Devil-12-aCTLA-4, and preparation method and application thereof

Abstract

The application discloses a DEV@IL-12-aCTLA-4 as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The application uses the IL-12 and anti-CTLA-4 antibody modified dendritic cell-derived extracellular vesicles as a cancer vaccine, which can induce specific anti-tumor immunity in vivo, inhibit tumor growth, and improve the tumor microenvironment, and meanwhile, the cancer treatment effects of IL-12 and the anti-CTLA-4 antibody are exerted, the adverse reactions of IL-12 and the anti-CTLA-4 antibody are reduced, and combined immunotherapy is achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a DEV@IL-12-aCTLA-4 and a preparation method and application thereof. BACKGROUND

[0002] Due to the characteristics of unlimited proliferation and invasion and metastasis of cancer cells, cancer can rapidly deteriorate the functional state of the patient's body, thereby leading to death.

[0003] The generation of cancer is mainly due to the occurrence of gene mutations in normal cells of the body. These mutated genes can encode abnormal proteins, leading to the transformation of their phenotypes into malignant cancer cells. Cancer cells have the potential for unlimited proliferation, which can consume a large amount of energy of the body, and cancer cells can also metastasize and spread throughout the body, leading to abnormalities in various parts of the body. Under normal circumstances, the body's own immune system can recognize abnormal proteins produced by gene mutations and play an immune surveillance role to clear away abnormal cells. However, as mutations accumulate and an immunosuppressive microenvironment is formed, cancer often develops immune escape.

[0004] Traditional cancer treatment methods include radiotherapy, chemotherapy, and surgical treatment. These treatment methods can only target early-stage tumors, and it is difficult to completely cure advanced cancer that has undergone extensive metastasis. In recent years, the new immunotherapy has revolutionized the treatment of many advanced cancers, and a part of cancer patients have achieved complete cure through immunotherapy, even in the advanced stage of cancer. Cancer vaccines, as a new immunotherapy method, are also expected to be applied to cancer treatment. Cancer vaccines induce the body's own immune system to produce specific anti-tumor immune responses to fight cancer. A successful anti-tumor immune response requires first recognizing tumor antigens by antigen-presenting cells (APCs), which take up, process, and present antigens, and then provide APCs with antigen / MHC complexes, CD80 / CD86 / CD40, and other activated immune signals to promote T cell activation, induce strong and sustained CD4+ helper T cells and cytotoxic T lymphocyte (CTL) responses, and activate T cells to migrate to tumor sites to kill tumor cells expressing specific antigens. This close tumor immune loop is crucial for anti-tumor immunity, and cancer vaccines can play a role by promoting the smooth progress of the tumor immune loop. Although cancer vaccines have been developed for cancer treatment, their treatment response rate and treatment effect still need to be further improved to promote their clinical application.

[0005] Extracellular vesicles (EVs) are lipid bilayer membrane vesicles released by cells into the extracellular space, which are involved in physiological processes such as cell communication and material exchange. EVs can be loaded with some bioactive substances or chemotherapeutic drugs by incubation, electroporation, etc. and used for the treatment of cancer, and the lipid bilayer membrane on the surface of EVs can also be modified to target tumor cells and improve therapeutic effect. Moreover, EVs are substances derived from the body itself, which have good biocompatibility. Based on these good properties, EVs are developed as a biological delivery platform for the treatment of cancer. Dendritic cells (DCs) are antigen-presenting cells that initiate specific immune responses, which can present tumor antigens to T cells and provide immune stimulation signals to activate anti-tumor immunity. Dendritic cell-derived extracellular vesicles (DEVs) retain the bioactive molecules on the surface of DCs and can also function to initiate specific immune responses. Therefore, DEVs can be designed to construct cancer vaccines and be applied to cancer immunotherapy, and as a non-cellular component, DEVs are easier to store and transport, and do not need to consider the problems such as the decline of cell activity and the influence of immune suppression microenvironment after infusion into the body. Despite these characteristics and advantages, the clinical effect of DEVs-based cancer vaccines is still poor, and only a few cancer patients can stimulate specific anti-tumor immunity. How to further develop DEVs to design more effective cancer vaccines still has great challenges.

