An enhanced tumor vaccine adjuvant

Abstract

The present invention discloses an enhanced tumor vaccine adjuvant, and specifically relates to the field of vaccine adjuvants, the adjuvant comprising granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-9 (IL-9), gamma interferon (IFN-γ) and at least one additional immunomodulatory factor, wherein the immunomodulatory factor is selected from tumor-specific antigen WT1, CD40 ligand (CD40L), Toll-like receptor agonist or a combination thereof. The tumor vaccine adjuvant of the present invention can enhance the body's immune response to the tumor vaccine and improve the prevention and treatment effect of the tumor vaccine on tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine adjuvants, and more particularly to an enhanced tumor vaccine adjuvant. Background Art

[0002] With the rapid development of immunotherapy technology, tumor vaccines, as an emerging treatment method, have shown great potential in tumor prevention and treatment. However, existing tumor vaccines are often difficult to efficiently activate cellular immune responses due to their weak immunogenicity, which limits their clinical application.

[0003] Traditional vaccine adjuvants such as aluminum hydroxide, Corynebacterium brevis, lipopolysaccharide, etc., although they can enhance the immune response to a certain extent, have limited effects on promoting cellular immunity, especially in tumor vaccines that are mainly based on cellular immunity. In recent years, cytokines, as an important class of immunoregulatory molecules, have received extensive attention in tumor immunotherapy. Among them, cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-9 (IL-9) and interferon-γ (IFN-γ) have shown excellent effects in activating and regulating the functions of immune cells.

[0004] Therefore, an enhanced tumor vaccine adjuvant is proposed. Summary of the invention

[0005] In order to overcome the above-mentioned defects of the prior art, the present invention provides an enhanced tumor vaccine adjuvant to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solution: an enhanced tumor vaccine adjuvant, the tumor vaccine adjuvant comprising granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-9 (IL-9) and gamma interferon (IFN-γ), and at least one adjuvant component (such as WT1 peptide, CpG ODN, etc.). By introducing these additional adjuvant components, the tumor vaccine adjuvant of the present invention can further activate and regulate immune cells, thereby enhancing the ability to recognize and kill tumor cells.

[0007] Preferably, the content of GM-CSF is 10-20wt%, preferably 13-17wt%, and more preferably 15wt%, based on the weight of the tumor vaccine adjuvant; the content of IL-2 is 20-35wt%, based on the weight of the tumor vaccine adjuvant; the content of IL-9 is 20-35wt%, based on the weight of the tumor vaccine adjuvant; the content of IFN-γ is 15-35wt%, based on the weight of the tumor vaccine adjuvant. In addition, the content of the additional adjuvant components introduced should be adjusted according to their specific effects to ensure the best immune enhancement effect of the overall adjuvant.

[0008] The second aspect of the present invention provides a method for preparing the tumor vaccine adjuvant according to the first aspect of the present invention, the method comprising: mixing GM-CSF, IL-2, IL-9, IFN-γ and at least one adjuvant component to obtain the enhanced tumor vaccine adjuvant. The mixing process can be carried out under appropriate conditions, such as appropriate pH value, temperature and stirring speed, to ensure that the components are fully mixed and maintain activity.

[0009] The third aspect of the present invention provides a use of the tumor vaccine adjuvant as described in the first aspect of the present invention in preparing a tumor vaccine composition, which not only comprises the tumor vaccine adjuvant of the present invention, but also comprises a tumor vaccine, a tumor antigen, and a carrier acceptable to the vaccine, etc. By combining these components together, a tumor vaccine composition with excellent immune effect can be prepared.

[0010] Preferably, the tumor vaccine includes a protein vaccine, a gene vaccine (such as a DNA vaccine, an RNA vaccine, etc.) or a combination thereof, and the tumor antigen can be mixed with a tumor vaccine adjuvant in a certain proportion to further improve the targeting and immune effect of the vaccine. The carrier acceptable to the vaccine can be used to improve the solubility, stability and administration efficiency of the vaccine.

[0011] The enhanced tumor vaccine adjuvant proposed by the present invention has the following beneficial effects:

[0012] The adjuvant of the present invention can significantly enhance the body's immune response to tumor vaccines and improve the preventive and therapeutic effects of tumor vaccines on tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a schematic diagram of the amount of division of immune cells in the experimental organism;

[0014] Figure 2 This is a graph showing the immunization rates of five experimental mice after being vaccinated.

