Construction and application of enhanced neutrophil drug

Abstract

The invention relates to construction and application of an enhanced neutrophile granulocyte drug, in particular to a construction method for obtaining enhanced neutrophile granulocytes, which comprises the following steps: inducing and culturing neutrophile granulocytes in a culture medium containing any one or more of a component A, a component B and a component C, the component A is prepared from one or more of IFN gamma, GM-CSF, G-CSF, IFN-beta, IFN-alpha, TNF alpha, LPS, IL1beta, IL6, MCP-1, IL-23, BCG or beta-glucan; the component B is prepared from one or more of duvaluzumab, abamectin monoclonal antibody, atelizumab, ipimumab or texistimumab; and the component C is prepared from one or more of Q-VD-Oph, DFO, Hsp70, NAC (N-Activated Carbon) or Nec-1. According to the construction method provided by the invention, the in-vitro lifetime of the neutrophil is prolonged, and the tumor killing activity, the immune activation ability and / or the anti-infection ability are enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the construction and application of an enhanced neutrophil drug. Background Art

[0002] Neutrophils are the most abundant white blood cells in human blood and are the body's first line of defense against foreign substances. In addition to being able to effectively kill pathogens, neutrophils also exhibit unique advantages in tumor treatment, including: (1) Neutrophils can quickly respond to inflammatory factors released by tumor sites, actively move to tumor sites, and can extravasate from tumor blood vessels and penetrate into the deep layers of the tumor, highly infiltrating in a variety of tumors. 1 (2) Neutrophils have the ability to kill tumors by releasing reactive oxygen species (ROS), nitric oxide (NO), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). (3) Neutrophils have an immune regulatory effect and can recruit and activate T cells or NK cells by releasing cytokines. 2 Based on this, many studies in recent years have reported neutrophil-based anti-tumor therapies, which can be summarized as follows:

[0003] (1) Researchers have used the infusion of healthy unrelated donors’ white blood cells to treat solid tumors (phase I / II, NCT00900497). White blood cells from 4-5 donors were collected by apheresis and 2×10 10-11 The patients are infused with a dose of 10 ...

[0004] (2) Some researchers have shown that the tumor-killing efficacy of neutrophils is genetically defined. Therefore, they screened healthy people with neutrophils with high tumor-killing activity, isolated hematopoietic stem cells from these donors, and used some cytokines or vitamins to differentiate them into neutrophils with similar high tumor-killing activity. The researchers provided a culture of hematopoietic stem cells differentiated into neutrophils, including hematopoietic stem cells, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), growth hormone, serotonin, vitamin C, vitamin D, glutamine (Glutamine, Gln), arachidonic acid, AGE-albumin, interleukin (IL), tumor necrosis factor (TNF) α, Flt-3 ligand, thrombopoietin and fetal bovine serum (CN111278968A). The method of using donors of neutrophils with high tumor killing activity as a source of hematopoietic stem cells and then differentiating hematopoietic stem cells into neutrophils with similar high tumor killing activity for tumor treatment has the following defects: 1) A large number of people need to be screened to find donors of neutrophils with high tumor killing activity, and the number of donors is limited; 2) The induction of hematopoietic stem cells into neutrophils requires a long induction cycle and a large amount of cytokines, which is time-consuming and expensive; 3) The purity of the induced neutrophils is less than 80%, which contains monocytes and macrophages that also express CD11b and CD15, and is mixed with 4%-10% hematopoietic stem cells, which poses a risk of transfusion-related acute lung injury caused by monocytes and graft-versus-host disease caused by hematopoietic stem cells.

[0005] (3) Based on the plasticity of neutrophils 3 The researchers used different cytokines to stimulate neutrophils to exert a certain degree of anti-tumor effect. For example, the use of a single inducer, interferon (IFN)-γ, can prolong the life of neutrophils. 500U / mL of IFNγ can maintain the activity of neutrophils at more than 80% for 48 hours. 4 , and can upregulate CD64, ICAM-1 and HLA-DR, which may be beneficial for tumor killing 5; The researchers also optimized the concentration of a single inducer, IFNα, and obtained an induction method to increase the activity of neutrophils, which can convert N2-type (pro-tumor) neutrophils into N1-type (anti-tumor) neutrophils, thereby enhancing the tumor killing effect (CN110066766A); In early but not advanced non-small cell lung cancer, 50pg / mL-20ng / mL IFNγ and 50pg / mL GM-CSF have been shown to drive neutrophils to differentiate into APC-like MHCII neutrophils expressing co-stimulatory molecules OX40 ligand, CD86 and 4-1BB ligand, and this APC-like MHCII neutrophil can stimulate and enhance the anti-tumor effector T cell response; Some researchers pre-stimulated neutrophils with 1nM phorbol 12-myristate 13-acetate (PMA) for 30 minutes, and then added 40μg / mL of oxidized low-density lipoprotein (Oxidized Low Density Lipoprotein) Lipoprotein (ox-LDL) stimulation for 12 hours, the neutrophils with antigen presenting cell phenotype can promote T cell proliferation and produce IFN-γ (CN114045261A). The technology of cytokine-induced neutrophil anti-tumor has the following limitations: 1) There are differences in the time and concentration dependence of the effects of different cytokines on neutrophils, but there is no systematic study so far; 2) Cytokines have multiple effects on neutrophils. Although they can enhance tumor killing effects, they may also cause immunosuppression. The anti-tumor effect of neutrophils induced by cytokines alone has not been effectively verified in vivo, and the therapeutic effect is questionable; 3) Considering that clinical applications require longer neutrophil lifespan and higher tumor killing efficacy, there is currently a lack of combined inducers for both.

