CN110575537A

CN110575537A - Composition of DC vaccine and NKG2A antagonist and application of composition in resisting breast cancer or liver cancer

Info

Publication number: CN110575537A
Application number: CN201910842048.XA
Authority: CN
Inventors: 刘慧宁
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-09-06
Filing date: 2019-09-06
Publication date: 2019-12-17

Abstract

The invention provides a composition of a DC vaccine and an NKG2A antagonist and application of the composition in resisting breast cancer or liver cancer. The DC vaccine in the composition is different from the early traditional DC vaccine loaded with the tumor cell whole antigen, has a specific target antigen, can more accurately treat tumors, and reduces the killing side effect on normal cells; the composition also contains a novel immune checkpoint inhibitor NKG2A antagonist, so that tumor cells with CD94-NKG2A/HLA-E receptor-ligand on the surface can be recognized and killed by NK cells and T cells, the antigen presenting function of dendritic cells is enhanced, part of cells which are not presented with antigens by the DC vaccine are captured by an immune system again, the treatment effect on breast cancer and liver cancer is enhanced, the effect is quicker and the duration is longer than that of the common DC vaccine, and the immunotherapy process is promoted.

Description

composition of DC vaccine and NKG2A antagonist and application of composition in resisting breast cancer or liver cancer

Technical Field

the invention relates to the technical field of biological immunity, in particular to a composition of a DC vaccine and an NKG2A antagonist and application thereof in resisting breast cancer or liver cancer.

Background

Breast cancer is a malignant tumor that occurs in mammary gland epithelial tissue, with 99% occurring in women. The number of new breast cancer cases worldwide stably lives the first of female malignant tumors every year, the incidence of breast cancer in China in recent years is high year by year, and the number of breast cancer cases is 43/10 ten thousands in 2017. Breast cancer is currently clinically classified into 4 types: luminal type A (ER +/PR +, HER-2+), Luminal type B (ER +/PR +, HER-2+), HER-2+ -type (ER-/PR-/HER-2+), and triple negative breast cancer (ER-/PR-/HER-2 +). The current situation of triple negative breast cancer accounts for 15% -23.8% of all breast cancer pathological types, about 40-70 thousands of patients exist in China, the onset age of the patients is early, the cell differentiation is poor, the patients are highly invasive, the transfer is easy, and the recurrence is easy; in addition, the heterogeneity is high, the curative effect is poor, and the drug resistance is easy to generate; the disease-free survival period is short, the overall survival rate is poor, the disease mutation frequency is high, and the disease mechanism is difficult to study; at present, a few medicines for treating breast cancer exist, and no approved targeted treatment means and specific diagnostic reagent exist in China. There is therefore an urgent need in the art to develop drugs for the treatment of breast cancer.

the statistics in 2018 report that liver cancer is the fifth cancer with the world morbidity rate and the third cancer with the mortality rate, new liver cancer and death cases in China account for more than half of the world, and the liver cancer is the second highest cancer with the mortality rate in China. Liver cancer can be divided into primary and secondary categories. The primary liver malignant tumor originates from the epithelium or mesenchymal tissue of the liver, the former is called primary liver cancer, which is a highly-developed malignant tumor with great harm in China; the secondary liver cancer is named sarcoma, and refers to the invasion of malignant tumor of multiple organ origins in the whole body to the liver. The etiology and the exact molecular mechanism of primary liver cancer are not completely clear at present, the pathogenesis of the primary liver cancer is influenced by various factors, and Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) infection, aflatoxin, drinking water pollution, alcohol, liver cirrhosis, sex hormone, nitrosamine substances and the like are related to the pathogenesis of the liver cancer. At present, aiming at the serious defects of medicines and means for liver cancer, the improvement of the medical level is urgently needed to help liver cancer patients get rid of the suffering of diseases.

The body performs immune response, has the function of identifying and eliminating antigenic foreign bodies, and is a system consisting of immune organs, immune cells and immune molecules, which is called an immune system. The immune system plays an important role in identifying and eliminating foreign invaded pathogenic microorganism antigens, tumor cells mutated in vivo, senescent cells, dead cells or other harmful components and the like, and is coordinated with other systems of the organism to jointly maintain the functions of stable environment and physiological balance in the organism.