[0015] Figure 3 This is a diagram of the proliferation of CD4+T cells in the spleen and lymph nodes of mice detected by flow cytometry.

[0016] Figure 4 This is a diagram of the proliferation of CD8+T cells in the spleen and lymph nodes of mice detected by flow cytometry.

[0017] Figure 5 It is the tumor growth after mice are inoculated with specific tumor cells. DETAILED DESCRIPTION

[0018] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only 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.

[0019] Example 1

[0020] 1. Materials and Methods

[0021] Material

[0022] Granulocyte-macrophage colony-stimulating factor (GM-CSF): purity ≥ 95%.

[0023] Interleukin-2 (IL-2): purity ≥ 98%.

[0024] Interleukin-9 (IL-9): purity ≥ 97%.

[0025] Interferon-γ (IFN-γ): purity ≥ 96%.

[0026] Tumor vaccine antigens (such as WT1 antigen): derived from specific tumor cell lines and used after purification.

[0027] Suitable animal models: such as C57BL / 6 mice, are used to evaluate the effects of adjuvants.

[0028] Preparation of tumor vaccine adjuvant

[0029] GM-CSF, IL-2, IL-9 and IFN-γ were mixed in the following proportions: GM-CSF 15wt%, IL-2 25wt%, IL-9 25wt%, IFN-γ 35wt%, based on the total adjuvant weight.

[0030] The above ingredients are fully mixed under sterile conditions and subjected to appropriate sterile filtration to obtain a uniform tumor vaccine adjuvant solution.

[0031] Preparation of tumor vaccine compositions

[0032] The prepared tumor vaccine adjuvant is mixed with the WT1 antigen at a weight ratio of 1:5, and an appropriate amount of a pharmaceutically acceptable carrier (such as phosphate buffer, trehalose, etc.) is added to prepare a vaccine composition suitable for injection.

[0033] Animal experiments

[0034] C57BL / 6 mice were randomly divided into experimental and control groups, with 10 mice in each group.

[0035] The mice in the experimental group were injected with the vaccine containing the tumor vaccine composition of the present invention, and the mice in the control group were injected with the vaccine containing only the WT1 antigen (without adjuvant).

[0036] Multiple immunizations were performed according to the predetermined immunization schedule, and the immune response and tumor growth of the mice were monitored.

[0037] 2. Results

[0038] Immune response assessment

[0039] like Figure 3 and Figure 4 As shown, the proliferation of CD4+T cells and CD8+T cells in the spleen and lymph nodes of mice was detected by flow cytometry, and it was found that the T cell proliferation of mice in the experimental group was significantly higher than that in the control group.

[0040] The levels of GM-CSF, IL-2, IL-9 and IFN-γ in mouse serum were tested, and the results showed that the cytokine levels of mice in the experimental group were significantly increased, indicating that the adjuvant effectively enhanced the body's immune response.

[0041] Tumor growth inhibition

[0042] like Figure 5 As shown, at a certain time point after immunization, mice are inoculated with specific tumor cells (such as melanoma cells expressing WT1 antigen).

[0043] By monitoring the tumor growth of mice, it was found that the tumor growth rate of the experimental group mice was significantly slower than that of the control group, and the tumors of some mice completely regressed.

[0044] Conclusion

[0045] This example demonstrates the effect of the tumor vaccine adjuvant of the present invention in practical application. By optimizing the ratio of the adjuvant components and combining them with specific tumor antigens, the adjuvant of the present invention can significantly enhance the body's immune response to the tumor vaccine and improve the tumor vaccine's preventive and therapeutic effects on tumors.

[0046] Example 2

[0047] As attached Figure 1 and 2As shown, the cells were incubated in a 37°C humidity incubator until the cells showed signs of viral infection. The infected cells were repeatedly frozen and thawed to cause cell lysis. Due to the binding of flagellin to its ligand TLR5, downstream NF-κB, MEKK-p38MAPK and other signaling pathways were activated. Flagp96 was co-cultured in DC (Dendritic Cells), and the cells were harvested at different time points. Proteins were extracted and MAPK and its phosphorylation were detected by immunohistochemistry. The results showed that Flagp96 can activate MAPK and its phosphorylation in DC.