[0006] (4) Our team previously used the phagocytic effect of neutrophils to construct neutrophil drugs for tumor treatment (CN104225609B). Although the neutrophil drug delivery system can significantly improve the targeting effect and therapeutic effect of drugs, the anti-tumor effect of neutrophils as drug carriers is not enough to effectively inhibit tumor growth.

[0007] References:

[0008] 1Jaillon,S.et al.Neutrophil diversity and plasticity in tumor progression and therapy.

[0009] Nature Reviews Cancer 20,485-503(2020).

[0010] 2Ustyanovska Avtenyuk,NV,Nienke Bremer,Edwin Wiersma,Valerie R.The Neutrophil:

[0011] The underdog that packs a punch in the fight againstcancer. International Journal of Molecular Sciences 21,7820-7878(2020).

[0012] 3Giese, MA, Hind, LE & Huttenlocher, A. Neutrophil plasticity in the tumor microenvironment. Blood 133, 2159-2167 (2019).

[0013] 4Colotta F, RF, Polentarutti N, Sozzani S, Mantovani A. Modulation of granulocyte survival and programmed cell death by cytokines and bacterial products. Blood 80, 2012-2020 (1992).

[0015] 5Ricardo, G.-B., Tarik, EN, S. Hackert & Felix, ARS, A. Jelinsky Olha, Halyabar Alexandra. Ageing and interferon gamma response drive the phenotype of neutrophils in the inflamed joint. Annals of the Rheumatic Diseases 81, 805-814 (2022). Summary of the invention

[0016] In view of the above-mentioned defects of the prior art, the object of the present invention is to provide a construction method for obtaining enhanced neutrophils to prolong the in vitro lifespan of neutrophils and enhance their tumor killing activity and immune activation ability.

[0017] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0018] The first object of the present invention is to provide a method for obtaining enhanced neutrophils, the method comprising: inducing and culturing neutrophils in a culture medium comprising any one or more of component A, component B and component C,

[0019] wherein the A component comprises one or more of IFNγ, GM-CSF, G-CSF, IFN-β, IFN-α, TNFα, LPS, IL1β, IL6, MCP-1, IL-23, BCG or β-glucan;

[0020] The B component includes one or more of durvalumab, avelumab, atezolizumab, ipilimumab or tesimumab;

[0021] The C component includes one or more of Q-VD-Oph, DFO, Hsp70, NAC or Nec-1.

[0022] Preferably, the A component is selected from IFNγ and\or GM-CSF.

[0023] Preferably, the B component is selected from durvalumab.

[0024] Preferably, the C component is selected from Q-VD-Oph.

[0025] Preferably, the concentration of component A is 1-200 ng / mL, preferably 1-100 ng / mL, more preferably 1-50 ng / mL, particularly preferably 1-30 ng / mL, and most preferably 20 ng / mL.

[0026] Preferably, the concentration of component B is 0.1-10 μg / mL, preferably 0.5-8 μg / mL, more preferably 0.5-5 μg / mL, particularly preferably 1-3 μg / mL, and most preferably 2 μg / mL.

[0027] Preferably, the concentration of the C component is 1-100 μM, preferably 10-80 μM, more preferably 10-60 μM, particularly preferably 20-60 μM, and most preferably 50 μM.

[0028] Preferably, the culture medium is 1640 culture medium or DMEM culture medium.

[0029] Preferably, the method comprises incubating neutrophils in the culture medium in an aerobic or anaerobic environment.

[0030] Preferably, the incubation temperature is controlled to be 35-40°C, preferably 36-40°C, more preferably 37°C.

[0031] Preferably, the incubation time is controlled to be 2-48 h, preferably 2-36 h, more preferably 4-24 h, and most preferably 4-18 h.

[0032] Preferably, the incubation is controlled to be carried out under 5% CO2 conditions.

[0033] Preferably, the neutrophils are derived from healthy people or tumor patients.

[0034] The second object of the present invention is to provide an enhanced neutrophil obtained by the construction method as described in any one of the above items.

[0035] The third object of the present invention is to provide a pharmaceutical composition, which comprises the enhanced neutrophils obtained by any of the construction methods described above or the enhanced neutrophils as described above.

[0036] Preferably, the pharmaceutical composition further comprises nanomedicine.

[0037] Further preferably, the nanomedicine is selected from anti-tumor drugs, immunomodulatory drugs and antibacterial drugs.

[0038] More preferably, the anti-tumor drug is selected from paclitaxel, doxorubicin, 5-fluorouracil and camptothecin.

[0039] More preferably, the immunomodulatory drug is selected from STING modulators, TGF-β modulators and HIF-1α modulators.

[0040] More preferably, the antibacterial drug is selected from amphotericin B, colistin, azithromycin, clavulanic acid, ampicillin, ciprofloxacin and vancomycin.

[0041] The fourth object of the present invention is to provide a method for preparing a pharmaceutical composition, the method comprising: mixing the enhanced neutrophils obtained by any of the construction methods described above or the enhanced neutrophils as described above with nanomedicines, and incubating them in an aerobic or anaerobic environment.

[0042] Preferably, the incubation temperature is controlled to be 35-40°C, preferably 36-40°C, more preferably 37°C.

[0043] Preferably, the incubation time is controlled to be 0.1-12 h, preferably 0.2-10 h, more preferably 0.2-6 h, most preferably 0.2-3 h.

[0044] Preferably, the incubation is controlled to be carried out under 5% CO2 conditions.

