Application of enhancer or inhibitor of MARCKS protein

Abstract

The invention discloses application of a reinforcing agent or an inhibitor of MARCKS protein, and belongs to the technical field of medicines. It is proved for the first time that by applying an enhancer of MARCKS and targeting MARCKS protein, the infection capacity of HIV virus particles is inhibited, the neutralizing capacity of a broad-spectrum neutralizing antibody to HIV is enhanced, the limitation of an existing antiviral treatment scheme can be effectively overcome, and a more effective treatment scheme is provided for HIV infection. The MARCKS inhibitor is applied to target MARCKS protein, so that the combination of PD-1 and PD-L1 is inhibited, the killing capability of CTL cells on target cells is enhanced, and the effectiveness of tumor immunotherapy is improved. The content disclosed by the invention shows good potential in treatment of AIDS and malignant tumors, and provides a new strategy for treatment of AIDS and malignant tumors.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the application of enhancers or inhibitors of MARCKS protein. More specifically, it relates to the application of enhancers / inhibitors targeting myristoylated alanine rich protein kinase C substrate (MARCKS) in anti-human immunodeficiency virus infection and anti-tumor immunotherapy. Background Art

[0002] Human immunodeficiency virus (HIV) infection is a major public health problem worldwide. Although existing antiretroviral therapy (ART) can effectively control viral replication and prolong the survival of patients, it cannot completely cure AIDS. Broadly neutralizing antibodies (bNAbs) have been widely studied in recent years as an immunotherapy strategy for AIDS. Broadly neutralizing antibodies recognize and bind to envelope protein antigens on the surface of HIV, thereby preventing the virus from invading and infecting host cells. However, due to the frequent escape mutations of the virus, it can evade the attack of the immune system, resulting in a decrease in the neutralizing efficacy of broadly neutralizing antibodies. At the same time, there are differences in the neutralizing ability of broadly neutralizing antibodies against different virus strains, which limits their therapeutic effect and thus affects their application in the clinical treatment of AIDS.

[0003] The incidence and mortality of malignant tumors have been increasing year by year. In recent years, immunotherapy has become an important part of the treatment of malignant tumors and has shown good efficacy in many types of malignant tumors. Among them, immune checkpoint blockade therapy targeting the PD-1 / PD-L1 pathway has been widely used. By blocking the binding of PD-1 and PD-L1, the activity of cytotoxic T lymphocytes is restored, thereby enhancing the body's anti-tumor immune response. Although existing immune checkpoint inhibitors (such as anti-PD-1 / PD-L1 antibodies) have shown good efficacy in most types of tumors (such as melanoma, non-small cell lung cancer, renal cell carcinoma, etc.), most patients still do not respond to PD-1 / PD-L1 inhibitor treatment, and even develop drug resistance, and may induce immune-related adverse reactions. Therefore, there is an urgent need to find more effective immune checkpoints to overcome the limitations of existing immune checkpoints.

[0004] As an important cell membrane-bound protein, MARCKS is widely involved in various life activities such as cell signal transduction, cell movement and membrane structure regulation. So far, there has been no report on the functional mechanism of MARCKS in HIV infection and anti-tumor immunotherapy. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to provide a new strategy for treating AIDS or malignant tumors using enhancers or inhibitors of MARCKS protein. This strategy can effectively inhibit the infectivity of HIV virus particles through MARCKS enhancers (such as recombinant proteins), while enhancing the neutralization ability of broad-spectrum neutralizing antibodies on HIV virus particles, thereby improving the therapeutic effect. MARCKS inhibitors regulate the killing ability of cytotoxic T lymphocytes (CTL) on tumor cells by inhibiting the binding of PD-1 and PD-L1, providing a new strategy for the treatment of malignant tumors.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0007] The invention discloses application of a reagent for increasing the expression level of MARCKS in preparing a medicine for preventing and / or treating HIV infection.

[0008] The present invention also discloses a pharmaceutical composition, characterized in that the pharmaceutical composition comprises an agent for up-regulating the expression level of MARCKS and a broad-spectrum neutralizing antibody.

[0009] The present invention also discloses a sensitizer for broad-spectrum neutralizing antibodies for preventing and / or treating HIV infection, characterized in that the sensitizer is an agent for upregulating the expression level of MARCKS.