[0048] Naive Pmel CD8+T cells (2×106) were injected into C57BL / 6 mice (5 mice per group) through the tail vein. Mice were immunized with 1×106 DC loaded with gp96, Flagp96 and melanoma antigen peptide gp10025-33 complex, OVA, and Flagellin-DC loaded with gp10025-33 through the tail vein. At different time points, spleen and refluxing lymph nodes were collected, and flow cytometry was used to detect the proliferation of Pmel-specific CD8+T cells. In the presence of Golgi blockers, after 4 hours of stimulation with gp10025-33 peptide, intracellular staining was used to analyze the secretion of GM-CSF, IL-2, IL-9, and IFN-γ by CD8+T cells, which significantly improved the anti-tumor immune effect of DNA vaccines. Cancer cell amorphous test (vaccine protection): Normal mice were first given vaccine preparations to stimulate the immune effect, and then cancer cells were inoculated. The mice did not grow cancer cells.

[0049] The changes in tumor growth of mice in the implementation group and the control group were recorded. Through the experiment, the mice in the implementation group given the vaccine adjuvant containing GM-CSF, IL-2, IL-9 and IFN-γ were still alive after 100 days of immunization, and no tumor masses were seen in the body; the mice in the control group that were not given the vaccine adjuvant containing GM-CSF, IL-2, IL-9 and IFN-γ had severe tumor spread within the 50th day. In addition, compared with the unencapsulated mcGMCSF-293 cell line (dose: 1×106 cells) immunization group, the encapsulated mcGMCSF-293 cell line (dose: 1×105 cells) immunization group only needed less GM-CSF, IL-2, IL-9 and IFN-γ vaccine adjuvant to achieve complete protection. Cancer cell non-formation test (vaccine protection): Normal mice were first given the vaccine preparation to stimulate the immune effect, and then inoculated with cancer cells, and the mice did not grow cancer cells.

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An enhanced tumor vaccine adjuvant, characterized in that: The adjuvant comprises granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-9 (IL-9), gamma interferon (IFN-γ) and at least one additional immunomodulatory factor, wherein the immunomodulatory factor is selected from tumor-specific antigen WT1, CD40 ligand (CD40L), Toll-like receptor agonist or a combination thereof.

2. An enhanced tumor vaccine adjuvant according to claim 1, characterized in that: The content of GM-CSF is 10-20wt%, preferably 13-17wt%, and more preferably 15wt%, based on the weight of the adjuvant; the content of IL-2 is 20-35wt%, based on the weight of the adjuvant; the content of IL-9 is 20-35wt%, based on the weight of the adjuvant; the content of IFN-γ is 15-35wt%, based on the weight of the adjuvant; and the content of the at least one additional immunomodulatory factor is 5-20wt%, based on the weight of the adjuvant.

3. The method for preparing an enhanced tumor vaccine adjuvant according to claim 1, characterized in that: The method comprises: mixing GM-CSF, IL-2, IL-9, IFN-γ and at least one additional immunomodulatory factor to obtain the enhanced tumor vaccine adjuvant.

4. Use of an enhanced tumor vaccine adjuvant according to claim 1 in the preparation of a tumor vaccine composition.

5. The use according to claim 4, characterized in that The vaccine includes vaccines for treating and / or preventing tumors, and the tumor-specific antigen WT1 is used to enhance the immune response to a specific type of tumor.

6. A tumor vaccine composition, characterized in that: The composition comprises the enhanced tumor vaccine adjuvant as claimed in claim 1 and a tumor vaccine, wherein the tumor vaccine comprises but is not limited to the tumor-specific antigen WT1, a protein vaccine, a gene vaccine or a combination thereof.

7. A tumor vaccine composition according to claim 6, characterized in that: The composition also includes a pharmaceutically or vaccine acceptable carrier, and the dosage form of the composition is a solid preparation, a liquid preparation or a semi-solid preparation, including but not limited to an oral preparation, an external preparation or an injection preparation.

8. Use of the tumor vaccine composition according to claim 6, for preparing a preparation for preventing and / or treating tumors, especially for tumor types expressing WT1 antigen.

9. A method for preventing and / or treating tumors, characterized in that: The method comprises administering the tumor vaccine composition of claim 6 to a subject in need thereof, thereby preventing and / or treating a tumor, wherein the subject is preferably a human being, and the tumor comprises a human tumor.

10. The method according to claim 9, characterized in that The administration method includes intravenous injection, intramuscular injection, subcutaneous injection or oral administration, and the dosage administered is a safe and effective amount, which is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 8 mg / kg body weight.

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