[0045] The fifth object of the present invention is to provide the use of the enhanced neutrophils obtained by any of the construction methods described above, the enhanced neutrophils described above, the pharmaceutical composition described above, or the pharmaceutical composition obtained by the preparation method described above in preventing and / or treating tumors, infections or immune diseases.

[0046] Compared with the prior art, the present invention has the following technical effects:

[0047] The enhanced neutrophils obtained by the construction method provided by the present invention have a longer lifespan, stronger anti-tumor and immunoregulatory abilities, and also have the advantages of simple operation, mild conditions, and short time consumption.

[0048] The enhanced neutrophil pharmaceutical composition obtained by the construction method provided by the present invention can jointly exert the therapeutic effects of enhanced neutrophils and nanomedicines, and can break through the limitation that neutrophils are only used as drug carriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The flowchart of the construction of the enhanced neutrophil and pharmaceutical composition of the present invention;

[0050] Figure 2 This is a bar graph showing the effects of different concentrations of a single inducer on the in vitro lifespan of neutrophils in Example 1 of the present invention;

[0051] Figure 3 This is a bar graph showing the effects of different combined inducers on the in vitro lifespan of neutrophils in Example 2 of the present invention;

[0052] Figure 4 This is a bar graph showing the effects of different combined inducers on the in vitro tumor killing ability of neutrophils in Example 3 of the present invention;

[0053] Figure 5 This is a comparison chart of the viability of neutrophils treated with a combined inducer and a single inducer in Example 4 of the present invention, wherein the Control group is untreated neutrophils;

[0054] Figure 6 The killing ability of the enhanced neutrophils of the present invention in Example 5 on different tumor cells, wherein HELA: human cervical cancer cells; A549: human non-small cell lung cancer cells; PANC-1: human pancreatic cancer cells; MCF7: human breast cancer cells; T24: human bladder transitional cell cancer cells; SW480: human colon cancer cells; A-375: human malignant melanoma cells; U251: human brain glioma cells;

[0055] Figure 7This is a bar graph of the tumor cell killing ability of enhanced neutrophils induced by neutrophils from different sources in Example 6, where young people are 18-25 years old, middle-aged people are 30-45 years old, and elderly people are over 60 years old;

[0056] Figure 8 It is a bar graph of the ability of the enhanced neutrophils of the present invention to recruit and activate T cells in Example 7;

[0057] Fig. 9 It is a bar graph of the recruitment and activation of NK cells by the enhanced neutrophils of the present invention in Example 7;

[0058] Fig.10 This is the survival curve of mice with brain glioma treated with the pharmaceutical composition of the present invention in Example 8;

[0059] Fig.11 This is the survival curve of mice with triple-negative breast cancer lung metastasis treated with the pharmaceutical composition of the present invention in Example 9;

[0060] Fig.12 This is the colony forming unit value of the brain of mice with invasive fungal infection treated with the pharmaceutical composition of the present invention in Example 10. DETAILED DESCRIPTION

[0061] In order to make the technical solutions and beneficial effects of the present invention more clearly understandable, the following is a detailed description by listing specific embodiments. The drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0062] In view of the limitation that neutrophils are only used as delivery carriers in neutrophil drug delivery systems and cannot effectively exert their anti-tumor effects, the inventors have proposed a method for constructing enhanced neutrophils after extensive research and experiments (see the principle for details). Figure 1), which specifically includes inducing and culturing neutrophils in a culture medium comprising any one or more of component A, component B and component C, wherein component A is a cytokine, including but not limited to IFN-γ, GM-CSF, G-CSF, IFN-β, IFN-α, lipopolysaccharides (LPS), interleukin (IL), monocyte chemoattractant protein-1 (MCP-1), Bacillus Calmette-Guérin (BCG), and cytokine. Calmette-Guérin, BCG), TNFα, β-glucan and other cytokines; B component is immune checkpoint inhibitors, including but not limited to Durvalumab, Avelumab, Atezolizumab, Ipilimumab, Tremelimumab / Tremelimumab; C component is anti-apoptotic drugs, including but not limited to broad-spectrum caspase inhibitors (Q-VD-Oph), deferoxamine (Deferoxamine, DFO), heat shock protein 70 (Heatshockprotein7, HSP70), N-acetylcysteine ​​(N-Acetylcysteine, NAC), necroptosis inhibitor Necrostatin-1 (Nec-1). The enhanced neutrophils obtained by the construction method provided by the present invention have the advantages of longer lifespan, stronger anti-tumor and immunomodulatory abilities.

[0063] The first object of the present invention is to provide a method for obtaining enhanced neutrophils, the method comprising: inducing and culturing neutrophils in a culture medium comprising any one or more of component A, component B and component C,

[0064] wherein the A component comprises one or more of IFNγ, GM-CSF, G-CSF, IFN-β, IFN-α, TNFα, LPS, IL1β, IL6, MCP-1, IL-23, BCG or β-glucan;

[0065] The B component includes one or more of durvalumab, avelumab, atezolizumab, ipilimumab or tesimumab;

[0066] The C component includes one or more of Q-VD-Oph, DFO, Hsp70, NAC or Nec-1. The present invention obtains enhanced neutrophils with long life span, high tumor killing activity and immune activation ability by optimizing the combined inducer components, the concentration of each component, and the action time. The construction method provided by the present invention prolongs the in vitro life span of neutrophils and facilitates quality control.

[0067] In certain embodiments, the method comprises: inducing culturing neutrophils in a culture medium comprising component A, component B, and component C.

[0068] In certain embodiments, the A component is selected from IFNγ and\or GM-CSF.