[0010] Furthermore, the above-mentioned pharmaceutical composition and the above-mentioned sensitizer are used in the preparation of drugs for preventing and / or treating HIV infection.

[0011] Furthermore, the reagents for upregulating the expression level of MARCKS in the above-mentioned uses, pharmaceutical compositions and sensitizers include recombinant plasmid vectors containing the MARCKS gene, MARCKS recombinant proteins and agonists for the function of MARCKS.

[0012] The invention also discloses the use of an inhibitor of the expression level of the MARCKS protein in preparing a medicine for treating malignant tumors.

[0013] Furthermore, the malignant tumors include human lung adenocarcinoma and cervical cancer.

[0014] The present invention also discloses the use of an inhibitor of MARCKS protein expression level in the preparation of products related to PD-1 / PD-L1 immune checkpoint inhibitors.

[0015] Furthermore, any of the above uses is characterized in that the inhibitor of MARCKS protein expression level includes MANS peptide, MARCKS siRNA, MARCKS shRNA, MARCKS neutralizing antibody, etc.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0017] 1. The present invention proposes for the first time to target the MARCKS protein by using an enhancer of MARCKS, thereby inhibiting the infectivity of HIV virus particles and enhancing the neutralizing ability of broad-spectrum neutralizing antibodies against HIV, which can effectively overcome the limitations of existing antiviral treatment options and provide a more effective treatment option for HIV infection.

[0018] 2. The present invention proposes for the first time to target MARCKS protein by using MARCKS inhibitors, thereby inhibiting the binding of PD-1 and PD-L1, enhancing the killing ability of CTL cells on target cells, and improving the effectiveness of tumor immunotherapy.

[0019] 3. Compared with the single use of PD-1 monoclonal antibody or PD-L1 monoclonal antibody, the present invention enhances the therapeutic effect of PD-1 monoclonal antibody or PD-L1 monoclonal antibody by applying MARCKS inhibitor, effectively avoids the immune escape of tumor cells, and significantly improves the response rate of treatment and the efficacy of immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a comparison chart of the effects of MARCKS on the ability of HIV virus particles to infect target cells.

[0021] Figure 2 This is a comparison chart of the effects of MARCKS on the production of HIV virus particle core antigen p24.

[0022] Figure 3 This is a comparison chart of the effects of MARCKS on the content of genomic RNA associated with HIV virus particles.

[0023] Figure 4 This is a comparison chart of the effects of MARCKS on the ability of HIV virus particles with different tropisms to infect target cells.

[0024] Figure 5 This is a comparison chart of the effect of MARCKS on the ability of broadly neutralizing antibodies to neutralize HIV.

[0025] Figure 6This is a comparison chart of the effects of MANS peptide, an inhibitor of MARCKS, on the ability of HIV virus particles to infect target cells.

[0026] Figure 7 This is a comparison chart of the effects of MARCKS on the binding rates of PD-1 and PD-L1.

[0027] Figure 8 This is a comparison chart of the effects of the MARCKS inhibitor MANS peptide on the binding rates of PD-1 and PD-L1.

[0028] Fig. 9 This is a comparison chart of the effects of MANS peptide, an inhibitor of MARCKS, on the ability of CTL to kill human lung adenocarcinoma cell line NCI-H1975 and cervical cancer cell line HeLa. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0030] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0031] Example 1.

[0032] MARCKS can effectively inhibit the infection ability of HIV virus particles. In order to verify the role of MARCKS, 293T cells with different MARCKS protein expression levels were treated with HIV virus vectors. It was found that compared with 293T cells that did not express MARCKS protein, the HIV infection ability of these 293T cells containing MARCKS protein was significantly reduced. The specific experimental steps are as follows: 1×10 6 293T cells were plated at a density of 100 cells / well and incubated at 37°C with 5% CO 2 Culture under static conditions. For each transfection sample, dilute 0.5µg / 1.0µg / 1.5µg of pcDNA3.1-MARCKS-3Flag or Mock plasmid and 0.5µg of pNL4.3 plasmid to a total volume of 50µL using DMEM and mix gently; at the same time, dilute 6µL of LipoD293™ transfection reagent to 50µL using DMEM, add the diluted LipoD293™ reagent to the diluted plasmid, mix well and incubate at room temperature for 10 minutes to form DNA-liposome complexes. Drop 100µL of DNA-liposome complexes into 1mL of cell culture medium. After 6h of transfection, replace 1mL of fresh culture medium. After 48h of transfection, take 200µL of supernatant to infect 2×10 5TZM-bL reporter cells were used to obtain the infection level of the virus. The background RLU ( Figure 1 ).

