CN114470195A

CN114470195A - Antifungal preparation, inhibitor for inhibiting expression of cell PD-L1 and application

Info

Publication number: CN114470195A
Application number: CN202210059044.6A
Authority: CN
Inventors: 贾鑫明; 于垚; 王蓉蓉; 鲁向冉
Original assignee: Tongji University
Current assignee: Tongji University
Priority date: 2022-01-19
Filing date: 2022-01-19
Publication date: 2022-05-13

Abstract

The invention discloses an antifungal preparation, an inhibitor for inhibiting the expression of cell PD-L1 and application thereof; the antifungal preparation comprises anti-PD-L1 antibody, dimethylamino michelia lactone fumarate; the inhibitor comprises dimethylamino michelia lactone fumarate; dimethylamino michelia lactone fumarate can obviously inhibit the expression of fungi infected host neutrophilic granulocyte PD-L1, recover the function of chemotaxis of host bone marrow neutrophilic granulocyte to infected organs, reduce the number of bone marrow neutrophilic granulocyte, increase the number of infected organ neutrophilic granulocyte, obviously reduce the bacterial load of host infected organs and obviously increase the survival rate; in conclusion, the antifungal preparation, the inhibitor for inhibiting the expression of the cell PD-L1 and the application have important significance and clinical application value for better preventing and treating fungal infection diseases.

Description

Antifungal preparation, inhibitor for inhibiting expression of cell PD-L1 and application

Technical Field

The invention relates to the technical field of medicines, in particular to an antifungal preparation, an inhibitor for inhibiting the expression of cell PD-L1 and application thereof.

Background

The wide use of broad-spectrum antibiotics, chemotherapeutics and immunosuppressants and the mass development of organ transplantation operations have increased the incidence of fungal infection year by year, and according to the statistics of the world health organization, the number of newly-increased invasive mycosis reaches 435 million every year, the mortality rate reaches 45-75%, and the disease becomes the fourth clinical major bloodstream infectious disease. The currently clinically available antifungal drugs have limited species, narrow antibacterial spectrum and serious drug resistance of fungi, so that the clinical treatment of antifungal infection is very difficult. With the research on the antifungal immune response mechanism of the host, molecular targets for regulating and controlling host immunity are continuously discovered, and the development of targeted antibody drugs becomes a novel immunotherapy method and is expected to become an effective means for treating fungal infection.

C-type lectin receptors (CLRs) are key pattern recognition receptors for the body to immunize against fungal infections, and CLRs use the extracellular carbohydrate binding domain to recognize a variety of pathogen-associated molecular patterns on fungal surfaces. Neutrophils are important effector cells of the innate immune system and play an important role in the anti-fungal immune response. At present, researches on signaling pathways of CLRs in neutrophils are relatively few, the molecular mechanism of the CLRs for regulating the functions of the neutrophils is not clear, and a new strategy for developing precise immunotherapy for fungal infection based on the neutrophils is not reported.

Dimethylamino michelia lactone fumarate (ACT001) is a high-activity compound which is discovered on the basis of screening of natural product michelia lactone derivatives and can selectively eliminate cancer stem cells, is identified as an orphan drug for treating glioblastoma by FDA, and has been clinically tested in multiple countries (stage I: ACTRN12616000228482, Australia, New Zealand clinical trial registration number; stage I: ChiCTR-OIC-17013604, Chinese clinical trial registration number).

The expression of PD-L1 by neutrophils is related to various diseases, but the action mechanism and meaning of PD-L1 expression by neutrophils are unclear. For example, there are related papers that show that the expression of PD-L1 in the peripheral blood neutrophils of Systemic Lupus Erythematosus (SLE) patients is abnormal, and the mechanism is still unclear, which is related to the disease activity and severity and the antibody production. In addition, there are studies showing that PD-L1 is highly expressed in peripheral blood neutrophils of septic mice, but the mechanism thereof has not been studied in depth.

Disclosure of Invention

The invention provides an antifungal preparation, an inhibitor for inhibiting expression of cell PD-L1 and application. Has important significance and clinical application value for better preventing and treating diseases and inhibiting fungi.

To achieve the purpose, the invention provides the following technical scheme:

in a first aspect of the invention, an antifungal agent is provided, which comprises an anti-PD-L1 antibody and dimethylamino michelia lactone fumarate.