[0069] In certain embodiments, the B component is selected from durvalumab.

[0070] In certain embodiments, the C component is selected from Q-VD-Oph.

[0071] In certain embodiments, the concentration of component A is 1-200 ng / mL, for example, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, etc.

[0072] In certain embodiments, the concentration of component A is 1-100 ng / mL.

[0073] In certain embodiments, the concentration of component A is 1-50 ng / mL.

[0074] In certain embodiments, the concentration of component A is 1-30 ng / mL.

[0075] In certain embodiments, the concentration of component A is 20 ng / mL.

[0076] In certain embodiments, the concentration of component B is 0.1-10 μg / mL, for example, 0.5 μg / mL, 1.0 μg / mL, 1.5 μg / mL, 2.0 μg / mL, 2.5 μg / mL, 3.0 μg / mL, 3.5 μg / mL, 4.0 μg / mL, 4.5 μg / mL, 5.0 μg / mL, 5.5 μg / mL, 6.0 μg / mL, 6.5 μg / mL, 7.0 μg / mL, 7.5 μg / mL, 8.0 μg / mL, 8.5 μg / mL, 9.0 μg / mL, 9.5 μg / mL, 10 μg / mL, etc.

[0077] In certain embodiments, the concentration of the B component is 0.5-8 μg / mL.

[0078] In certain embodiments, the concentration of the B component is 0.5-5 μg / mL.

[0079] In certain embodiments, the concentration of the B component is 1-3 μg / mL.

[0080] In certain embodiments, the concentration of the B component is 2 μg / mL.

[0081] In certain embodiments, the concentration of component C is 1-100 μM, for example, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, 100 μM, etc.

[0082] In certain embodiments, the concentration of the C component is 10-80 μM.

[0083] In certain embodiments, the concentration of the C component is 10-60 μM.

[0084] In certain embodiments, the concentration of the C component is 20-60 μM.

[0085] In some embodiments, the concentration of the C component is 50 μM. The present invention optimizes the combined inducer components, the concentration of each component, and the action time to obtain enhanced neutrophils with a long life span, high tumor killing activity and immune activation ability. The construction method provided by the present invention prolongs the in vitro life span of neutrophils and facilitates quality control.

[0086] In the present invention, the culture medium is selected from Dulbecco's modified Eagle medium (DMEM) and RPMI-1640 culture medium. Commercially available DMEM and RPMI-1640 culture media well known in the art can be used.

[0087] In certain embodiments, the culture medium contains 0.5-5 wt % penicillin-streptomycin and 5-20 vol % serum or plasma.

[0088] In certain embodiments, the culture medium is 1640 medium or DMEM medium containing 0.5-2 wt % penicillin-streptomycin and 5-15 vol % serum.

[0089] In certain embodiments, the culture medium is 1640 medium or DMEM medium containing 1 wt % penicillin-streptomycin and 10 vol % serum.

[0090] In certain embodiments, the culture medium may also be a serum-free culture medium.

[0091] In certain embodiments, the method comprises: incubating neutrophils in the culture medium in an aerobic or anaerobic environment. The construction method provided by the present invention is simple, gentle and time-saving.

[0092] In certain embodiments, the incubation temperature is controlled to be 35-40°C, such as 36°C, 37°C, 38°C, 39°C, etc.

[0093] In certain embodiments, the incubation temperature is controlled at 36-40°C.

[0094] In certain embodiments, the incubation temperature is controlled at 37°C.

[0095] In certain embodiments, the incubation time is controlled to be 2-48 hours, such as 2 hours, 4 hours, 6 hours, 10 hours, 12 hours, 18 hours, 20 hours, 24 hours, 32 hours, 36 hours, 48 ​​hours, etc. The present invention obtains enhanced neutrophils with long life span, high tumor killing activity and immune activation ability by optimizing the combined inducer components, the concentration of each component and the action time.

[0096] In certain embodiments, the incubation time is controlled to be 2-36 hours.

[0097] In certain embodiments, the incubation time is controlled to be 4-24 hours.

[0098] In certain embodiments, the incubation time is controlled to be 4-18 hours.

[0099] In certain embodiments, the incubation is controlled to be performed under 5% CO2 conditions.

[0100] In certain embodiments, the neutrophils are derived from healthy individuals or tumor patients.

[0101] The construction method for obtaining enhanced neutrophils provided by the present invention is not restricted by the activity of donor neutrophils and can be derived from neutrophils of different ages, genders, healthy people or even patients, thus expanding the donor source.

[0102] The second object of the present invention is to provide an enhanced neutrophil obtained by the construction method as described in any one of the above items.

[0103] The enhanced neutrophils provided by the present invention can be further prepared into neutrophil combination drugs with chemotherapeutics, immunomodulators or anti-infective drugs, etc., which can not only respond to the chemokines released by the tumor microenvironment, but also quickly enrich and target the tumor in situ and metastatic tumors or infection sites, thereby increasing the enrichment of the loaded drug in the target site and improving its efficacy; at the same time, the neutrophils in the disease microenvironment can also play their own tumor killing, immune activation or bactericidal effects, increase the intratumoral infiltration and antitumor efficacy or bactericidal ability of T cells and NK cells; and finally play a combined therapeutic role, significantly improving the therapeutic effect of solid tumors and infections. At the same time, the doses of the two can be relatively reduced to better meet clinical needs.

[0104] The third object of the present invention is to provide a pharmaceutical composition, which comprises the enhanced neutrophils obtained by any of the construction methods described above or the enhanced neutrophils as described above.