[0033] In the case of overexpression of MARCKS protein, there was no significant difference in the content of core antigen p24 of viral particles and genomic RNA of viral particles in the culture supernatant. The specific experimental steps are as follows: using the pretreatment steps similar to those in this example, the supernatant was taken for p24 enzyme-linked immunosorbent assay (ELISA) ( Figure 2 ), and specific probe-based qPCR measurement of viral particle-associated genomic RNA (Virion RNA) ( Figure 3 ).

[0034] Example 2.

[0035] MARCKS protein has a very strong ability to inhibit HIV infection, and it depends on the tropism of HIV. The specific experimental steps are as follows: 1×10 6 293T cells were plated at a density of 100 cells / well and incubated at 37°C with 5% CO 2 For each transfection sample, 0.5 µg of pcDNA3.1-MARCKS-3Flag or Mock plasmid, 0.5 µg of pNL4.3∆E-GFP plasmid, and 0.5 µg of pCMV-gp160 plasmids with different tropisms [pCMV-VSV-G (positive control), pCMV-DoL-gp160 (CXCR4 tropic), pCMV-SF162-gp160 (CCR5 tropic), pCMV-AD8-gp160 (CCR5 tropic), pCMV-89.6-gp160 (double tropic)] were diluted to a total volume of 50 µL with DMEM and gently mixed; at the same time, 6 µL of LipoD293™ transfection reagent was diluted to 50 µL with DMEM, and the diluted LipoD293™ reagent was added to the diluted plasmid, mixed, and incubated at room temperature for 10 min to form DNA-liposome complexes. 100 µL of DNA-liposome complex was added dropwise to 1 mL of cell culture medium. After 6 h of transfection, 1 mL of fresh culture medium was replaced. After 48 h of transfection, 200 µL of supernatant was used to infect 2 × 10 5 TZM-bL reporter cells were used to obtain the infection level of the virus. The background RLU ( Figure 4 ).

[0036] Example 3.

[0037] MARCKS protein can enhance the neutralization ability of broad-spectrum neutralizing antibodies against HIV. In order to verify the effect of MARCKS on the neutralization of HIV virus particles by broad-spectrum neutralizing antibodies, 293T cells expressing MARCKS protein or Mock were treated with HIV virus vectors, and the supernatant was incubated with broad-spectrum neutralizing antibodies. It was found that the viral infection level was significantly reduced after treatment with broad-spectrum neutralizing antibodies. The specific experimental steps are as follows: 1×10 6 293T cells were plated at a density of 100 cells / well and incubated at 37°C with 5% CO 2 Culture under the conditions. For each transfection sample, 0.5µg of pcDNA3.1-MARCKS-3Flag or Mock plasmid and 0.5µg of pNL4.3 plasmid were diluted to a total volume of 50µL using DMEM and gently mixed; at the same time, 6µL of LipoD293™ transfection reagent was diluted to 50µL using DMEM, and the diluted LipoD293™ reagent was added to the diluted plasmid, mixed and incubated at room temperature for 10min to form a DNA liposome complex. 100µL of DNA liposome complex was dropped into 1mL of cell culture medium. After 6h of transfection, 1mL of fresh culture medium was replaced. After 48h of transfection, 200µL of supernatant was incubated with HIV broad-spectrum neutralizing antibodies VRC01, 3BNC117, and N6 at 37°C for 1h. After the incubation, 2×10 5 TZM-bL reporter cells were used to obtain the infection level of the virus. The background RLU ( Figure 5 ).

[0038] Example 4.