Preferably, the anti-PD-L1 antibody includes, but is not limited to, alemtuzumab, avilummab, and dulvacizumab.

Preferably, the fungi include, but are not limited to, candida, aspergillus, cryptococcus, pneumocystis, trichoderma, penicillium marnanense.

Experiments show that the fungal infection can up-regulate the expression of host neutrophil PD-L1 molecule, inhibit the chemotactic function of neutrophil, and make neutrophil accumulate in bone marrow and incapable of migrating to infected organs. When the antibody is adopted to block the PD-L1 function or the host PD-L1 gene deletion, the chemotaxis of the host bone marrow neutrophilic granulocyte to the infected organ can be recovered, the number of the bone marrow neutrophilic granulocyte is reduced, the number of the infected organ neutrophilic granulocyte is increased, the host infected organ bacterium loading amount is obviously reduced, and the survival rate is obviously improved. The results of this study make targeted inhibition of neutrophil PD-L1 expression a new strategy to achieve precise immunotherapy of fungal infections.

Experiments further prove that the natural product michelia lactone derivative, dimethylamino michelia lactone fumarate (ACT001) has the effect of remarkably inhibiting the expression of the neutrophil PD-L1, the ACT001 treatment can also restore the function of chemotaxis of the host neutrophil from bone marrow to the infected organ, the quantity of the neutrophil in the bone marrow of the mouse infected by the fungus is remarkably reduced, the quantity of the neutrophil in the infected organ is remarkably increased, and further, the quantity of the host infected organ with the lotus bacterium is remarkably reduced, and the survival rate is remarkably increased.

The experiments prove that the anti-PD-L1 antibody and dimethylamino michelia lactone fumarate have effective antifungal effect, can reduce the bacterial load of host infected organs, and increase the survival rate of the host.

In a second aspect of the invention, there is provided the use of an antifungal formulation according to the invention in the preparation of an antifungal medicament.

Preferably, the antifungal preparation comprises anti-PD-L1 antibody and dimethylamino michelia lactone fumarate.

In a third aspect of the invention, an inhibitor for inhibiting the expression of cell PD-L1 is provided, wherein the inhibitor comprises dimethylamino michelia lactone fumarate.

Preferably, the cells comprise neutrophils.

Experiments further prove that dimethylamino michelia lactone fumarate (ACT001) inhibits the expression of neutrophil PD-L1 by inhibiting a neutrophil JAK-STAT signal passage, so that the function of chemotaxis of the neutrophil from bone marrow to infected organs is recovered. Therefore, dimethylamino michelia lactone fumarate can be used as an inhibitor for inhibiting the expression of PD-L1, and the signal pathway for inhibiting the expression of PD-L1 is as follows: JAK-STAT.

In a fourth aspect of the invention, the invention provides an application of the inhibitor in preparing a medicament for inhibiting the expression of cell PD-L1.

Preferably, the inhibitor comprises dimethylamino michelia lactone fumarate.

Preferably, the cells comprise neutrophils.

In a fifth aspect of the invention, a pharmaceutical composition for preventing, treating or adjunctively treating diseases is provided, which comprises the inhibitor of the invention.

Preferably, the disease includes immune regulation related diseases, fungal infections.

Preferably, the pharmaceutical composition may also be used for modulating the expression level of JAK2 protein, and/or modulating the phosphorylation level of JAK2 protein, and/or modulating the expression level of STAT3 protein, and/or modulating the phosphorylation level of STAT3 protein.

Preferably, the immune modulation related diseases include: PD-L1 up-regulation results in diseases associated with neutrophil immune regulation.

Preferably, the immune modulation related diseases include, but are not limited to: tumor, inflammation, systemic lupus erythematosus, sepsis. Further preferably, the tumor is selected from the group consisting of a tumor positive for PD-L1, and the tumor is independent of a tumor under T cell regulation.

Preferably, a pharmaceutically acceptable carrier is also included.

Preferably, the dosage of the medicine is 10-200 mg/kg.