Dendritic Cells (DCs) are the most powerful antigen presenting cells in the immune system, recognizing bacteria, virus-infected cells, self-cancerous cells, etc. invading the body, and presenting antigen signals to other immune cells through MHC molecules of the major histocompatibility complex, activating naive T cells, initiating, regulating and maintaining immune responses. Mature dendritic cells are less than 1% in number of peripheral blood mononuclear cells, but have abundant antigen-presenting molecules (e.g., MHC-I and MHC-II), co-stimulatory factors (CD80/B7-1, CD86/B7-2, CD40, etc.) and adhesion factors (ICAM-1, ICAM-2, ICAM-3, LFA-1, LFA-3), etc., on their surface. Dendritic cells are important natural immune cells and professional antigen presenting cells, and play a key role in activating immune response of an organism, maintaining autoimmune tolerance, and connecting natural immunity with acquired immunity. In addition, co-stimulatory molecules (CD80/B7-1, CD86/B7-2, CD40 and the like) highly expressed on the surface of dendritic cells provide a second signal necessary for T cell activation, and the dendritic cells can directly supply CD8 with the co-stimulatory molecules⁺T cells present antigenic peptides, at CD4⁺Presentation of CD8 under T cell help⁺T cells are activated, and the activated dendritic cells can secrete a large amount of IL-12, IL-18, Chemotactic factors (CCK) and the like to activate the proliferation of the T cells; finally, the dendritic cells can also activate perforin P granzyme B and FasL/Fas mediated ways to enhance natural killer cell toxicity so as to enhance the anti-tumor immune response of organisms and eliminate tumors. Dendritic cells can be used as a natural immunologic adjuvant to improve the immunity of the body by secreting various cytokines, and can also enhance the immune response of various vaccines and vaccine adjuvants (such as oligonucleotides and the like), and the dendritic cells with relevant antigen information and the vaccine function are generally called dendritic cell vaccines (DC vaccines).

Natural Killer (NK) cells are a type of cytotoxic lymphocytes, with rapid onset of killing action (4 hours in vivo) after acting on target cells, and are called natural killer activity because NK cells have no MHC restriction for killing activity and do not rely on antibodies. The target cells of the NK cells mainly comprise tumor cells, virus infected cells, certain self tissue cells, parasites and the like, are related to the occurrence and development of anti-tumor, anti-virus infection and immunoregulation, hypersensitivity reaction and autoimmune diseases of the body, can recognize the target cells and secrete killing media (such as perforin, NK cytotoxic factors, TNF and the like), and are important members of the immune system. T cells are the main components of lymphocytes, have various biological functions, such as direct killing of target cells, assistance of B cells in antibody production, participation in mediated cellular immunity, play an important role in eliminating pathogens and tumors, and maintaining the steady state and health of organisms, and are important components of the immune system.

To prevent hyperfunction from damaging self-organs and tissues, the immune system utilizes immune checkpoints (Checkpoint) to regulate immune response and maintain systemic balance. In normal conditions, T lymphocytes such as dendritic cells, macrophages, natural killer cells (NK cells), CD4, and CD8 in vivo can distinguish normal tissues, cancer tissues, infected bacteria and viruses, and foreign tissues by recognizing immune Checkpoint (Checkpoint) ligands on the surface of normal cells, thereby preventing false attack on self-cells. However, many cancer cells derive checkpoint molecules that allow them to evade the immune system, for example, CTLA-4, PD-1, etc. protein molecules exist on the surface of T lymphocytes, and some cancer cells express PD-L1, which binds to PD-1 of T lymphocytes to inhibit the function of T lymphocytes, thereby preventing T cells from killing cancer cells. Immune checkpoint inhibitors (monoclonal checkpoint inhibitors) developed in recent years with monoclonal antibodies can effectively relieve the inhibition of cancer cells on T lymphocytes, prevent the immune escape of cancer cells, and kill cancer cells.