[0006] Cytokines as a traditional therapy have been explored for cancer treatment as early as last century, and many cytokines have been shown to play a role in cancer treatment by promoting anti-tumor immunity. For example, interleukin-12 (IL-12) as a cytokine mainly secreted by DCs has been shown to promote anti-tumor Th1 immunity and has great anti-tumor potential, and is one of the typical representatives of the application of cytokines in cancer treatment, but the clinical application of cytokines is limited by the severe systemic toxicity caused by the therapeutic dose. How to target the delivery of cytokines is the core bottleneck in this field.

[0007] The significant clinical efficacy of immune checkpoint inhibitors (ICIs) has changed the prognosis of many patients with advanced cancer. Anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) antibodies as a typical ICI have been used to treat a variety of advanced cancers. Despite its excellent efficacy, the overall clinical response rate of ICIs is less than 15%. How to expand the benefit population of ICIs is a core problem that needs to be solved in this field.

[0008] Cancer vaccine combined with cytokine or ICI is considered as a potential optimization scheme of cancer immunotherapy, because cytokine can promote the effect of cancer vaccine, and ICI can effectively reverse the immunosuppressive microenvironment, so that the anti-tumor immunity induced by cancer vaccine can play a role smoothly. However, how to realize these combined treatment schemes, and how to realize these combined treatment schemes is still not conclusive. Moreover, these combined treatment schemes are only one-sided improvement of cancer vaccine, and how to realize the optimization of multiple treatment methods in the process of combined treatment to develop the best cancer vaccine treatment scheme still needs to be explored. SUMMARY

[0009] One of the purposes of the present application is to provide a preparation method of DEV@IL-12-aCTLA-4, which comprises the following steps:

[0010] (1) Culturing DC cells, after stable growth, adding tumor neoantigen peptide OVA, lipopolysaccharide LPS and cytokine IFN-γ into the culture medium, and stimulating for 22-26 hours;

[0011] (2) Replacing the culture medium, and after the cells grow to fullness, irradiating the DC cells with ultraviolet rays, and then incubating and culturing for 22-26 hours;

[0012] (3) After incubation, collecting the DEVs in the supernatant of the cells;

[0013] (4) Resuspending 20-40 μg of DEVs with 150-250 μl of PBS, then adding 1-2 μL of DSPE-PEG-NHS solution, and incubating at 3-5°C for 22-26 hours;

[0014] (5) Removing the excess DSPE-PEG-NHS, then adding 30-50 ng of IL-12 and 350-450 ng of anti-CTLA-4 antibody, and incubating at 2-5°C for 22-26 hours, removing the excess IL-12 and anti-CTLA-4 antibody, and then DEV@IL-12-aCTLA-4 can be obtained.

[0015] Preferably, the DC cells in step (1) are DC2.4 cells or BMDC cells.

[0016] More preferably, the stimulation time in step (1) is 24 hours.

[0017] More preferably, the culture time in step (2) is 24 hours.

[0018] More preferably, the concentration of the DSPE-PEG-NHS aqueous solution in step (4) is 3.5 mg / ml.

[0019] More preferably, the incubation time in step (4) is 24 hours.

[0020] More preferably, the incubation time in step (5) is 24 hours.

[0021] More preferably, in steps (4) and (5), 30 μg of DEVs is resuspended with 200 μl of PBS, 1.5 μL of DSPE-PEG-NHS solution is added, and after incubation, 40 ng of IL-12 and 400 ng of anti-CTLA-4 antibody are added.

[0022] The second object of the present application is to provide DEV@IL-12-aCTLA-4 prepared by the above method.

[0023] The third object of the present application is to provide the use of the above DEV@IL-12-aCTLA-4 in the preparation of a cancer vaccine product.

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

[0025] (1) The IL-12 and anti-CTLA-4 antibody modified dendritic cell-derived extracellular vesicles are used as a cancer vaccine, and the preparation process is relatively simple, while retaining the functions of biocompatibility, safety, targeting, etc.

[0026] (2) The DEV@IL-12-aCTLA-4 as a new type of cancer vaccine induces specific anti-tumor immunity in vivo, inhibits tumor growth, and improves the tumor microenvironment.