The invention discovers that dimethylamino michelia lactone fumarate (ACT001) can inhibit PD-L1 expression by inhibiting a neutrophil JAK-STAT signal path for the first time. The down-regulation of PD-L1 expression is related to a plurality of diseases related to immune regulation, so the invention can prove that dimethylamino michelia lactone fumarate (ACT001) can be used for treating the diseases related to immune regulation. In addition, the invention also proves that the fungus surface beta-glucan mediates the activation of a neutrophil JAK-STAT pathway through activating a neutrophil Dectin-1 receptor, and dimethylamino michelia lactone fumarate (ACT001) can inhibit the neutrophil JAK-STAT signal pathway. Therefore, dimethylamino michelia lactone fumarate (ACT001) inhibits the expression of neutrophil PD-L1 by inhibiting a neutrophil JAK-STAT signal path, restores the function of chemotaxis of the neutrophil to an infected organ, and further remarkably reduces the bacterial load of the infected organ of a host and remarkably increases the survival rate.

Compared with the prior art, the invention has the beneficial effects and remarkable progresses that:

1. the invention firstly provides that the anti-PD-L1 antibody and dimethylamino michelia lactone fumarate can be used for antifungal immunotherapy. The inhibition principle may be: the anti-PD-L1 antibody and dimethylamino michelia lactone fumarate can reduce the expression of the neutrophil PD-L1, and experiments prove that the beta-glucan on the surface of the fungus activates a neutrophil Dectin-1 receptor to mediate the JAK-STAT pathway activation of the neutrophil, so that the PD-L1 molecular expression is up-regulated. The anti-PD-L1 antibody and dimethylamino michelia lactone fumarate can be used for inhibiting the damage of fungi to host organs by down-regulating the expression of neutrophil PD-L1.

2. The invention firstly provides that dimethylamino michelia lactone fumarate can inhibit the expression of neutrophil PD-L1. The signal path suppressed by the method is as follows: the JAK-STAT signaling pathway.

3. The invention provides a new therapeutic drug for diseases related to immunoregulation and fungal infection caused by PD-L1 up-regulation, namely dimethylamino michelia lactone fumarate.

Drawings

To more clearly illustrate the technical solution of the present invention, the drawings required for the embodiment of the present invention will be briefly described below.

It should be apparent that the drawings in the following description are only drawings of some embodiments of the invention, and that other drawings can be obtained by those skilled in the art without inventive exercise, and the other drawings also belong to the drawings required by the embodiments of the invention.

FIG. 1A shows bone marrow neutrophils (CD11 b) according to example 1 of the present invention⁺Ly-6G⁺) Middle PD-L1⁺The content of (a);

FIG. 1B shows bone marrow and kidney neutrophils (CD 11B) from mice at different treatment times according to example 1 of the present invention⁺Ly-6G⁺)；

FIG. 2A is a photograph showing the neutrophils in the bone marrow and kidney (CD11 b) of the mice treated with the infection according to example 2 of the present invention⁺Ly-6G⁺) Content (c);

FIG. 2B is a graph showing survival and renal load of infection-treated mice according to example 2 of the present invention;

FIG. 2C shows neutrophils (CD) from bone marrow and kidney of infected, antibody-treated mice according to example 2 of the present invention11b⁺Ly-6G⁺) Content (c);

FIG. 2D is a graph showing survival and kidney load of infected, antibody-treated mice according to example 2 of the present invention;

FIG. 3A illustrates curdlan processing WT and Clec7a according to embodiment 3 of the present invention^-/-The content of mouse bone marrow-derived neutrophils;

FIG. 3B illustrates curdlan processing WT and Clec7a according to embodiment 3 of the present invention^-/-JAK2 and STAT3 phosphorylation levels of mouse bone marrow derived neutrophils and human blood neutrophils;

FIG. 3C illustrates curdlan processing WT and Clec7a according to embodiment 3 of the present invention^-/-JAK2 and STAT3 phosphorylation levels of mouse bone marrow-derived neutrophils;

FIG. 4A is a graph of the phosphorylation levels of JAK2 and STAT3 of bone marrow-derived neutrophils treated with curdlan and ACT001 according to example 4 of the present invention;

FIG. 4B is a graph of JAK2 and STAT3 phosphorylation levels of bone marrow derived neutrophils and human blood neutrophils treated with curdlan and ACT001 of example 4 of the present invention;

FIG. 4C shows the bone marrow and renal neutrophils (CD11 b) of an infected, gavage mouse of example 4 of the present invention⁺Ly-6G⁺) Middle PD-L1⁺The content of (A);

FIG. 4D shows the bone marrow and renal neutrophils (CD11 b) of an infected, gavage mouse of example 4 of the present invention⁺Ly-6G⁺) Content (c);

FIG. 4E is a graph showing survival and renal load of infected, gavage treated mice of invention 4.

