.

[0062] Table 3 Statistical results of mouse survival rate

[0063]

[0064] As can be seen from Table 3, compared with the blank group, the tumor vaccines provided by Examples 1 and 2 of the present invention can significantly prolong the survival of mice; as can be seen from the comparison between the examples and normal saline, when the technical solution of the present invention is not adopted, there is no obvious effect of prolonging the survival of tumor-bearing mice.

[0065] Experimental Example 3

[0066] The present invention uses LLC lung cancer-bearing mice to evaluate the in vivo immune activation ability of the tumor vaccines prepared in Examples 1 and 2 and Comparative Examples 1-4, specifically comprising the following steps:

[0067] Seventy mice bearing LLC lung cancer were divided into seven groups, with 10 mice in each group;

[0068] The tumor vaccines obtained in Examples 1 and 2 and Comparative Examples 1-4 were injected in the following manner: the seven groups were injected with the tumor vaccines obtained in Examples 1 and 2 and Comparative Examples 1-4 and a normal saline control group, respectively.

[0069] On days 4, 6, and 8 after tumor inoculation, mice were injected intramuscularly with 100 μL of the tumor vaccines from Examples 1 and 2 and Comparative Examples 1-4 (six experimental groups). A blank control group was injected with an equal volume of saline. Tumor growth was observed on days 6, 8, 10, 12, 14, 16, and 18 after injection. On day 18, mice were sacrificed and organs, including mice, tumors, and spleens, were collected for subsequent analysis. Tumor-infiltrating T cells were labeled for CD3, CD4, and CD8, and statistically analyzed.

[0070] Specifically, equal amounts of tumor tissue were collected and ground to extract tumor cells. Red blood cells in the tumor were treated with red blood cell lysis buffer, stained with antibodies, and analyzed by flow cytometry. Mouse spleen cells were seeded at 100,000 per well in a 96-well ELISPOT plate. After stimulation with 10 μg / mL LLC tumor lysate for 24 hours, antigen-specific T cells were analyzed using ELISPOT. The results are shown in Table 4.

[0071] Table 4 T cell analysis results

[0072]

[0073] As can be seen from the above table, compared with the blank group, the tumor vaccines provided by Examples 1 and 2 of the present invention can significantly increase the infiltration of CD8+ T cells in the tumor and increase the number of tumor-specific T cells in the spleen.

[0074] Example 1 exceeds that of Comparative Example 1 by more than 63%, and exceeds that of Comparative Example 2 by more than 108%. This shows that the anti-tumor vaccine prepared by selecting PLVAP in the present invention has great immune activity and is significantly better than other antigens expressed on vascular endothelial cells.

[0075] Examples 1, 2, and Comparative Examples 3 and 4 were significantly higher than the blank group, and Example 1 exceeded Comparative Example 3 by more than 128%, and Example 2 exceeded Comparative Example 4 by more than 207%. This shows that the anti-tumor nanoparticles prepared by the present invention have a high immune activation ability. On this basis, the cationic lipid SM102 has better immune activation ability.

[0076] 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 make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. An anti-tumor nanoparticle, characterized in that: include: nucleic acids encoding plasma membrane vesicle-associated proteins, cationic lipids, and noncationic lipids; The cationic lipid is SM102; The non-cationic lipids include: cholesterol, phospholipids and PEG lipids; The nucleic acid encoding the plasma membrane vesicle-associated protein is mRNA obtained by in vitro transcription of the nucleotide sequence shown in SEQ ID NO.

2.

2. The anti-tumor nanoparticle according to claim 1, characterized in that The molar ratio of the cationic lipid to the non-cationic lipid is 1:(1-2).

3. The anti-tumor nanoparticle according to claim 1 or 2, characterized in that The non-cationic lipid is composed of cholesterol, phospholipid and PEG lipid in a molar ratio of 1: (3-5): (0.1-0.3).

4. The anti-tumor nanoparticle according to claim 1 or 2, characterized in that Also includes: Nucleic acid encoding MUC1.

5. An anti-tumor vaccine, characterized in that: include: The antitumor nanoparticles according to any one of claims 1 to 4.

6. Use of the anti-tumor nanoparticles according to any one of claims 1 to 4, or the anti-tumor vaccine according to claim 5, in the preparation of a medicament for preventing or treating cancer; the cancer is lung cancer.

7. The use according to claim 6, characterized in that The prevention or treatment of cancer includes: reducing tumor volume, or improving survival rate.

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