[0105] In certain embodiments, the pharmaceutical composition further comprises a nanodrug.

[0106] In certain embodiments, the nanodrug is selected from anti-tumor drugs, immunomodulatory drugs and antibacterial drugs.

[0107] In certain embodiments, the anti-tumor drug is selected from paclitaxel, doxorubicin, 5-fluorouracil and camptothecin.

[0108] In certain embodiments, the immunomodulatory drug is selected from a STING modulator, a TGF-β modulator, and a HIF-1α modulator. The "super" neutrophil combination drug constructed by the present invention has a synergistic therapeutic effect (see the principle Figure 1 ).

[0109] In certain embodiments, the antibacterial drug is selected from amphotericin B, colistin, azithromycin, clavulanic acid, ampicillin, ciprofloxacin and vancomycin.

[0110] The fourth object of the present invention is to provide a method for preparing a pharmaceutical composition, the method comprising: mixing the enhanced neutrophils obtained by any of the construction methods described above or the enhanced neutrophils as described above with nanomedicines, and incubating them in an aerobic or anaerobic environment.

[0111] In certain embodiments, the incubation temperature is controlled to be 35-40°C, such as 36°C, 37°C, 38°C, 39°C, etc.

[0112] In certain embodiments, the incubation temperature is controlled at 36-40°C.

[0113] In certain embodiments, the incubation temperature is controlled at 37°C.

[0114] In certain embodiments, the incubation time is controlled to be 0.1-12 h, for example, 0.1 h, 0.2 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, etc.

[0115] In certain embodiments, the incubation time is controlled to be 0.2-10 h.

[0116] In certain embodiments, the incubation time is controlled to be 0.2-6h.

[0117] In certain embodiments, the incubation time is controlled to be 0.2-3 h.

[0118] In certain embodiments, the incubation is controlled to be performed under 5% CO2 conditions.

[0119] The fifth object of the present invention is to provide the use of the enhanced neutrophils obtained by any of the construction methods described above, the enhanced neutrophils described above, the pharmaceutical composition described above, or the pharmaceutical composition obtained by the preparation method described above in preventing and / or treating tumors, infections, or immune diseases.

[0120] Preferably, the tumor includes but is not limited to breast cancer, cervical cancer, non-small cell lung cancer, pancreatic cancer, bladder cancer, colon cancer, melanoma and brain glioma.

[0121] Preferably, the infection includes but is not limited to viral infection, bacterial infection, fungal infection, parasitic infection, bacterial infection or fungal infection associated with chemotherapy-induced agranulocytosis, invasive fungal infection, sepsis, new coronavirus infection, etc.

[0122] Preferably, the immune disease includes but is not limited to rheumatoid arthritis, systemic lupus erythematosus, allergic asthma, etc.

[0123] definition

[0124] In the present invention, the term "comprising" or "comprises" is used with respect to compositions, methods and corresponding components thereof that can be used in embodiments, and it is still an open term that includes unspecified elements, whether or not the elements are useful. It should be understood by those skilled in the art that, in general, the terms used herein are generally intended to be "open" terms (e.g., the term "comprising" should be interpreted as "including but not limited to", the term "having" should be interpreted as "at least having", the term "including" should be interpreted as "including but not limited to", etc.).

[0125] In the present invention, the term "cytokine" is a molecule produced by various cells (such as monocytes and macrophages) as a mediator of inflammatory processes. In a specific and preferred embodiment, the cytokine includes but is not limited to IFN-γ, GM-CSF, G-CSF, IFN-β, IFN-α, lipopolysaccharide (LPS), interleukin (IL), monocyte chemoattractant protein-1 (MCP-1), Bacillus Calmette-Guérin (BCG), TNFα, β-glucan, etc.

[0126] In the present invention, the term "immune checkpoint inhibitor" refers to a drug that blocks certain proteins produced by certain types of immune cells (such as T cells) and certain cancer cells. These proteins help control immune responses and can prevent T cells from killing cancer cells. When these proteins are blocked, the brakes on the immune system are released, and T cells are better able to kill cancer cells. Examples of immune checkpoint inhibitors include, but are not limited to, PD-1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors, TIM-3 inhibitors, BTLA inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, B7-1 inhibitors, B7-2 inhibitors, galectin-9 inhibitors, and HVEM inhibitors. Immune checkpoint inhibitors can be small molecules, peptides, proteins (such as antibodies), nucleic acids, etc.

[0127] In the present invention, "PD-L1 inhibitor" refers to an antibody or other molecule that inhibits the function of programmed death ligand 1 (PDL1). In a specific embodiment, the antibody is Durvalumab, Avelumab, or Atezolizumab.

[0128] In the present invention, "CTLA4 inhibitor" refers to an antibody or other molecule that inhibits the function of cytotoxic T lymphocyte-associated antigen 4 (CTLA4). In a specific embodiment, the antibody is Ipilimumab and Tremelimumab / Tremelimumab.

[0129] In the present invention, the term "TGF-β" is a type of growth factor, which is produced in almost all cells such as kidneys, bone marrow, platelets, etc. There are five subtypes of TGF-β (β1 to β5). It is known that TGF-β promotes the proliferation of osteoblasts and the synthesis and proliferation of connective tissues such as collagen, and has an inhibitory effect on the proliferation of epithelial cells and osteoclasts. The term "TGF-β modulator" is a substance that prevents or inhibits, for example, TGF-β from binding to TGF-β receptors, and is an agent that binds to TGF-β to form a complex for neutralizing TGF-β activity. Including but not limited to, one or more of A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494 and SJN-2511.