Detailed Description

In order to make the objects, technical solutions, advantageous effects and significant progress of the embodiments of the present invention clearer and more complete, the technical solutions in the embodiments of the present invention will be described below with reference to the drawings provided in the embodiments of the present invention.

It is to be understood that all of the described embodiments are merely some, and not all, embodiments of the invention; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

It should be further noted that the following embodiments may be combined with each other, and the same or similar concepts or processes may not be repeated in some embodiments.

The technical means of the present invention will be described in detail below with specific examples.

The experimental protocols of the invention are approved by the ethical committee of experimental animals of the university of the same university.

Example 1

1.1 obtaining experimental samples, C57BL/6 mice tail vein injection SC5314(2 x 10)⁵CFU), mice were sacrificed 4 days later, and mouse tissues were taken;

1.2, obtaining mouse bone marrow neutrophils: taking the thighbone and the tibia of the hind leg of the mouse, and collecting bone marrow flushing fluid; adding 2mL of IB solution for resuspension; percoll extracts the cell layer between the 67% and 76% concentration gradient and washes the cells twice (500g, 10min, 4 ℃) with 5ml PBS buffer; discarding the supernatant, lysing erythrocytes, adding 2mL of medical sterile water for injection, gently blowing for 20s with a sterile pasteur tube, adding equal volume of 2 Xcalcium-magnesium-free HBSS and IB flushing fluid, centrifuging (500g, 7min, 4 ℃), and discarding the supernatant to obtain purified bone marrow neutrophils;

1.3, flow cytometry detection: placing appropriate amount of single cell (bone marrow neutrophil) suspension in 1.5mL EP tube, centrifuging (500g, 10min, 4 deg.C), and discarding supernatant; the cells were washed 2 times with 1ml PBS, the live cell dye was diluted with PBS (ratio: 1/100), 100. mu.L of the diluted dye was added to each sample, staining was carried out for 30min (4 ℃ C.), centrifuged (500g, 10min, 4 ℃ C.), and the supernatant was discarded; preparing an antibody diluent (PBS buffer + 2% FBS), diluting the antibody according to the proportion, resuspending the cells with 100. mu.L of the antibody diluent, staining for 30min (4 ℃), centrifuging (500g, 10min, 4 ℃), discarding the supernatant, and washing the cells 2 times with 1ml of the antibody diluent; resuspending the cells in 500. mu.L of antibody diluent, filtering through 200 mesh wire gauze into a flow tube, and detecting the PD-L1 expression of bone marrow neutrophils by flow cytometry;

1.4 obtaining experimental samples, C57BL/6 mice tail vein injection SC5314(2 x 10)⁵CFU), mice were sacrificed 0 days, 1 day, 2 days, 3 days, 4 days, 5 days and 6 days after treatment, respectively, and mouse tissues were taken;

1.5, obtaining mouse bone marrow neutrophils: the experiment was carried out according to the method described previously.

1.6, obtaining mouse kidney neutrophils: taking out the kidney of the dead mouse, cutting the kidney by using surgical scissors, adding 5mL of 1mg/mL collagenase D solution, shaking the table at 37 ℃, and digesting the kidney for 1h under the oscillation of 200 rpm; filtering the digestive juice and the ground solution of the broken tissue with 70 μm filter membrane to obtain kidney single cell suspension, centrifuging (500g, 10min, 4 deg.C), and removing supernatant; percoll extracts the cell layer between the 40% and 80% concentration gradient and washes the cells twice (500g, 10min, 4 ℃) with 5ml PBS buffer; adding 2mLRBC buffer solution, breaking red for 5min, adding 10mL1640 culture medium, centrifuging (500g, 10min, 4 ℃), and resuspending cells with 1mL 1640 culture medium to obtain the single cell suspension of mouse kidney immune cells.

1.7, flow cytometry detection: experiments were performed according to the methods described previously to detect bone marrow and kidney neutrophil numbers and PD-L1 expression.

The experimental results are shown in fig. 1A, and the expression of mouse bone marrow neutrophil PD-L1 is significantly increased by fungal infection. As shown in fig. 1B, fungal infection increased the number of neutrophils in the bone marrow of mice and decreased the number of neutrophils in the kidney of the infected organ. This result demonstrates that fungal infection causes a large amount of host neutrophils to accumulate in the bone marrow, and the chemotactic ability toward the kidney of the infected organ is significantly reduced.