NKG2A is a recently identified novel immune checkpoint inhibitor molecule belonging to the C-type lectin superfamily, with a relative molecular mass of 43000, and consisting of 233 amino acids, and is a cell surface receptor, in CD56hi NK cells and CD8⁺The alpha beta T cell surface is expressed, and the alpha beta T cell surface has 135 amino acids outside the cell, has a Carbohydrate-recognizing domain CRD (Carbohydrate-recognition domain, usually consisting of 115-130 amino acids, containing 2-3 disulfide bonds, and having 2-3N-linked glycosylation sites, and the ligand recognition process is Ca²⁺Dependent)And can be combined with CD94 to form a dimer complex to recognize and bind to MHC-I molecule HLA-E. CD94 is also known as Kp43, belongs to the C-type lectin superfamily, is expressed on the surface of most NK cells, γ δ T cells and α β T cells, has a relative molecular mass of 30000, is a type II transmembrane molecule, has a similar structure to NKG2A, and contains CRD. The non-classical MHC-I molecule HLA-E is a main ligand of CD94/NKG2A, can inhibit TCR gamma/delta-CD 3-mediated cytolytic activity, plays a key role in the process of inhibitory signal transduction, is mainly expressed in most tissues of human bodies, and can be combined with signal peptides from the classical MHC-I molecules HLA-A, HLA-B, HLA-C and HLA-G to form a stable conformation among proteins to influence immune response.

The combination of NKG2A-CD94 and HLA-E can lead to tyrosine phosphorylation of cytoplasmic immunoreceptors, transduce inhibitory signals into cells, play a role in negative regulation on killing of NK cells and T cells, and inhibit the functions of the T cells and the NK cells. Like other immune checkpoint receptor-ligands (e.g., PD-1/PD-L1), CD94-NKG2A/HLA-E pairs of immune checkpoints are also utilized by cancer cells to evade attack by the immune system. French researchers used cetuximab (cetuximab), an EGFR Inhibitor, and the Anti-NKG2A antibody, monoclonal (a first-in-class humanized IgG4 antibody that prevents the binding of NKG2A-CD94 to HLA-E) in combination to treat 31 patients with squamous cell head and neck cancer (SCCHN), with good efficacy over cetuximab alone, better drug tolerance and no serious side effects (PascaleAndr et al. In addition, researches report that in breast cancer and liver cancer, tumor cells abnormally and highly express HLA-E, so that the inhibition effect of NKG2A-CD94 receptors on the surfaces of NK cells and T cells cannot be effectively exerted, and cancer cells are promoted to avoid the recognition and killing of the NK cells and the T cells. Therefore, the NKG2A antagonist is a potential immune checkpoint inhibitor and can promote the anti-tumor immunity of T cells and NK cells.

The defects and shortcomings of the prior art are as follows:

1. At present, DC vaccine is mostly prepared by adopting the technology of loading dendritic cells such as tumor cell lysate and the like or fusing the tumor cells and the dendritic cells. Since most of the cells in the tumor are derived from the human body, and some tumor cells express molecules on the surface which can not be recognized by the immune system (such as immune check points), the immune response caused by most DC vaccines can not kill the tumor successfully, and the treatment effect is limited.

2. The targeted antigen of most DC vaccines is expressed on cancer cells and a few normal cells, and the immune system has a certain probability of attacking the normal cells.

3. The existing DC vaccine is slow in effect and longer in treatment process compared with other immunotherapy such as CAR-T and the like because the DC is required to present antigen to an immune system, and the immunity is gradually started.

4. in addition, in vivo, many tumor cells can secrete a plurality of cytokines for inhibiting dendritic cell maturation, so that the number of dendritic cells existing in a tumor part is relatively small, the expression of MHC molecules on the surface of the dendritic cells is influenced, the antigen presentation function of the dendritic cells is weakened, the anti-tumor immune response induced by the dendritic cells cannot play a remarkable treatment effect in a host, and most of the anti-tumor immune response can only achieve partial relief.

5. At present, the clinical application still lacks of very effective reagents and medicines aiming at the breast cancer and the liver cancer, and a new way for solving or partially solving the pain of the breast cancer or the liver cancer patient is urgently needed to be developed and searched.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a composition of a DC vaccine and an NKG2A antagonist and application of the composition in resisting breast cancer or liver cancer.