[0027] (3) The DEV@IL-12-aCTLA-4 also exhibits the cancer treatment effects of IL-12 and anti-CTLA-4 antibody, reduces their adverse reactions, and realizes combined immunotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The figure is a schematic diagram of the preparation process of the DEV@IL-12-aCTLA-4 of the present application.

[0029] Figure 2 The figure is a transmission electron microscope image of the DEV@IL-12-aCTLA-4 in Example 2.

[0030] Figure 3 The figure is a schematic diagram of the size distribution (left) and particle concentration (right) of the DEV@IL-12-aCTLA-4 in Example 2.

[0031] Figure 4 The figure is a Western blot (WB) showing the expression results of the DEV@IL-12-aCTLA-4 related membrane protein markers in Example 2.

[0032] Figure 5 Figure (left) and anti-CTLA-4 antibody content (right) of DEV@IL-12-aCTLA-4 by enzyme-linked immunosorbent assay (ELISA) in Example 2.

[0033] Figure 6 Figure of the results of in vitro activation of tumor immune T cells in Example 3, in which (A) is the expression of CD69 (marker of activation) of CD4 + T and CD8 + T cells by flow cytometry; (B) is the expression of IFN-γ (marker of activation) of CD4 + T and CD8 + T cells by flow cytometry; (C) is the secretion of IFN-γ in the supernatant of T cell culture medium by ELISA; (D) is the proliferation of T cells by MTS assay.

[0034] Figure 7 Figure of the activation of anti-tumor immunity in vivo in Example 4, in which (A) is the expression of CD69 of CD4 + T and CD8 + T cells by flow cytometry; (B) is the expression of IFN-γ of CD4 + T and CD8 + T cells by flow cytometry.

[0035] Figure 8 Figure of the change in tumor volume in mice in Example 4, in which (A) is the tumor volume of B16-OVA mouse model; (B) is the tumor volume of LLC-OVA mouse model.

[0036] Figure 9 Figure of the naked tumor in mice in Example 4.

[0037] Figure 10 Figure of the proportion of Th1 cells (IFN-γ + CD4 + T cells) at the tumor site by flow cytometry in Example 5.

[0038] Figure 11 Figure of the proportion of regulatory T cells (Foxp3 + CD4 + T cells) at the tumor site by flow cytometry in Example 5.

[0039] Figure 12 Figure of the proportion of exhausted CD8 + T cells (PD-1 + CD8 + T cells) at the tumor site by flow cytometry in Example 5.

[0040] Figure 13 For example, in Example 5, flow cytometry was used to detect cytotoxic T cells (IFN-γ) at the tumor site. + CD8 + T cells and Granzyme B + CD4 + The proportion of T cells. Detailed Implementation

[0041] Example 1: Preparation of DEVs modified with IL-12 and anti-CTLA-4 antibodies (DEV@IL-12-aCTLA-4)

[0042] DC2.4 cells (dendritic cell line) were cultured adherently. After stable growth, DMEM medium was added to a final concentration of 100 μg / ml OVA, 10 ng / ml IFN-γ, and 1 μg / ml LPS for 24 hours. The medium was then replaced with fresh DMEM medium. Once the cells reached confluence, they were exposed to ultraviolet light (300 μm). -2 Cells were irradiated for 30 minutes, then incubated in a cell culture incubator for 24 hours. After 24 hours, the cell supernatant was collected, and extracellular vesicles (DEVs) derived from DC2.4 were obtained by gradient centrifugation. The cell supernatant was centrifuged at 800 rpm for 10 minutes to remove excess cell pellet, followed by centrifugation at 2000 g for 30 minutes to remove excess cell debris. The resulting supernatant was centrifuged at 16000 g for 60 minutes, and the precipitate was the DEVs. 30 μg of DEVs were resuspended in 200 μl of PBS, and 1.5 μL of DSPE-PEG-NHS aqueous solution (3 kDa, 3.5 mg / ml) was added. The mixture was thoroughly vortexed and incubated at 4°C for 24 hours. After centrifugation to wash away excess DSPE-PEG-NHS, 40 ng of IL-12 and 400 ng of anti-CTLA-4 antibody were added, and the mixture was thoroughly vortexed and incubated at 4°C for 24 hours. Centrifugation washes away excess IL-12 and anti-CTLA-4 antibody to obtain DEVs modified with IL-12 and anti-CTLA-4 antibody (DEV@IL-12-aCTLA-4).

