Anti-tumor vaccine and application thereof

Abstract

The invention relates to the technical field of medical materials, in particular to an anti-tumor vaccine and application thereof. The anti-tumor vaccine is prepared from anti-tumor nanoparticles, the anti-tumor nanoparticles comprise nucleic acid for coding plasma membrane vesicle associated protein, cationic lipid and non-cationic lipid. Research finds that the anti-tumor vaccine prepared based on the antigen molecule (plasma membrane vesicle associated protein) specifically expressed on tumor vascular endothelial cells can efficiently activate the immunosuppression microenvironment of tumor patients and induce generation of a large number of killer T cells; the polypeptide can recognize and kill tumor vascular endothelial cells with PLVAP protein expression in a targeted manner, efficiently destroys tumor blood vessels, and has an important value in the field of anti-tumor biological materials.

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 are an effective way to replace or supplement traditional cancer therapies because they can induce effective anti-tumor responses and have low systemic toxicity. They are also one of the hot topics in the field of tumor research in recent years. However, since the first tumor vaccine Provenge was used to treat prostate cancer, no new therapeutic tumor vaccine products have been approved for marketing. At present, the market for vaccines that can be used to treat tumors is almost 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 great investment value. However, due to the existence of an immunosuppressive tumor microenvironment, patients' clinical response rates to tumor vaccines and more general immunotherapy are generally low. Tumor blood vessels are crucial to maintaining tumor growth and metastasis, and targeted killing of tumor blood vessels can lead to rapid necrosis of hypervascular 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 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 antigen molecules that are highly expressed on tumor cells. There are many types of tumor-associated antigens, and their expression levels in tumor cells are usually higher than those in normal cells. Although some studies have applied tumor-associated antigens to anti-tumor treatment, most tumor-associated antigens are actually not suitable for use as immunogenic substances 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 of 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 effects. In addition, not all tumor-associated antigens have sufficient immunogenicity to stimulate an effective immune response. Some tumor-associated antigens may not be able to effectively activate the immune system due to immune tolerance or other mechanisms.

[0006] After a large number of research experiments, the present invention discovered 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 induce 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; the molar ratio of the cationic liposome to the non-cationic lipid is 1:(1-2).

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

[0010] Furthermore, the plasma membrane vesicle-associated protein comprises an amino acid sequence as shown in SEQ ID NO.1.

[0011] SEQ ID NO.1: MGLAMEHGGSYARAGGSSRGCWYYLRYFFLFVSLIQFLIILGLVLFMVYGNVHVSTESNLQATERRAEGLYSQLLGLTASQSNLTKELNFTTRAKDAIMQMWLNARRDLD RINASFRQCQGDRVIYTNNQRYMAAIILSEKQCRDQFKDMNKSCDALLFMLNQKVKTLEVEIAKEKTICTKDKESVLLNKRVAEEQLVECVKTRELQHQERQLAKEQLQKV QALCLPLDKDKFEMDLRNLWRDSIIPRSLDNLGYNLYHPLGSELASIRRACDHMPSLMSSKVEELARSLRADIERVARENSDLQRQKLEAQQGLRASQEAKQKVEKEAQAR EAKLQAECSRQTQLALEEKAVLRKERDNLAKELEEKKREAEQLRMELAIRNSALDTCIKTKSQPMMPVSRPMGPVPNPQPIDPASLEEFKRKILESQRPPAGIPVAPSSG.

[0012] Furthermore, the nucleic acid encoding the plasma membrane vesicle-associated protein is mRNA or DNA, preferably comprising a nucleotide sequence as shown in SEQ ID NO.2.

[0013] SEQ ID NO.2:

[0014] Furthermore, it also includes: a nucleic acid encoding MUC1.

[0015] In a second aspect, the present invention provides an anti-tumor vaccine, comprising: the anti-tumor nanoparticles described above.

[0016] In a third aspect, the present invention provides the use of the anti-tumor nanoparticles in the preparation of drugs for preventing or treating cancer.

[0017] The present invention further provides the use of the anti-tumor nanoparticles or the anti-tumor vaccine in preventing or treating cancer.

