DEV@il-12-actla-4, preparation method therefor and use thereof

Abstract

Provided are a DEV@IL-12-aCTLA-4, a preparation method therefor and a use thereof, belonging to the technical field of biomedicine. Extracellular vesicles derived from dendritic cells modified with IL-12 and anti-CTLA-4 antibodies are used as a cancer vaccine, which can induce specific anti-tumor immunity in vivo, inhibit tumor growth, and improve the tumor microenvironment, as well as take advantage of the cancer treatment effects of the IL-12 and anti-CTLA-4 antibodies, reduce adverse reactions thereto, and implement combined immunotherapy.

Description

A DEV@IL-12-aCTLA-4 and its preparation method and application Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to DEV@IL-12-aCTLA-4 and its preparation method and application. Background Art

[0002] Cancer remains one of the major threats to human life and health. The latest 2020 global cancer burden data released by the World Health Organization's International Agency for Research on Cancer (IARC) shows that China experienced 4.57 million new cancer cases and 3 million cancer deaths in 2020. Due to the unlimited proliferation and invasive metastasis of cancer cells, cancer can rapidly deteriorate a patient's functional status, leading to death.

[0003] Cancer primarily arises from gene mutations in normal cells. These mutated genes encode abnormal proteins, leading to phenotypic transformation into malignant cancer cells. Cancer cells have the potential for unlimited proliferation, consuming significant amounts of the body's energy. They can also metastasize and spread throughout the body, causing abnormalities in various parts of the body. Under normal circumstances, the body's immune system can recognize the abnormal proteins produced by gene mutations, exerting immune surveillance and eliminating abnormal cells. However, with the continuous accumulation of mutations and the formation of an immunosuppressive microenvironment, cancer often escapes the immune system.

[0004] Traditional cancer treatments include radiotherapy, chemotherapy, and surgery. These treatments are only effective for early-stage tumors and are unlikely to completely cure advanced cancers with widespread metastasis. In recent years, the emergence of immunotherapy has revolutionized the treatment of many advanced cancers, with some cancer patients achieving complete cures through immunotherapy, even in the late stages. Cancer vaccines, as an emerging immunotherapy approach, also hold promise for cancer treatment. They work by inducing the body's own immune system to generate a specific anti-tumor immune response. A successful anti-tumor immune response requires antigen-presenting cells (APCs) to recognize tumor antigens, take up, process, and present them. APCs then provide T cells with activation signals, such as antigen / MHC complexes and CD80 / CD86 / CD40, to promote T cell activation, inducing a strong and sustained CD4+ helper T cell and cytotoxic T lymphocyte (CTL) response. Activated T cells then migrate to the tumor site and kill tumor cells expressing specific antigens. This tight tumor-immune system is crucial for anti-tumor immunity, and cancer vaccines can be effective by promoting its smooth function. Although cancer vaccines have been developed for cancer treatment, their therapeutic response rate and therapeutic 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, participating in physiological processes such as cell communication and material exchange. EVs can be loaded with bioactive substances or chemotherapeutic drugs through incubation or electroporation for cancer treatment. Their surface lipid bilayer membrane can also be modified to target tumor cells and enhance therapeutic efficacy. Furthermore, EVs are biologically derived and possess excellent biocompatibility. Based on these favorable properties, EVs have been developed as a biodelivery platform for cancer treatment. Dendritic cells (DCs) are antigen-presenting cells that initiate specific immune responses. They can present tumor antigens to T cells and provide immunostimulatory signals to activate anti-tumor immunity. Dendritic cell-derived extracellular vesicles (DEVs) retain the bioactive molecules on the DC surface and can also play a role in initiating specific immune responses. Therefore, DEVs can be designed to construct cancer vaccines for use in cancer immunotherapy. Furthermore, as a non-cellular component, DEVs are easier to store and transport, and after infusion, there is no need to worry about the effects of decreased cell activity on the immunosuppressive microenvironment. Despite these properties and advantages, the clinical efficacy of DEV-based cancer vaccines remains poor, stimulating specific anti-tumor immunity in only a small number of cancer patients. Further development of DEVs to design more effective cancer vaccines remains a major challenge.

[0006] Cytokines, a traditional therapeutic approach, have been explored for cancer treatment since the last century, and many cytokines have been shown to play a therapeutic role by promoting anti-tumor immunity. For example, interleukin-12 (IL-12), a cytokine primarily secreted by DCs, has been shown to promote anti-tumor Th1 immunity and possesses significant anti-tumor potential, making it a prime example of cytokine applications in cancer therapy. However, the clinical application of cytokines is limited by the severe systemic toxicity induced by therapeutic doses. Targeted delivery of cytokines remains a key bottleneck in this field.