In order to achieve the above objects, the present invention provides a composition of DC vaccine and NKG2A antagonist for treating breast cancer and liver cancer, i.e. a composition of DC vaccine and NKG2A antagonist as novel immune checkpoint inhibitor in a specific content ratio for treating tumor. Extracting and inducing dendritic cells, culturing and maturing in vitro, loading specific breast cancer or liver cancer antigens to dendritic cells extracted from a patient, enabling the surfaces of the dendritic cells to be provided with corresponding antigens, forming a composition with an NKG2A antagonist, and infusing the composition back to the patient to stimulate natural immunity, induce NK cells and T lymphocytes to generate an acquired immune response, and strengthen the immune effect of the NK cells and the T lymphocytes to treat breast cancer or liver cancer.

The dendritic cell function and the whole immune system of most breast cancer or liver cancer patients are damaged, in the process of most tumors, if a large number of mature or immature dendritic cells which can normally play the roles of antigen presentation and immune adjuvant exist to start acquired immunity, and NK cells and T cells can normally identify and kill tumor cells, the proliferation growth and the diffusion of the tumor cells can be completely inhibited and eliminated to a certain extent, and side effects like chemotherapeutic drugs cannot occur, so that the tumor cell proliferation and the tumor cell proliferation are safer and more effective. The invention provides a method for enhancing the immune response of an organism, which can effectively treat breast cancer and liver cancer, can efficiently inhibit the growth of tumor cells for a long time and eliminate the tumor cells by improving the immunity of the organism.

The present invention comprises providing an NKG2A antagonist in combination with a DC vaccine loaded with a tumor specific antigen to form a composition for enhancing the immune competence of the body against breast and liver cancer.

In some aspects, the subject treated according to embodiments is a mammalian subject. The subject is a primate, e.g., human, monkey; or a non-human mammal, such as a mouse, rabbit, dog, cat, horse, cow, goat, pig or other animal.

In some embodiments, the antigen-primed dendritic cell population is an immunogenic composition, the dendritic cell population having been loaded with a corresponding antigen. Specific antigens include polypeptide antigens, or nucleic acids (DNA or RNA) and proteins encoding antigens, tumor cell surface specific antigens.

In other aspects of the invention, the application of the composition of the DC vaccine and the NKG2A antagonist in resisting breast cancer or liver cancer is provided. The DC vaccine loaded with tumor specific antigens, in some particular embodiments a DC vaccine loaded with only relevant first antigens, in other particular embodiments a DC vaccine loaded with relevant second antigens, or even multiple antigens simultaneously, in some particular embodiments the DC vaccine loaded with first specific antigens is applied simultaneously with dendritic cells loaded with second antigens, dendritic cells loaded with third antigens or more.

In certain aspects, the DC vaccine may comprise a first adjuvant (Ploy (I: C), etc.) or other therapeutic co-adjuvant cytokines such as IL-2, TNF- α, IL-12, etc.

Wherein said NKG2A antagonists include NKG2A antibodies and other small molecule antagonists. The NKG2A antibody is in some particular embodiments a recombinant monoclonal antibody (human, humanized or deimmunized), in some particular embodiments a polyclonal antibody; antagonists include inhibitory nucleic acid RNAs (e.g., small interfering RNAs (sirnas), short hairpin RNAs (shrnas), micrornas (mirnas), double-stranded RNAs (dsrnas), and other chemically synthesized small molecule inhibitors (e.g., oligonucleotides).

wherein the NKG2A antibody is a monoclonal or polyclonal antibody, the NKG2A binding antibody is IgA, IgG (e.g., IgG1, IgG2, IgG3 or IgG4), a genetically modified IgG isotype, or an antigen-binding fragment thereof in some particular embodiments; in some particular embodiments are Fab ', F (ab ')2, F (ab ')3, monovalent scFv, bivalent scFv, bispecific, nanobody, or single domain antibody; in other specific embodiments are human, humanized or deimmunized antibodies.

In embodiments, the NKG2A antagonist and the DC vaccine are co-administered in a composition formed in specified amounts, the DC vaccine and NKG2A antagonist composition being administered three times, each one or two weeks apart; the cell amount of dendritic cells in each time of cell return is 5 x 10⁶-5*10⁸In each case, the NKG2A antagonist (preferably NKG2A antibody, monoclonal) is injected at a dose of 10 ug/kg-10 mg/kg (preferably 10ug/kg-1 mg/kg).

in some aspects, administering the DC vaccine and NKG2A antagonist combination therapy modality to the subject comprises intravenous, intradermal, intratumoral, intramuscular, intraperitoneal, subcutaneous, or topical administration.