[0018] Furthermore, the prevention or treatment of cancer includes: reducing tumor volume, or increasing survival rate.

[0019] The present invention has the following beneficial effects: The present invention has found that the antigen molecules (plasma membrane vesicle-associated proteins) specifically expressed on tumor vascular endothelial cells can effectively stimulate the body's anti-tumor immune response and specifically destroy tumor blood vessels when used as immunogenic components to prepare vaccines. In addition, the tumor vaccine can induce the generation of memory T cells, thereby destroying tumor blood vessels in a long-term and effective manner. After the tumor blood vessels are destroyed, it can not only block the nutrition and oxygen supply of the tumor tissue, but also relieve the immunosuppression caused by the tumor vascular endothelial cells, and transform "cold" tumors with poor immunotherapy response into "hot" tumors that can respond to immunotherapy, thereby significantly improving the patient's response and efficacy to tumor vaccines.

[0020] 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

[0021] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces 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 creative work.

[0022] 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

[0023] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0025] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources, for example: Example 1 How to mine specific antigen molecules on the surface of tumor vascular endothelial cells and find safe and effective tumor vascular endothelial antigen proteins is the top priority of this project. The previous single-cell sequencing results of this invention found that compared with normal tissue blood vessels, a variety of blood-rich solid tumor tumor tissues highly expressed vascular endothelial specific antigen molecules plasma membrane vesicle associated protein PLVAP, and its expression was verified by flow cytometry ( Figure 1 ).

[0026] 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. The mRNA is obtained by in vitro transcription method as follows: First, PLVAP-expressing Escherichia coli was constructed, cultured and amplified; then, plasmid DNA was extracted and purified; again, a linear DNA template was prepared; then, T7 based in vitro transcription was performed; then, nucleoside modification (N1-Methylpseudo-UTP), enzymatic capping: (Cap1) and template tailing: 110 nt polyA tail were performed, and finally, the template was digested with DNase I and the mRNA was purified using the magnetic beads method.

[0027] The E. coli expressing PLVAP as described above 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 it into E. coli.

[0028] This embodiment further provides an anti-tumor vaccine, which is prepared by the following method: (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; (2) The mRNA component encoding PLVAP was then dissolved in a PBS solution at a concentration of 0.5 mg / mL, and the aqueous phase and the organic phase were passed through a microfluidic chip at a speed ratio of 3:1 to obtain LNP nanoparticles carrying PLVAP mRNA; then, 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.

[0029] Example 2 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.

[0030]

[0031] Comparative Example 1 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 nucleolin (also expressed on vascular endothelial cells).

[0032] Comparative Example 2 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 by an equal amount of VEGF protein (also expressed on vascular endothelial cells).

[0033] Comparative Example 3 The comparative example of the present invention provides a tumor vaccine, which is the same as Example 1, except that the cationic lipid SM-102 is replaced by an equal amount of modRNA-LNP (MC3).

[0034] Comparative Example 4 The comparative example of the present invention provides a tumor vaccine, which is the same as Example 2, except that the cationic lipid SM-102 is replaced by an equal amount of modRNA-LNP (MC3).

[0035] Experimental Example 1 The particle size, particle size distribution coefficient (PDI) and Zeta potential value of the tumor vaccine prepared in Test Example 1-2 of the present invention, wherein the particle size and particle size distribution coefficient are measured by dynamic light scattering, and the Zeta potential is measured by a surface potential meter, and the test results are shown in the following table: Table 1 Particle size, particle size distribution index (PDI) and Zeta potential value of tumor vaccines

[0036] It can be seen from Table 1 that the tumor vaccine prepared by the preparation method of the present invention has uniform particle size and excellent dispersibility.