[0007] The remarkable clinical efficacy of immune checkpoint inhibitors (ICIs) has transformed the prognosis of many patients with advanced cancer. Anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) antibodies, a typical ICI, have been used to treat a variety of advanced cancers. Despite their excellent efficacy, the overall clinical response rate of ICIs is less than 15%. Expanding the population that can benefit from ICIs is a core issue that needs to be addressed in this field.

[0008] Cancer vaccines combined with cytokines or ICIs are considered potential cancer immunotherapy optimization options, as cytokines can promote the effects of cancer vaccines, while ICIs can effectively reverse the immunosuppressive microenvironment, allowing the anti-tumor immunity induced by cancer vaccines to function smoothly. However, how to implement these combined treatments and how to achieve the optimal solution for these combined treatments remain unresolved. Moreover, these combined treatments are only unilateral improvements to cancer vaccines, and how to optimize multiple treatment methods during combined treatment to develop the best cancer vaccine treatment plan still needs to be explored.

[0009] Summary of the Invention

[0010] One of the objectives of the present invention is to provide a method for preparing DEV@IL-12-aCTLA-4, which comprises the following steps:

[0011] (1) Cultivating DC cells. After stable growth, adding tumor neoantigen peptide OVA, lipopolysaccharide LPS, and cytokine IFN-γ to the culture medium for stimulation for 22-26 hours;

[0012] (2) Replace the culture medium. When the cells are almost full, irradiate the DC cells with ultraviolet light and then incubate for 22-26 hours.

[0013] (3) After incubation, DEVs were collected from the cell supernatant;

[0014] (4) Resuspend 20-40 μg of DEVs in 150-250 μl PBS, then add 1-2 μl of DSPE-PEG-NHS solution and incubate at 3-5°C for 22-26 hours;

[0015] (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°C for 22-26 hours, remove excess IL-12 and anti-CTLA-4 antibody, and obtain DEV@IL-12-aCTLA-4.

[0016] Preferably, the DC cells in step (1) are DC2.4 cells or BMDC cells.

[0017] More preferably, the stimulation time in step (1) is 24 hours.

[0018] More preferably, the culture time in step (2) is 24 hours.

[0019] More preferably, the concentration of the DSPE-PEG-NHS aqueous solution in step (4) is 3.5 mg / ml.

[0020] More preferably, the incubation time in step (4) is 24 hours.

[0021] More preferably, the incubation time in step (5) is 24 hours.

[0022] More preferably, in steps (4) and (5), 30 μg of DEVs are resuspended in 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.

[0023] A second object of the present invention is to provide DEV@IL-12-aCTLA-4 prepared by the above method.

[0024] A third object of the present invention is to provide the use of the above-mentioned DEV@IL-12-aCTLA-4 in the preparation of cancer vaccine products.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) Using extracellular vesicles derived from dendritic cells modified with IL-12 and anti-CTLA-4 antibodies as cancer vaccines has a relatively simple preparation process and retains functions such as biocompatibility, safety, and targeting.

[0027] (2) DEV@IL-12-aCTLA-4, as a new cancer vaccine, induces specific anti-tumor immunity in vivo, inhibits tumor growth, and improves the tumor microenvironment.

[0028] (3) DEV@IL-12-aCTLA-4 also simultaneously exerts the cancer treatment effects of IL-12 and anti-CTLA-4 antibodies, reduces their adverse reactions, and achieves combined immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 is a schematic diagram of the preparation process of DEV@IL-12-aCTLA-4 of the present invention.

[0030] Figure 2 is a transmission electron micrograph of DEV@IL-12-aCTLA-4 in Example 2.

[0031] Figure 3 is a schematic diagram of the DEV@IL-12-aCTLA-4 size distribution (left) and particle concentration (right) in Example 2.

[0032] FIG4 is a Western blot (WB) showing the expression results of DEV@IL-12-aCTLA-4 related membrane protein markers in Example 2.

[0033] Figure 5 is a diagram showing the IL-12 content (left) and anti-CTLA-4 antibody content (right) on DEV@IL-12-aCTLA-4 detected by enzyme-linked immunosorbent assay (ELISA) in Example 2.

[0034] Figure 6 is a diagram showing the results of an in vitro activation experiment of tumor immune T cells in Example 3, wherein (A) is a flow cytometry assay for CD4 + T and CD8 + The expression of CD69 (activation marker) on T cells; (B) flow cytometry detection of CD4 + T and CD8 + The expression of IFN-γ (activation marker) in T cells; (C) ELISA detection of IFN-γ secretion in the supernatant of T cell culture medium; (D) MTS experiment detection of T cell proliferation.