[0130] In the present invention, the term "HIF-1α" refers to hypoxia-inducible factor. In cells, the HIF signaling cascade is affected by hypoxia. Under hypoxia, cells usually continue to differentiate. However, hypoxia promotes angiogenesis, which is very important for the vascular system in embryos and cancer tumors. The term "HIF-1α modulator" refers to a compound or composition that can be used to inhibit the activity of HIF-1α. Including but not limited to one or more of BAY87-2243, KC7F2, LW 6, PX-4782HCI.

[0131] In the present invention, the term "STING" is an abbreviation for "stimulator of interferon genes", and "STING modulator" refers to a substance that activates or inhibits STING receptors in vitro or in vivo, including but not limited to cyclic dinucleotide (CDN) or non-cyclic dinucleotide agonists.

[0132] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, cell culture, and related fields in the technical field. These techniques are well described in the literature, see Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (P. M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (P. M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999; INMOLECULARBIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0133] Unless otherwise stated, the materials, reagents, etc. used in the present invention can be obtained from commercial sources or synthesized according to known methods unless otherwise specified.

[0134] Example 1 Effects of different concentrations of a single inducer on the viability of neutrophils

[0135] Human neutrophils were incubated with different concentrations of a single inducer in 1640 medium for 8 h. The single inducers at different concentrations included:

[0136] Inducer 1 (IFNγ): 0, 10, 20, 30, 50, 100 ng / mL;

[0137] Inducer 2 (GM-CSF): 0, 10, 20, 30, 50, 100 ng / mL;

[0138] Inducer 3 (TNFα): 0, 10, 20, 30, 50, 100 ng / mL;

[0139] Inducer 4 (durvalumab): 0.1, 0.5, 1, 2, 3, 5, 8, 10 μg / mL;

[0140] Inducer 5 (avelumab): 0.1, 0.5, 1, 2, 3, 5, 8, 10 μg / mL;

[0141] Inducer 6 (Q-VD-Oph): 1, 10, 20, 50, 60, 80, 100 μM;

[0142] Inducer 7 (Nec-1): 1, 10, 20, 50, 60, 80, 100 μM;

[0143] The culture conditions were 37°C and 5% CO2. After removing the inducer and continuing the culture for 24 hours, the apoptosis of neutrophils was detected by flow cytometry. The cell viability results are shown in Figure 2 .

[0144] Figure 2 The results showed that among the inducers 1-3, which are all cytokines, both inducers 1 and 2 could significantly prolong the life span of neutrophils and the effect was better than that of inducer 3, with the concentration of 20 ng / mL having the best effect. Among the inducers 4-5, which are all PD-L1 inhibitors, both could increase the viability of neutrophils, with 2 μg / mL having the best effect. Among the inducers 6-7, which are all apoptosis inhibitors, both could increase the viability of neutrophils, with 50 μM having the best effect.

[0145] Example 2 demonstrates that combined treatment with inducers can increase the activity of neutrophils

[0146] The composition of combined inducer 1 is: 20 ng / mL IFNγ+20 ng / mL GM-CSF.

[0147] The composition of combined inducer 2 was: 2 μg / mL durvalumab + 50 μM Q-VD-Oph.

[0148] The components of combined inducer 3 are: 20 ng / mL IFNγ+2 μg / mL durvalumab+50 μM Q-VD-Oph.

[0149] The components of combined inducer 4 are: 20 ng / mL GM-CSF+2 μg / mL durvalumab+50 μM Q-VD-Oph.

[0150] The components of combined inducer 5 are: 20 ng / mL IFNγ+20 ng / mL GM-CSF+2 μg / mL durvalumab+50 μM Q-VD-Oph.

[0151] The composition of combined inducer 6 is: 20ng / mL IFNγ+50μM Q-VD-Oph. Human neutrophils were incubated with different combined inducers in 1640 medium for 8h, and the culture conditions were 37℃, 5% CO2. After removing the inducer and continuing to culture for 24h, neutrophil apoptosis was detected by flow cytometry. Cell viability results are shown in Figure 3 .

[0152] Figure 3 The results showed that neutrophils treated with the combined inducer 3 had a longer lifespan in vitro. In particular, after the inducer was removed, the neutrophils could survive in vitro for at least 24 hours, and the cell viability was above 90%.

[0153] Example 3 demonstrates that combined inducer treatment can enhance the tumor killing ability of neutrophils

[0154] Human neutrophils were incubated with the above-mentioned different combined inducers in 1640 culture medium for 8 hours, and the culture conditions were 37°C and 5% CO2. Subsequently, the cells were washed three times with PBS, centrifuged at 1800 rpm, 4°C for 3 minutes, and the supernatant was discarded to obtain "super" neutrophils. The "super" neutrophils were co-incubated with human breast cancer cells MCF7 at a target-effect ratio of 5:1 at 37°C and 5% CO2 for 24 hours, wherein MCF7 cells were stably transfected with lentivirus to express mcherry. After washing twice with PBS, the fluorescence of mcherry was detected by a microplate reader. The tumor killing ability was calculated as follows: [(fluorescence value of the experimental well - fluorescence value of the blank well) / (fluorescence value of the control well - fluorescence value of the blank well)] × 100%, and the results are shown in Figure 4 .

[0155] Figure 4 It was shown that the combined inducer 3 could more significantly enhance the tumor killing ability of neutrophils.