[0037] Experimental Example 2 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: (1) Thirty mice bearing LLC lung cancer were divided into three groups, with 10 mice in each group; (2) Injection of the tumor vaccine prepared in Examples 1 and 2, the injection method is as follows: The blank group was intramuscularly injected with 100 μL of saline, and the other two groups were intramuscularly injected with 10 μL of the vaccine in Examples 1 and 2, wherein the mRNA content was 10 μg. The tumor volume of the mice was measured on the 6th, 8th, 10th, 12th, 14th, 18th, and 20th day after tumor inoculation, and the average volume was recorded. The tumor volume was measured by multiplying the length by the square of the width divided by 2. The tumor volume of the mice was greater than 2000 mm. 3 The mice were judged dead after the experiment. The weight and survival period of the mice were observed. The survival rate was 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.

[0038] Table 2 Statistical results of tumor volume

[0039] It can be seen from Table 2 that, 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.

[0040] Table 3 Statistical results of mouse survival rate

[0041] It can be seen from Table 3 that, compared with the blank group, the tumor vaccines provided in Examples 1 and 2 of the present invention can significantly prolong the survival of mice; from the comparison between the examples and normal saline, it can be seen that when the technical solution of the present invention is not adopted, there is no obvious effect of prolonging the survival of tumor mice.

[0042] Experimental Example 3 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: Seventy mice bearing LLC lung cancer were divided into seven groups, with 10 mice in each group; The tumor vaccines obtained in Examples 1, 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, 2 and Comparative Examples 1-4 and a normal saline control group, respectively. 100 μL of the tumor vaccine of Examples 1, 2 and Comparative Examples 1-4 (six experimental groups) were injected intramuscularly on the 4th, 6th and 8th days after tumor inoculation, and the blank group was injected with an equal amount of saline. The growth of the tumor was observed on days 6, 8, 10, 12, 14, 16 and 18 after injection. On the 18th day, the mice were sacrificed and the organs such as mice, tumors and spleen were taken for subsequent analysis, and the tumor-infiltrating T cells were marked with CD3, CD4, CD8, etc. for statistical analysis.

[0043] Specifically, equal amounts of tumor tissue were taken and ground to extract tumor cells. After the red blood cells in the tumor were treated with red blood cell lysis solution, the cells were stained with antibodies and then measured by flow cytometry. At the same time, for mouse spleen cells, 100,000 spleen cells were inoculated into a 96-well plate for ELISPOT test at each well. After stimulating 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; Table 4 T cell analysis results

[0044] It can be seen from the above table that, compared with the blank group, the tumor vaccine 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.

[0045] Example 1 exceeds that of Comparative Example 1 by more than 63%, and exceeds that of Comparative Example 2 by more than 108%. This indicates 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.

[0046] Examples 1, 2 and comparative examples 3, 4 are significantly higher than the blank group, and Example 1 exceeds Comparative Example 3 by more than 128%, and Example 2 exceeds 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 a better immune activation ability.

[0047] 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 embodiments of the present invention.

Claims

1. An anti-tumor nanoparticle, characterized in that: include: Nucleic acids, cationic lipids and non-cationic lipids encoding plasma membrane vesicle-associated proteins.

2. The anti-tumor nanoparticle according to claim 1, characterized in that: The cationic lipid is SM102 and / or Lipid5; the molar ratio of the cationic liposome 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 lipids include: one or more of cholesterol, phospholipids or PEG lipids; 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).

4. The anti-tumor nanoparticle according to any one of claims 1 to 3, characterized in that: The plasma membrane vesicle-associated protein comprises the amino acid sequence shown in SEQ ID NO.

1.

5. The anti-tumor nanoparticle according to claim 4, characterized in that: 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.

6. The anti-tumor nanoparticle according to any one of claims 1 to 5, characterized in that: Also includes: Nucleic acids encoding MUC1.

7. An anti-tumor vaccine, characterized in that: include: The antitumor nanoparticles according to any one of claims 1 to 6.

8. Use of the anti-tumor nanoparticles according to any one of claims 1 to 6 in the preparation of a drug for preventing or treating cancer.

9. Use of the anti-tumor nanoparticles according to any one of claims 1 to 6, or the anti-tumor vaccine according to claim 7 in preventing or treating cancer.

10. The use according to claim 9, characterized in that: The prevention or treatment of cancer includes: reducing tumor volume, or improving survival rate.

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