[0035] FIG7 shows the activation of anti-tumor immunity in vivo in Example 4, wherein (A) is the flow cytometry detection of CD4 + T and CD8 + The expression of CD69 in T cells; (B) Flow cytometry was used to detect CD4 + T and CD8 + Expression of IFN-γ in T cells.

[0036] FIG8 is a curve showing the change of subcutaneous tumor volume in mice in Example 4, wherein (A) is the tumor volume of the B16-OVA mouse model; and (B) is the tumor volume of the LLC-OVA mouse model.

[0037] FIG9 is a diagram showing nude tumors in mice in Example 4.

[0038] Figure 10 is a flow cytometry analysis of Th1 cells (IFN-γ) in the tumor site in Example 5. + CD4 + T cell) ratio.

[0039] FIG11 is a flow cytometry analysis of regulatory T cells (Foxp3 + CD4 + T cell) ratio.

[0040] FIG. 12 is a flow cytometry analysis of exhausted CD8 in the tumor site in Example 5. + T cells (PD-1 + CD8 + T cell) ratio.

[0041] Figure 13 is a flow cytometry analysis of the cytotoxic T cells (IFN-γ) in the tumor site in Example 5. + CD8 + T cells and Granzyme B + CD4 + T cell) ratio. DETAILED DESCRIPTION

[0042] Example 1 Preparation of DEVs Modified with IL-12 and Anti-CTLA-4 Antibody (DEV@IL-12-aCTLA-4)

[0043] DC2.4 cells (dendritic cell line) were cultured on the wall. After the cells grew stably, 100 μg / ml OVA, 10 ng / ml IFN-γ, and 1 μg / ml LPS were added to the DMEM medium and stimulated for 24 hours. Then, the medium was replaced with new DMEM medium. After the cells were fully grown, ultraviolet light (300 Jm -2) irradiated the cells for 30 minutes, then placed them in a cell culture incubator and incubated for 24 hours. After 24 hours, the cell supernatant was collected and DC2.4-derived extracellular vesicles (DEVs) were obtained by gradient centrifugation. The cell supernatant was centrifuged at 800 rpm for 10 minutes, and the excess cell pellet was discarded. Then, the excess cell debris pellet was discarded and the supernatant was centrifuged at 16,000 g for 60 minutes. The resulting precipitate was DEVs. 30 μg of DEVs were resuspended in 200 μl PBS, and 1.5 μL of DSPE-PEG-NHS aqueous solution (3 kDa, 3.5 mg / ml) was added. The mixture was thoroughly shaken and incubated at 4°C for 24 hours. The excess DSPE-PEG-NHS was washed away by centrifugation, and 40 ng of IL-12 and 400 ng of anti-CTLA-4 antibody were added. The mixture was thoroughly shaken and incubated at 4°C for 24 hours. Excess IL-12 and anti-CTLA-4 antibodies were washed away by centrifugation to obtain DEVs modified with IL-12 and anti-CTLA-4 antibodies (DEV@IL-12-aCTLA-4).

[0044] Example 2 Identification of DEV@IL-12-aCTLA-4

[0045] 1. DEV@IL-12-aCTLA-4 size and morphology

[0046] The shape and size of the extracellular particles of DEV@IL-12-aCTLA-4 prepared in Example 1 were identified using transmission electron microscopy and nanoparticle tracking (NTA) analysis, as shown in Figures 2 and 3. Under the electron microscope, DEV@IL-12-aCTLA-4 appeared cup-shaped with an average particle size of 174 nm.

[0047] 2. Membrane surface protein labeling of DEV@IL-12-aCTLA-4

[0048] The surface of DCs-derived extracellular vesicles has classic protein markers such as Alix, CD80, CD86, MHC-I, and CD63. Immunoblotting experiments showed that DEV@IL-12-aCTLA-4 expressed the above proteins (see Figure 4), retaining the characteristics of DCs-derived vesicles.

[0049] 3. Quantification of IL-12 and anti-CTLA-4 antibodies on DEV@IL-12-aCTLA-4

[0050] To identify the amount of IL-12 and anti-CTLA-4 antibodies on DEV@IL-12-aCTLA-4, we used enzyme-linked immunosorbent assay (ELISA) and deduced that every 25 μg of DEV@IL-12-aCTLA-4 contained 24.5 ng of IL-12 and 205.7 ng of anti-CTLA-4 antibodies ( FIG5 ).

[0051] Example 3: DEV@IL-12-aCTLA-4 induces anti-tumor immunity at the cellular level

[0052] In order to verify the activation effect of DEV@IL-12-aCTLA-4 on anti-tumor immune T cells prepared in Example 1, we first conducted experiments at the cellular level. 5 Mouse spleen T cells were co-cultured in vitro, and the results of flow cytometry, ELSIA, and cell proliferation experiments showed that DEV@IL-12-aCTLA-4 successfully activated T cells (Figure 6).