[0156] Example 4 demonstrates that combined inducers can enhance neutrophil viability more than single inducers

[0157] Human neutrophils were incubated in 1640 medium containing 20 ng / mL IFNγ, 2 μg / mL Durvalumab, 50 μM Q-VD-Oph, or a combination of the three for 8 h at 37°C and 5% CO2. After the inducer was removed and cultured for 24 h, neutrophil apoptosis was detected by flow cytometry. The results are shown in Figure 5 .

[0158] Figure 5 It was shown that neutrophils treated with the combined inducer had a longer in vitro lifespan than those treated with a single inducer, and could survive in vitro for at least 24 hours with 90% cell viability.

[0159] Example 5 demonstrates that the tumor killing ability of neutrophils treated with combined inducers is stronger than that of neutrophils treated with single inducers

[0160] Human neutrophils were incubated in 1640 medium containing 20ng / mL IFNγ or 2μg / mL Durvalumab or 50μM Q-VD-Oph or a combined inducer of the three for 8 hours at 37°C and 5% CO2. The cells were then washed three times with PBS, centrifuged at 1800rpm, 4°C for 3 minutes, and the supernatant was discarded to obtain "super" neutrophils. The "super" neutrophils were co-incubated with various tumor cells at a 5:1 effector-target ratio at 37°C and 5% CO2 for 24 hours. The tumor cells were all transfected with lentivirus to stably express mcherry. After washing twice with PBS, the fluorescence of mcherry was detected by an enzyme plate reader. The tumor killing ability was calculated as follows: [(fluorescence value of the experimental well - fluorescence value of the blank well) / (fluorescence value of the control well - fluorescence value of the blank well)]×100%, and the test results are shown in Figure 6 . Figure 6 It was shown that the tumor killing ability of neutrophils treated with combined inducers was stronger than that of neutrophils treated with single inducers.

[0161] Example 6 proves that this construction method can reshape neutrophils from different sources into neutrophils with stronger anti-tumor ability

[0162] Blood from volunteers of different ages and health conditions was collected and neutrophils were obtained by density gradient centrifugation. Human neutrophils were incubated in 1640 medium containing a combined inducer (20ng / mL IFNγ+2μg / mL durvalumab+50μM Q-VD-Oph) for 8 hours at 37°C and 5% CO2. Subsequently, the cells were washed three times with PBS, centrifuged at 1800rpm, 4°C for 3 minutes, and the supernatant was discarded to obtain "super" neutrophils. The "super" neutrophils were co-incubated with a variety of tumor cells at a target-effect ratio of 5:1 at 37°C and 5% CO2 for 24 hours. The tumor cells were all transfected with lentivirus to stably express mcherry. After washing twice with PBS, the fluorescence of mcherry was detected by an enzyme plate reader. The tumor killing ability was calculated as follows: [(fluorescence value of the experimental well-fluorescence value of the blank well) / (fluorescence value of the control well-fluorescence value of the blank well)]×100%, and the test results are shown in Figure 7 . Figure 7 It was shown that this construction method can enhance the tumor-killing ability of neutrophils from different sources.

[0163] Example 7 proves that "super" neutrophils can effectively activate the body's immune response

[0164] An orthotopic breast cancer mouse model was established by injecting 5×10 6 Neutrophils or "super neutrophils" induced by 20ng / mL IFNγ+2μg / mL durvalumab+50μM Q-VD-Oph were placed in mice. After 24 hours, the mouse tumor tissue was removed, washed with Hanks buffer, and cut into pieces with scissors with a volume of less than 1mm. 3 The cells were resuspended in Hanks buffer and washed three times, and then stained with flow cytometry antibodies. The proportion of T cells and NK cells in the tumor site, as well as the proportion of T cells and NK cells with high tumor killing activity (IFNγ+, GzmB+) in the tumor site were analyzed by flow cytometry. The results are shown in Table 1. Figure 8 and Fig. 9 . Figure 8-9 It was shown that compared with untreated neutrophils, "super" neutrophils have significantly stronger recruitment and activation effects on T cells and NK cells.

[0165] Example 8 proves that "super" neutrophil combination drugs can synergistically treat brain glioma

[0166] 1×10 induced with combined inducer (20 ng / mL IFNγ+2 μg / mL durvalumab+50 μM Q-VD-Oph) prepared according to Example 2 5 Super neutrophils (5×10 / mL) were mixed with 50 μg / mL paclitaxel albumin nanoformulation, incubated at 37°C, 5% CO2 for 50 min, then centrifuged at 1800 rpm, 4°C for 3 min, and the supernatant was discarded to obtain the "super" neutrophil combination drug. A mouse brain glioma model was established, and normal saline (200 μL), paclitaxel albumin nanoformulation (10 mg / kg, 200 μL), neutrophils (5×10 / mL) were injected into the tail vein. 6 , 200 μL), “super” neutrophils (5×10 6 , 200 μL) and “super” neutrophil combination drugs (5×10 6 , 200 μL) in mice, and the results are shown in Fig.10 . Fig.10 The results showed that the "super" neutrophil combination drug can significantly prolong the survival of mice and has a better therapeutic effect on brain glioma.

[0167] Example 9 demonstrates that "super" neutrophil combination drugs can synergistically treat triple-negative breast cancer lung metastasis

[0168] 6×10 cells induced with the combined inducer (20 ng / mL IFNγ+2 μg / mL durvalumab+50 μM Q-VD-Oph) prepared in Example 2 were 6 Super neutrophils were mixed with 20 μg / mL TGFβ inhibitor SB525334 liposomes, incubated at 37°C, 5% CO2 for 50 min, then centrifuged at 1800 rpm, 4°C for 3 min, and the supernatant was discarded to obtain the "super" neutrophil combination drug. A spontaneous triple-negative breast cancer lung metastasis model was established in mice, and normal saline (200 μL), TGFβ inhibitor liposomes (1 mg / kg, 200 μL), neutrophils (6×10 6 , 200 μL), “super” neutrophils (6×10 6 , 200 μL) and “super” neutrophil combination drugs (6×10 6 , 200 μL) in mice, and the results are shown in Fig.11 . Fig.11 The results showed that the "super" neutrophil combination drug can significantly prolong the survival of mice and has a better therapeutic effect on breast cancer lung metastasis.