[0043] Example 2 Identification of DEV@IL-12-aCTLA-4

[0044] 1. Size and shape of DEV@IL-12-aCTLA-4

[0045] The DEV@IL-12-aCTLA-4 prepared in Example 1 was analyzed for the shape and size of extracellular particles using transmission electron microscopy and a nanoparticle tracking system (NTA). The results are as follows: Figure 2 , 3As shown, DEV@IL-12-aCTLA-4 appears cup-shaped under an electron microscope, with an average particle size of 174 nm.

[0046] 2. Membrane surface protein labeling of DEV@IL-12-aCTLA-4

[0047] The extracellular vesicle membranes of DC-derived cells are marked with classic proteins such as Alix, CD80, CD86, MHC-I, and CD63. Immunoblotting experiments indicate that DEV@IL-12-aCTLA-4 expresses these proteins (see...). Figure 4 It retains the characteristics of DC-derived vesicles.

[0048] 3. Quantitative analysis of IL-12 and anti-CTLA-4 antibody on DEV@IL-12-aCTLA-4

[0049] To determine the amounts of IL-12 and anti-CTLA-4 antibody on DEV@IL-12-aCTLA-4, we used an enzyme-linked immunosorbent assay (ELISA) to infer that each 25 μg of DEV@IL-12-aCTLA-4 contained 24.5 ng of IL-12 and 205.7 ng of anti-CTLA-4 antibody. Figure 5 ).

[0050] Example 3: Cellular level DEV@IL-12-aCTLA-4 induces anti-tumor immunity

[0051] To verify the activating effect of DEV@IL-12-aCTLA-4 prepared in Example 1 on anti-tumor immune T cells, we first conducted experiments at the cellular level. 10 μg of DEV@IL-12-aCTLA-4 was mixed with 5 × 10⁻⁶ cells / mL of DEV@IL-12-aCTLA-4. 5 Mouse spleen T cells were co-incubated in vitro. Flow cytometry, ELISA, and cell proliferation assays showed that DEV@IL-12-aCTLA-4 successfully activated T cells. Figure 6 ).

[0052] Example 4: Animal-level DEV@IL-12-aCTLA-4 induces anti-tumor immunity to inhibit tumor growth.

[0053] In animal studies, we inoculated melanoma (B16-OVA cell line) and lung adenocarcinoma (LLC-OVA cell line) into the lower right flank of C57BL / 6 mice, respectively. The tumors were allowed to grow to a size of 80 mm. 3Subsequently, intervention with DEV@IL-12-aCTLA-4 was initiated. Mice were randomly divided into 5 groups of 5 mice each. The 5 groups were: PBS, DEV, DEV@IL-12, DEV@aCTLA-4, and DEV@IL-12-aCTLA-4. The preparation methods of DEV@IL-12 and DEV@aCTLA-4 were the same as in Example 1, except that aCTLA-4 and IL-12 were omitted. Injection dosage: the latter four groups were subcutaneously injected with 30 μg of DEV, DEV@IL-12, DEV@aCTLA-4, and DEV@IL-12-aCTLA-4 (dissolved in 50 μl of PBS), respectively. The PBS group was directly subcutaneously injected with 50 μl of PBS. Injection frequency and cycle: the first injection was given when the mouse tumor reached 80 mm. 3 The mice were injected three times, with each injection spaced five days apart. Two days after the third injection, the mice were tested, and the results showed that the DEV@IL-12-aCTLA-4 group produced anti-tumor immunity. Figure 7 Tumor growth was significantly inhibited. Figure 8 After three interventions, the subcutaneous tumors obtained through dissection were as follows: Figure 9 As shown.

[0054] Example 5: Animal-level DEV@IL-12-aCTLA-4 remodels the tumor immune microenvironment

[0055] For cancer vaccines to be effective, they not only need to induce specific anti-tumor immunity, but also need to overcome the tumor immunosuppressive microenvironment. Therefore, we investigated the local immune response in tumors to explore whether DEV@IL-12-aCTLA-4, as a cancer vaccine, can reshape the tumor immune microenvironment.