Example 2

2.1, C57BL/6 mice and PD-L1^-/-Mouse tail vein injection SC5314(2 x 10)⁵CFU), day 4 sacrifice, bone marrow and kidney were processed as single cell suspensions as per 1.2 and 1.6 methods, and FASC analysis for neutrophils (CD11 b)⁺Ly-6G⁺) The ratio of (A) to (B); as shown in FIG. 2A, in the PD-L1 gene-deficient mice, the number of bone marrow neutrophils decreased and the number of kidney neutrophils increased in the infected organs.

2.2, C57BL/6 mice and PD-L1^-/-The mice were the subjects to investigate the difference in survival rates of systemic candida albicans infected mice: the experiments were divided into control and experimental groups, each group consisting of 10 mice, and mice were injected with SC5314(2 x 10) via tail vein⁵CFU) to construct an infection model, recording the survival of mice in each group daily, observing for 15 days, and statistically analyzing the difference among the groups.

2.3, C57BL/6 mice and PD-L1^-/-The mice are experimental subjects to investigate the difference of the kidney load of the systemic candida albicans infected mice: the experiments were divided into control and experimental groups, 6 mice each, mice injected with SC5314(2 x 10) tail vein⁵CFU) constructing an infection model, taking the kidney of an infected organ of a mouse on the 4 th day after infection, weighing and grinding the organ, performing gradient dilution and plate coating on a grinding fluid, counting the number of bacterial colonies after plate coating so as to calculate the bacterial load of the mouse kidney, and performing statistical analysis on the difference among the groups. The results of the experiment are shown in FIG. 2B, PD-L1^-/-The amount of the mouse kidney lotus bacteria is obviously reduced, and the survival rate is obviously improved.

2.4, C57BL/6 mice tail vein injection SC5314(2 x 10)⁵CFU), day 2 intraperitoneal injection of anti-PD-L1 antibody and IgG antibody (200. mu.g/mouse), day 4 sacrifice, bone marrow and kidney processing as single cell suspension, FASC analysis of neutrophils (CD11 b)⁺Ly-6G⁺) The ratio of (A) to (B); the results of the experiment are shown in FIG. 2C, and the amount of neutrophils in the bone marrow of the mice is reduced and the amount of neutrophils in the kidney of the infected organs is increased by the treatment with the PD-L1 antibody.

2.5, C57BL/6 mice tail vein injection SC5314(2 x 10)⁵CFU), anti-PD-L1 antibody and IgG antibody (200 μ g/mouse) were intraperitoneally injected on day 2, and mouse survival was recorded as per experimental procedure in 2.2. The results of the experiment are shown in FIG. 2D.

2.6, C57BL/6 mice tail vein injection SC5314(2 x 10)⁵CFU), anti-PD-L1 antibody and IgG antibody (200. mu.g/mouse) were intraperitoneally injected on day 2, and the kidney load of the mice on day 4 of infection was calculated according to the experimental method in 2.3. The results of the experiment are shown in FIG. 2D.

The experimental results prove that the fungus-infected mouse can recover the function of neutrophils chemotactic from bone marrow to the kidney of an infected organ by the treatment of an anti-PD-L1 antibody or the deletion of a host PD-L1 gene, the quantity of the bone marrow neutrophils is reduced, the quantity of the kidney neutrophils is increased, the quantity of the infected organ kidney bacteria is obviously reduced, and the survival rate of the mouse is obviously improved. The experimental result proves that the targeted inhibition of the expression of the neutrophil PD-L1 can be used as a brand new strategy for the immunotherapy of fungal infection.

Example 3

3.1, 25 μ g curdlan treatment of C57BL/6 mice and Clec7a^-/-Mouse bone marrow-derived neutrophils for 12 hours, FASC detection of neutrophils (CD11 b)⁺Ly-6G⁺) Middle PD-L1⁺The ratio of (A) to (B); the experimental result is shown in fig. 3A, the neutrophil of the mice with the Clec7a gene deletion is stimulated by beta-glucan (curdlan) from fungi, and the expression of PD-L1 on the surface of the neutrophil can be obviously reduced; the neutrophil is proved to recognize beta-glucan on the surface of the fungus through a cell surface dectin-1 receptor, and then the expression of PD-L1 is up-regulated.