The invention has the following advantages and beneficial effects:

compared with the traditional DC vaccine, the antigen-loaded DC vaccine and the composition of the NKG2A antagonist which are used together for treating the breast cancer and the liver cancer have the following advantages:

1. Specifically targeting cancer cells. The selected antigen is specifically expressed on the surface of breast cancer or liver cancer cells through comprehensive analysis such as literature data comparison, high performance liquid chromatography, gene sequencing and the like, and after the antigen is identified by dendritic cells, an in vivo immune system only aims at the cancer cells, so that the safety and the effectiveness are obviously improved.

2. The antigen loaded DC vaccine and the novel anti-immune suppression check point NKG2A antagonist composition are combined, and compared with the antigen loaded DC vaccine or the novel anti-immune suppression check point NKG2A antagonist, the antigen loaded DC vaccine composition can effectively enhance the immune effect capability of an organism and improve the duration of immune response.

3. the composition of the DC vaccine and a novel immune check point inhibitor NKG2A can synergistically promote the interaction among dendritic cells, NK cells and T cells and enhance the function of immune cells, the DC vaccine can generate a large amount of dendritic cells, the dendritic cells can secrete a large amount of cytokines to promote the immune function of the NK cells and the T cells and enhance the killing effect of an organism on breast cancer or liver cancer cells besides presenting antigen signals to the T cells, the NKG2A antagonist can promote the interaction among the NK cells, the T cells and the cancer cells, so that tumor cells with CD94-NKG2A/HLA-E receptor-ligand on the surface can be recognized and killed by the NK cells and the T cells, part of cells which are not presented with antigens by the DC vaccine are captured by an immune system again, the recognition effect of the dendritic cells on the tumor cells is further enhanced, and the immune effect of the organism is enhanced, reduces the side effect of killing normal cells, has quicker effect and longer duration than the prior DC vaccine, and promotes the progress of immunotherapy.

Drawings

FIG. 1 shows the morphology of mouse dendritic cells under an optical microscope;

FIG. 2 flow-assay of the number of CD11c positive cells in the bone marrow of Balb/c mice;

FIG. 3 flow assay of expression of relevant cytokines;

FIG. 4. average percentage of dendritic cells for a particular surface marker molecule;

FIG. 5 Effect of DC vaccine in combination with anti-NKG2A antibody on growth of mouse breast cancer tumor;

FIG. 6 lymphocyte killing assay;

FIG. 7 Interferon gamma secretion levels;

FIG. 8 shows the number of CD11C positive cells in bone marrow of a liver cancer model C57BL/6J mouse detected by flow;

FIG. 9 shows the expression ratio of dendritic cell surface marker molecules;

FIG. 10 effects of DC vaccine and anti-NKG2A antibody combinations on growth of mouse liver cancer tumors;

FIG. 11 lymphocyte killing assay;

Figure 12 interferon gamma secretion levels.

Detailed Description

Further features and advantages of the present invention will be understood from the following detailed description. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.

Example one combination of DC vaccine and NKG2A antagonist for treatment of mouse Breast cancer

firstly, the method comprises the following steps: culture of dendritic cells with mouse bone marrow precursor cells

extraction, culture and identification of Balb/c mouse marrow-derived dendritic cells

1. Soaking a 6-7-week-old Balb/c mouse in 75% alcohol by volume fraction immediately after the mouse is subjected to cervical vertebra dislocation for 5min, taking out the femur and tibia under a sterile environment, placing the mouse in an RPMI-1640 culture medium, removing muscle tissues on a sterile gauze pad, placing the clean bone in a new flat dish containing 70% alcohol for soaking for 2min, and finally washing for 2 times by using the RPMI-1640 culture medium.