[0056] 1. Th1 cells

[0057] Th1 cells, as important helper T cells, assist in the generation and function of anti-tumor immune responses. Considering that DEV@IL-12-aCTLA-4 contains IL-12, and one of the main biological functions of IL-12 is to induce Th1-type immune responses, after the experiments in Example 4, we first examined the Th1 cell subset (IFN-γ) in melanoma sites. + CD4 + T cells), and found that the proportion of Th1 cells increased in the DEV@IL-12-aCTLA-4 group. Figure 10 ).

[0058] 2. Treg cells

[0059] Regulatory T cells (Tregs) are a subset of cells that exert immunosuppressive effects, negatively regulating tumor immunity and assisting tumors in immune escape. We examined Treg cells (Foxp3) in melanoma sites. + CD4 + T cells), the results showed that DEV@IL-12-aCTLA-4 could effectively reduce the number of Tregs at the tumor site (T cells). Figure 11 ).

[0060] 3. Exhausted CD8 + T cells

[0061] CD8 + T cells are the main effector cells in anti-tumor immunity, but they often become exhausted under chronic tumor stimulation, resulting in decreased tumor-killing function. Intervention with DEV@IL-12-aCTLA-4 can induce the depletion of CD8+ cells in melanoma sites. + T cells (PD-1) + CD8 + T cells decreased ( Figure 12 This suggests that DEV@IL-12-aCTLA-4 has the potential to reverse CD8 cell failure. + T cell capacity.

[0062] 4. Tumor-killing T lymphocytes (CTLs)

[0063] Tumor-killing T lymphocytes (CTLs) are a type of functional T cell with tumor cytotoxicity, capable of producing a strong tumor-suppressive effect. We examined CTLs (IFN-γ) in melanoma sites. + CD8 + T cells and Granzyme B + CD8 + T cells were found to be significantly increased in the DEV@IL-12-aCTLA-4 group. Figure 13 ).

[0064] Based on these results, we believe that DEV@IL-12-aCTLA-4 can effectively reverse the tumor immunosuppressive microenvironment after injection into tumor-bearing mice, providing favorable conditions for anti-tumor immunity.

[0065] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing DEV@IL-12-aCTLA-4, characterized in that, The preparation method includes the following steps: (1) After the DC cells are cultured and their growth is stable, tumor neoantigen peptide OVA, lipopolysaccharide LPS and cytokine IFN-γ are added to the culture medium and stimulated for 22-26 hours. (2) Change the culture medium. After the cells are almost fully grown, irradiate the DC cells with ultraviolet light and then incubate for 22-26 hours. (3) After incubation, collect the DEVs from the cell supernatant; (4) Resuspend 20-40 μg DEVs in 150-250 μl PBS, then add 1-2 μL LDSPE-PEG-NHS aqueous solution and incubate at 3-5℃ for 22-26 hours; (5) Remove excess DSPE-PEG-NHS, then add 30-50 ng IL-12 and 350-450 ng anti-CTLA-4 antibody, incubate at 2-5℃ for 22-26 hours to remove excess IL-12 and anti-CTLA-4 antibody, and you can get DEV@IL-12-aCTLA-4.

2. The preparation method according to claim 1, characterized in that, The DC cells in step (1) are DC2.4 cells or BMDC cells.

3. The preparation method according to claim 2, characterized in that, The stimulation time in step (1) is 24 hours.

4. The preparation method according to claim 3, characterized in that, The incubation time in step (2) is 24 hours.

5. The preparation method according to claim 4, characterized in that, In step (4), the concentration of the DSPE-PEG-NHS aqueous solution is 3.5 mg / ml.

6. The preparation method according to claim 5, characterized in that, The incubation time in step (4) is 24 hours.

7. The preparation method according to claim 6, characterized in that, The incubation time in step (5) is 24 hours.

8. The preparation method according to claim 7, characterized in that, In steps (4) and (5), 30 μg of DEVs were resuspended in 200 μl of PBS, 1.5 μL of DSPE-PEG-NHS aqueous solution was added, and after incubation, 40 ng of IL-12 and 400 ng of anti-CTLA-4 antibody were added.

9. DEV@IL-12-aCTLA-4 prepared by the preparation method according to any one of claims 1-8.

10. The use of DEV@IL-12-aCTLA-4 as described in claim 9 in the preparation of melanoma and / or lung adenocarcinoma vaccine products.

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