3.2, neutrophil protein extraction and western blotting: preparing protein lysate (1mL Mammali Cell Lysis Buffer + 1U)

Nuclean + 10. mu.L of Protease Inhibitor (100X)), 500. mu.L of protein lysate was resuspended at 5X 10⁶The neutrophils were shaken at 4 ℃ for 5 min. Centrifuging at 4 deg.C and 14000g for 10min, collecting supernatant, determining protein concentration by BCA method, adding 5 × loadingbuffer into the supernatant at a ratio of 1:4, mixing, and decocting at 95 deg.C for 10min for denaturation. The samples were stored at-20 ℃. A10% SDS-PAGE electrophoresis gel was prepared, and electrophoresis was performed at a constant pressure. After the electrophoresis is finished, the target protein is transferred from the gel to a PVDF membrane. After the membrane transfer is finished, 5% skimmed milk blocks the epitope for at least 1h at room temperature, and primary antibody is incubated overnight at 4 ℃. Washing the membrane 3 times and 10 min/time with 1 xTBS/T, incubating the secondary antibody, washing the membrane 5 times and 10 min/time with 1 xTBS/T at 37 ℃, performing exposure analysis, and storing the picture.

3.3, 25. mu.g of curdlan treated C57BL/6 mouse bone marrow-derived neutrophils for 1 hour, 50. mu.g of curdlan treated human blood neutrophils for 1 hour, and the phosphorylation levels of JAK2 and STAT3 were measured as Westernblot method 3.2. Results as shown in figure 3B, stimulation of mouse and human neutrophils by curdlan activates JAK2 and STAT3 phosphorylation.

3.4, flow intracellular staining: after the mouse bone marrow-derived neutrophils were stimulated by Curdlan for 30min, the cells were harvested by centrifugation, washed once with 2mL FACS Buffer and then incubated with stained surface proteins at 4 ℃ for 30min in the dark. After 1mL Buffer washing, the supernatant was discarded, 1mL Fix/permeabilizatin Buffer was added and cells were vortexed pulsed and incubated at 4 ℃ in the dark for 60 min. 2mL of 1X PermWash Buffer was added to each tube, centrifuged at 600g for 5min at room temperature, and the supernatant was discarded and repeated twice. Then intracellular protein staining is carried out, and incubation is carried out for 60min at 4 ℃ in the dark. 2mL of 1X Perm Wash Buffer was added to each tube, centrifuged at 600g for 5min at room temperature, the supernatant discarded, an appropriate amount of FACS Buffer was added to resuspend the stained cells, and the samples were analyzed using a flow cytometer.

3.5 mu.g curdlan treatment C57BL/6 and Clec7a as described in 3.4^-/-Mouse bone marrow derived neutrophils for 30min, FASC detected JAK2 and STAT3 phosphorylation levels. Results as shown in figure 3C, stimulation of mouse neutrophils by curdlan upregulates JAK2 and STAT3 phosphorylation.

The above experimental results prove that the beta-glucan on the surface of the fungus is up-regulated by Dectin-1/JAK2/STAT3 axis to the expression of PD-L1 of the neutrophil.

Example 4

4.1, dividing the C57BL/6 mouse bone marrow neutrophils into four groups: (1) blank control, (2) ACT001(40 μ M) treated group, (3)25 μ g curdlan treated group, (4)25 μ g curdlan + ACT001(40 μ M) treated group, FASC detected JAK2 and STAT3 phosphorylation levels as described in 3.4; as shown in fig. 4A, ACT001 significantly inhibited JAK2 and STAT3 phosphorylation.

4.2, 25 μ g of curdlan and ACT001(10, 20, 40, 60 μ M) Co-treatment of C57BL/6 mouse bone marrow-derived neutrophils for 12 hours, 50 μ g of curdlan and ACT001(20, 40, 60, 80 μ M) Co-treatment of human blood neutrophils for 12 hours, FASC detection of neutrophils (CD11 b)⁺Ly-6G⁺) Middle PD-L1⁺The ratio of (A) to (B); as shown in FIG. 4B, the inhibitory effect of ACT001 on the expression of mouse and human neutrophil PD-L1 was dose-dependent,the higher the dose of ACT001, the greater the ability to inhibit neutrophil PD-L1 expression.