[0169] The "super" neutrophil combination drug of the present invention can effectively treat in situ tumors and metastatic tumors, and can exert a better tumor treatment effect compared with free drugs, untreated neutrophils and simple "super" neutrophils.

[0170] Example 10 demonstrates that "super" neutrophil combination drugs can synergistically treat fungal infections

[0171] 1.2×10 7 100 / mL super neutrophils were mixed with 2 μg amphotericin B nanoparticles and incubated at 37°C, 5% CO2 for 50 min, then centrifuged at 1800 rpm, 4°C for 3 min, and the supernatant was discarded to obtain the "super" neutrophil combination drug. A mouse fungal infection model was established, that is, C57BL6 mice were injected with 1×10 5 Cryptococcus neoformans (H99) was inoculated with normal saline (250 μL), amphotericin B (2 μg / mL, 250 μL), and neutrophils (3×10 6 / mL, 250μL), "super" neutrophils (3×10 6 , 250 μL) and “super” neutrophil combination drugs (3×10 6, 250 μL) in mice, and the results are shown in Fig.12 . Fig.12 The results showed that the "super" neutrophil combination drug can significantly reduce the amount of bacteria in brain tissue and has a better anti-infection effect.

[0172] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. A method for obtaining enhanced neutrophils, characterized in that: The method comprises: inducing and culturing neutrophils in a culture medium comprising any one or more of component A, component B and component C, wherein the A component comprises one or more of IFNγ, GM-CSF, G-CSF, IFN-β, IFN-α, TNFα, LPS, IL1β, IL6, MCP-1, IL-23, BCG or β-glucan; The B component includes one or more of durvalumab, avelumab, atezolizumab, ipilimumab or tesimumab; The C component includes one or more of Q-VD-Oph, DFO, Hsp70, NAC or Nec-1.

2. The construction method according to claim 1, characterized in that: The A component is selected from IFNγ and / or GM-CSF; Preferably, the B component is selected from durvalumab; Preferably, the C component is selected from Q-VD-Oph; Preferably, the concentration of component A is 1-200 ng / mL, preferably 1-100 ng / mL, more preferably 1-50 ng / mL, particularly preferably 1-30 ng / mL, and most preferably 20 ng / mL; Preferably, the concentration of component B is 0.1-10 μg / mL, preferably 0.5-8 μg / mL, more preferably 0.5-5 μg / mL, particularly preferably 1-3 μg / mL, and most preferably 2 μg / mL; Preferably, the concentration of the C component is 1-100 μM, preferably 10-80 μM, more preferably 10-60 μM, particularly preferably 20-60 μM, and most preferably 50 μM.

3. The construction method according to claim 1, characterized in that: The culture medium is 1640 culture medium or DMEM culture medium.

4. The construction method according to claim 1, characterized in that: The method comprises: incubating neutrophils in the culture medium in an aerobic or anaerobic environment; Preferably, the incubation temperature is controlled to be 35-40°C, preferably 36-40°C, more preferably 37°C; Preferably, the incubation time is controlled to be 2-48 h, preferably 2-36 h, more preferably 4-24 h, and most preferably 4-18 h; Preferably, the incubation is controlled to be carried out under 5% CO2 conditions; Preferably, the neutrophils are derived from healthy people or tumor patients.

5. An enhanced neutrophil obtained by the construction method according to any one of claims 1 to 4.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the enhanced neutrophils obtained by the construction method according to any one of claims 1 to 4 or the enhanced neutrophils according to claim 5.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition also includes nanomedicine; Preferably, the nanomedicine is selected from anti-tumor drugs, immunomodulatory drugs and antibacterial drugs; Preferably, the anti-tumor drug is selected from paclitaxel, doxorubicin, 5-fluorouracil and camptothecin; Preferably, the immunomodulatory drug is selected from STING modulators, TGF-β modulators and HIF-1α modulators; Preferably, the antibacterial drug is selected from amphotericin B, colistin, azithromycin, clavulanic acid, ampicillin, ciprofloxacin and vancomycin.

8. A method for preparing a pharmaceutical composition, characterized in that: The method comprises: mixing the enhanced neutrophils obtained by the construction method of any one of claims 1 to 4 or the enhanced neutrophils according to claim 5 with a nano drug, and then incubating them in an aerobic or anaerobic environment.

9. The preparation method according to claim 8, characterized in that: Controlling the incubation temperature to be 35-40°C, preferably 36-40°C, more preferably 37°C; Preferably, the incubation time is controlled to be 0.1-12 h, preferably 0.2-10 h, more preferably 0.2-6 h, and most preferably 0.2-3 h; Preferably, the incubation is controlled to be carried out under 5% CO2 conditions.

10. Use of the enhanced neutrophils obtained by the construction method according to any one of claims 1 to 4, the enhanced neutrophils according to claim 5, the pharmaceutical composition according to claim 6 or 7, or the pharmaceutical composition obtained by the preparation method according to claim 8 or 9 in preventing and / or treating tumors, infections, and immune diseases.

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