[0039] MARCKS inhibitors enhance HIV infection by targeting MARCKS. The specific experimental steps are as follows: 1×10 6 293T cells were plated at a density of 100 cells / well and incubated at 37°C with 5% CO 2 Culture under the conditions. For each transfection sample, 0.5µg of pcDNA3.1-MARCKS-3Flag or Mock plasmid and 0.5µg of pNL4.3 plasmid were diluted to a total volume of 50µL using DMEM and gently mixed; at the same time, 6µL of LipoD293™ transfection reagent was diluted to 50µL using DMEM, and the diluted LipoD293™ reagent was added to the diluted plasmid, mixed and incubated at room temperature for 10min to form DNA-liposome complexes. 100µL of DNA-liposome complexes were dropped into 1mL of cell culture medium. After 6h of transfection, 1mL of fresh culture medium was replaced and treated with solvent control or MARCKS inhibitor MANS peptide (concentration of 10µM). After 48h of treatment, 200µL of supernatant was taken to infect 2×10 5TZM-bL reporter cells were used to obtain the infection level of the virus. The background RLU ( Figure 6 ).

[0040] Example 5.

[0041] The 293T cells expressing MARCKS protein were treated with recombinant human PD-1 Fc chimeraprotein (rhPD-1Fc), and it was observed that MARCKS could enhance the binding of PD-1 and PD-L1. The specific experimental steps are as follows: 1×10 6 293T cells were plated at a density of 100 cells / well and incubated at 37°C with 5% CO 2 Culture under 4% RT-PCR conditions. For each transfection sample, dilute 0.5µg of pcDNA3.1-MARCKS-3Flag or Mock plasmid and 0.5µg of pPD-L1 plasmid to a total volume of 50µL using DMEM and mix gently. At the same time, dilute 3µL of LipoD293™ transfection reagent to 50µL using DMEM, add the diluted LipoD293™ reagent to the diluted plasmid, mix well, and incubate at room temperature for 10 minutes to form DNA-liposome complexes. Drop 100µL of DNA-liposome complexes into 1mL of cell culture medium. Replace 1mL of fresh culture medium 6h after transfection. 24 hours after transfection, 1µg / mL rhPD-1Fc recombinant protein or bovine serum albumin (BSA) of the same concentration was added, and the cells were collected after incubation at 4°C in the dark for 1 hour, and washed three times with phosphate buffered saline (PBS) containing 2% fetal bovine serum (FBS) (1500 rpm, 4°C centrifugation for 5 min), and 2µL anti-human PD-L1-APC antibody and 2µL anti-human PD-1-PE antibody were added. The cells were stained on ice in the dark for 30 minutes, and washed three times with PBS containing 2% FBS (1500 rpm, 4°C centrifugation for 5 min), and the binding of PD-1 and PD-L1 was detected by flow cytometry ( Figure 7 ).

[0042] Example 6.

[0043] MARCKS inhibitors weaken the binding of PD-1 and PD-L1 by targeting MARCKS. The specific experimental steps are as follows: 1×10 6 293T cells were plated at a density of 100 cells / well and incubated at 37°C with 5% CO 2Culture under the conditions of 4% paraformaldehyde and 1% paraformaldehyde. For each transfection sample, 0.5µg / 1.0µg / 1.5µg of pcDNA3.1-MARCKS-3Flag or Mock plasmid and 0.5µg of PD-L1 plasmid were diluted to a total volume of 50µL using DMEM and gently mixed; at the same time, 6µL of LipoD293™ transfection reagent was diluted to 50µL using DMEM, and the diluted LipoD293™ reagent was dropped into the diluted plasmid, mixed and incubated at room temperature for 10min to form DNA-liposome complexes. 100µL of DNA-liposome complexes were added to 1mL of cell culture medium. After 6h of transfection, 1mL of fresh culture medium was replaced and treated with MARCKS inhibitor MANS peptide (concentration of 10µM). 24 hours after transfection, 1µg / mL rhPD-1Fc recombinant protein or BSA of the same concentration was added, and the cells were collected after incubation at 4°C in the dark for 1 hour, washed three times with PBS containing 2% FBS (1500rpm, 4°C centrifugation for 5min), added with 2µL anti-human PD-L1-APC antibody and 2µL anti-human PD-1-PE antibody, stained on ice in the dark for 30min, washed three times with PBS containing 2% FBS (1500rpm, 4°C centrifugation for 5min), and the binding of PD-1 and PD-L1 was detected by flow cytometry ( Figure 8 ).

[0044] Example 7.