4.3, C57BL/6 mice tail vein injection SC5314(2 x 10)⁵CFU), 1-3 days of infection, intragastrically administered PBS and ACT001(200mg/kg), sacrificed on day 4, bone marrow and kidney processed into single cell suspensions, and FASC detected neutrophils (CD11 b)⁺Ly-6G⁺) Middle PD-L1⁺And neutrophil granulocytes (CD11 b)⁺Ly-6G⁺) The ratio of (A) to (B); the experimental results are shown in fig. 4C and 4D, and the ACT001 treatment can significantly reduce the expression of PD-L1 in mouse bone marrow and kidney neutrophils, restore the chemotactic ability of mouse bone marrow neutrophils to the kidney of the infected organ, reduce the number of mouse bone marrow neutrophils, and increase the number of kidney neutrophils.

4.4, C57BL/6 mice tail vein injection SC5314(2 x 10)⁵CFU), intragastric PBS (200. mu.L/mouse) and ACT001(200mg/kg, 200. mu.L/mouse) were administered orally on day 2, respectively, and mouse survival was recorded as per the experimental protocol in 2.2.

4.5, C57BL/6 mice tail vein injection SC5314(2 x 10)⁵CFU), orally administered gavage PBS (200. mu.L/mouse) and ACT001(200mg/kg, 200. mu.L/mouse) on day 2, respectively, and the kidney load of the mice on day 4 of infection was calculated according to the experimental method in 2.3. The experimental results are shown in fig. 4E, and the ACT001 treatment significantly reduces the amount of the bacteria in the kidney of the infected organ of the mouse and significantly improves the survival rate of the mouse.

The experimental results prove that ACT001 has the effect of remarkably inhibiting the expression of the neutrophil PD-L1, the function of chemotaxis of the host neutrophil from bone marrow to an infected organ can be restored by adopting the ACT001 for treatment, the number of the neutrophil in the bone marrow is reduced, the number of the neutrophil in the infected organ is increased, and the treatment effect of fungal infection is remarkable.

During the description of the above description:

the description of the terms "this embodiment," "an embodiment of the invention," "as shown at … …," "further improved technical solution," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention; in this specification, the schematic representations of the terms used above are not necessarily for the same embodiment or example, and the particular features, structures, materials, or characteristics described, etc., may be combined or brought together in any suitable manner in any one or more embodiments or examples; furthermore, those of ordinary skill in the art may combine or combine features of different embodiments or examples and features of different embodiments or examples described in this specification without undue conflict.

Finally, it should be noted that:

the above embodiments are only used for illustrating the technical solution of the present invention, and not for limiting the same;

although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that various changes and modifications may be made, and equivalents may be substituted for elements thereof without departing from the scope of the embodiments of the present invention.

Claims

1. An antifungal preparation, which is characterized by comprising an anti-PD-L1 antibody and dimethylamino michelia lactone fumarate.

2. The antifungal formulation of claim 1 wherein the anti-PD-L1 antibody comprises astuzumab, avilummab, and dulvacizumab.

3. The antifungal formulation of claim 1 wherein the fungus comprises candida, aspergillus, cryptococcus, pneumocystis, trichoderma, penicillium marnanense.

4. Use of the antifungal formulation of any one of claims 1 to 3 in the preparation of an antifungal medicament.

5. An inhibitor for inhibiting the expression of PD-L1 in a cell, wherein the inhibitor comprises dimethylamino michelia lactone fumarate.

6. The inhibitor of the expression of PD-L1 in a cell according to claim 5, wherein the cell comprises a neutrophil.

7. Use of an inhibitor according to any one of claims 5 to 6 in the manufacture of a medicament for inhibiting the expression of PD-L1 in a cell.

8. A pharmaceutical composition for the prevention, treatment or co-treatment of a disease comprising an inhibitor according to any one of claims 5 to 6.

9. The pharmaceutical composition for the prevention, treatment, or co-treatment of diseases according to claim 8, wherein the diseases include immune modulation related diseases, fungal infections.

10. The pharmaceutical composition for the prevention, treatment, or co-treatment of a disease as claimed in claim 8, wherein the fungus comprises candida, aspergillus, cryptococcus, pneumocystis, trichoderma, penicillium marnanense.

11. The pharmaceutical composition for the prevention, treatment or co-treatment of a disease as claimed in claim 8, further comprising a pharmaceutically acceptable carrier.

12. The pharmaceutical composition for the prevention, treatment or adjuvant therapy of diseases according to claim 8, wherein the drug is administered in an amount of 10 to 200 mg/kg.