[0053] Example 4: DEV@IL-12-aCTLA-4 induces anti-tumor immunity to inhibit tumor growth in animals

[0054] At the animal level, we inoculated melanoma (B16-OVA cell line) and lung adenocarcinoma (LLC-OVA cell line) into the right lower back of C57BL / 6 mice. 3 After that, DEV@IL-12-aCTLA-4 was used for intervention. The mice were randomly divided into 5 groups, with 5 mice in each group. The 5 groups were: PBS, DEV, DEV@IL-12, DEV@aCTLA-4, 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 dose: The following 4 groups were subcutaneously injected with 30μg DEV, DEV@IL-12, DEV@aCTLA-4, DEV@IL-12-aCTLA-4 (dissolved in 50μl PBS respectively), and the PBS group was directly subcutaneously injected with 50μl PBS. Injection frequency and cycle: The first injection was when the mouse tumor reached 80mm 3 The mice in the DEV@IL-12-aCTLA-4 group were injected three times, with each injection separated by five days. Two days after the third injection, the mice were tested for anti-tumor immunity (Figure 7) and tumor growth was significantly inhibited (Figure 8). After the three interventions, the subcutaneous tumors were removed and shown in Figure 9.

[0055] Example 5: DEV@IL-12-aCTLA-4 reshapes the tumor immune microenvironment at the animal level

[0056] Cancer vaccines must not only induce specific anti-tumor immunity but also overcome the tumor's immunosuppressive microenvironment to be effective. Therefore, we investigated the local immune response in the tumor to explore whether DEV@IL-12-aCTLA-4, as a cancer vaccine, could reshape the tumor immune microenvironment.

[0057] 1. Th1 cells

[0058] Th1 cells are important helper T cells that can 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 immune responses, after completing the experiment in Example 4, we first detected the Th1 cell subsets (IFN-γ + CD4 + T cells), and found that the proportion of Th1 cells increased in the DEV@IL-12-aCTLA-4 group ( Figure 10 ).

[0059] 2. Treg cells

[0060] Regulatory T cells (Treg) are a type of cell subset that exerts an immunosuppressive effect. They negatively regulate anti-tumor immunity and can help tumors escape immune responses. We detected Treg cells (Foxp3 + CD4 + The results showed that DEV@IL-12-aCTLA-4 could effectively reduce the number of Tregs in the tumor site (Figure 11).

[0061] 3. Exhausted CD8 + T cells

[0062] CD8 + T cells are the main effector cells of anti-tumor immunity, but they often fail under chronic tumor stimulation and their tumor killing function decreases. The intervention of DEV@IL-12-aCTLA-4 can make the exhausted CD8 + T cells (PD-1 + CD8 + T cells) decreased (Figure 12), suggesting that DEV@IL-12-aCTLA-4 has the potential to reverse the exhaustion of CD8 + T cell capacity.

[0063] 4. Tumor-killing T lymphocytes (CTL)

[0064] Tumor-killing T lymphocytes (CTL) are a type of functional T cell with tumor cytotoxicity and can produce a strong tumor suppressor effect. We detected CTL (IFN-γ + CD8 + T cells and Granzyme B + CD8 + T cells), and it was found that CTLs were significantly increased in the DEV@IL-12-aCTLA-4 group ( Figure 13 ).

[0065] Based on these results, we believe that DEV@IL-12-aCTLA-4 can effectively reverse the tumor immunosuppressive microenvironment after being injected into tumor-bearing mice, providing favorable conditions for the anti-tumor immunity to take effect.

[0066] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection 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 comprises the following steps: (1) Cultivating DC cells, and after stable growth, adding tumor neoantigen peptide OVA, lipopolysaccharide LPS, and cytokine IFN-γ to the culture medium for stimulation for 22-26 hours; (2) Replace the culture medium, and when the cells are almost full, irradiate the DC cells with ultraviolet light, and then incubate them for 22-26 hours; (3) After incubation, DEVs in the cell supernatant were collected; (4) Resuspend 20-40 μg DEVs in 150-250 μl PBS, then add 1-2 μL DSPE-PEG-NHS aqueous solution and incubate at 3-5°C for 22-26 h; (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°C for 22-26 hours, remove excess IL-12 and anti-CTLA-4 antibody, and obtain 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 culture time in step (2) is 24 hours.

5. The preparation method according to claim 4, characterized in that: The concentration of the DSPE-PEG-NHS aqueous solution in step (4) 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 to 8.

10. Use of DEV@IL-12-aCTLA-4 as claimed in claim 9 in the preparation of cancer vaccine products.

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