[0045] MARCKS inhibitors can enhance CD8 + The killing effect of T cells on human lung adenocarcinoma cell line NCI-H1975 and cervical cancer cell line HeLa. The specific experimental steps are as follows: On day 0, 50 mL of peripheral blood from healthy subjects was collected, and peripheral blood mononuclear cells (PBMCs) were separated by polysucrose density gradient centrifugation. RPMI 1640 was used at 10×10 6 The cells were plated at a density of 10 cells / 3 mL and cultured in an incubator. After the mononuclear cells adhered to the wall for 2-3 hours, the cells were gently blown and washed repeatedly. The non-adherent cells were transferred to a new centrifuge tube and purified using EasySep™ Human CD8 + T Cell Enrichment Kit for CD8 + T lymphocytes, and 2×10 6 / mL was cultured in RPMI1640 medium containing 10% FBS; 2mL of RPMI 1640 medium (1× concentration medium) containing 10% FBS, 100ng / mL recombinant human interleukin-4 (rhIL-4) and 100ng / mL recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was added to the well plate, and monocytes were induced into dendritic cells. On the second day, 1mL of RPMI1640 medium (2× concentration medium) containing 10% FBS, 200ng / mL IL-4 and 200ng / mL GM-CSF was replaced; CD8 + Add 100 U / mL recombinant human interleukin-2 (rhIL-2) and 25 µL Dynabeads to T cells. ® Human T Activator CD3 / CD28 / 1×10 6 On the 4th day, 1 mL of 2× concentration culture medium was replaced. On the 5th day, monocytes were induced to differentiate into dendritic cells. Tumor antigen 50 µg / mL (tumor antigen derived from NCI-H1975 or HeLa cells) was added to the dendritic cells for stimulation for 16 hours to allow the dendritic cells to take up tumor antigens. On the 6th day, the culture medium of dendritic cells was replaced with RPMI1640 medium containing 10% FBS, and TNF-α (10 ng / mL) was added to continue culturing for 24 hours to induce them into mature dendritic cells. On the 7th day, CD8 + T cells were co-cultured with dendritic cells, and interleukin-7 (recombinant human interleukin-2, rhIL-7) was added at a final concentration of 10 ng / mL and cultured for 24 hours. On day 8, interleukin-10 (recombinant human interleukin-2, rhIL-10) was added at a final concentration of 10 ng / mL and cultured for 7 days. On day 15, NCI-H1975 and HeLa cells were treated with control solvent or MARCKS inhibitor MANS peptide (concentration of 10 µM) for 24 hours. On day 16, CD8 + T cells were mixed with NCI-H1975 or HeLa cells treated with control solvent or MARCKS inhibitor MANS peptide at a ratio of 4:1 and cultured for 48 h. CD8 +T cells killing target cells ( Fig. 9 ).

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of an agent for upregulating the expression level of MARCKS in the preparation of a drug for preventing and / or treating HIV infection.

2. A pharmaceutical composition, characterized in that The drug combination includes an agent that upregulates the expression level of MARCKS and a broad-spectrum neutralizing antibody.

3. A sensitizer for broad-spectrum neutralizing antibodies for preventing and / or treating HIV infection, characterized in that: The sensitizer is an agent that upregulates the expression level of MARCKS.

4. Use of the pharmaceutical composition according to claim 2 and the sensitizer according to claim 3 in the preparation of a medicament for preventing and / or treating HIV infection.

5. The reagent for upregulating the expression level of MARCKS in the use according to claim 1, the pharmaceutical composition according to claim 2 and the sensitizer according to claim 4 comprises a recombinant plasmid vector containing the MARCKS gene, a MARCKS recombinant protein and an agonist for the function of MARCKS.

6. Application of inhibitors of MARCKS protein expression level in the preparation of drugs for the treatment of malignant tumors.

7. The use according to claim 6, characterized in that: The malignant tumors include human lung adenocarcinoma and cervical cancer.

8. Application of inhibitors of MARCKS protein expression levels in the preparation of products related to PD-1 / PD-L1 immune checkpoint inhibitors.

9. The use according to any one of claims 6 to 8, characterized in that: The inhibitors of MARCKS protein expression level include MANS peptide, MARCKS siRNA, MARCKS shRNA, and MARCKS neutralizing antibody.

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