2. The bone was cut at both ends (epiphyses) with scissors and then transferred to another petri dish, and the marrow cavity was washed with 2mL of RPMI-1640 medium aspirated with a syringe to obtain bone marrow, and washed with 1640 until the marrow cavity became white. The epiphyses were trimmed in another petri dish. Mixing the chopped epiphyses and bone marrow, breaking the mass with a pipette, filtering the suspension through a 200 mesh screen, removing particles and collecting in a 50mL centrifuge tube.

3. erythrocytes were lysed by adding 5mL of ammonium chloride solution. After standing at room temperature for 3min, 300g was centrifuged for 10min, and the supernatant was discarded.

4. the bone marrow cells were washed 2 times with RPMI-1640 and centrifuged at 300g for 10min at room temperature. Trypan blue counts live cells. Cell concentration was adjusted to 1X 10 with RPMI-1640 complete medium⁶per ml of individual cells.

5. Adding mouse recombinant GM-CSF to a final concentration of 1000IU/mL and 20ug/mLmIL-4, RPMI-1640 medium containing 10% FBS and 1% streptomycin; the cell suspension was seeded at approximately 2 mL/well in six well plates.

6. Every 2 days, the old medium was removed each time and finally 2mL of fresh RPMI-1640 complete medium containing 1000IU/mL mGM-CSF and 20ug/mLmIL-4 was added.

7. And (3) adding 10ng/mL TNF-alpha to induce DC maturation at day 7, randomly dividing the DC maturation into two groups, adding the obtained breast cancer tumor specific antigen peptide into one group, and performing the following steps of 1: 3, mixing the two solutions in proportion, adding the solution into the other control group, continuously culturing for 2h, adding 2ml of PBS, washing twice, obtaining mature dendritic cell vaccine (Ag-DC vaccine) loaded with antigen from the experimental group, obtaining mature DC without the loaded antigen from the control group, and observing the mature DC without the loaded antigen under an optical microscope to obtain a graph 1.

8. Primitive bone marrow cells, normal mature DC cells without antigen stimulation and DC cells loaded with breast cancer antigen were taken, stained with anti-mouse CD11 c-PE antibody, and cell surface CD11c expression was analyzed by flow cytometry, as shown in FIG. 2.

While further flow assays were performed to confirm the mature DCs obtained, flow cytometry embodiment:

1. Respectively collecting partial immature dendritic cells and mature dendritic cell suspensions on the 7 th day by using a centrifuge tube, and centrifuging at the room temperature of 1000rpm for 5 minutes;

2. Discarding the supernatant, adding 10mL of PBS to resuspend the cells, and then centrifuging at 1000rpm for 5 minutes;

3. Discarding the supernatant, and repeating the operation process of the previous step;

4. A small amount of PBS was added to the centrifuge tube to suspend the cells, and the cells were counted to adjust the density to 3x l0⁵Every 200. mu.L of each cell was transferred into a 1.5mL EP tube;

5. Selecting 1 st tube cell as a blank control (without adding any antibody), sequentially adding a proper amount of anti-mouse APC-CD40 antibody, anti-mouse APC-CD86 antibody, FITC-CD80 antibody and anti-mouse FITC-MHC II antibody into each tube, and respectively adding anti-mouse isotype control antibodies marked by APC, PE and FITC dyes into other tube cells;

6. Incubating the cells in the tube for 40 minutes at 4 ℃ in a dark place;

7. Adding l mL of PBS containing 2% FBS into each tube, centrifuging at 2000rpm for 3 minutes, removing the supernatant, and avoiding light as much as possible;

8. Repeating the step (7) once;

9. 500 mul of 1% paraformaldehyde solution is added into each tube to fix the cells;

10. The tube cells were analyzed using a flow cytometer, and 3 independent experiments were repeated to obtain a flow chart of the expression level of the dendritic cell surface marker molecules (fig. 3), and the expression level data of the mature dendritic cell surface marker molecules was processed using GraphPad Prism software to obtain fig. 4.

selection and dosage of anti-NKG2A antibodies

The anti-NKG2A antibody, NKG 2A-blocking antibody (20D5) (Innate Pharma murine source) was used at a preferred dose of 20ug/kg, and commercially available NKG2A antagonists include NKG2A antibody, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, or interfere with the binding interaction of NKG2A/CD94 with HLA-E, and the NKG2A antibody may be NKG2A antibody-20D 5 (murine source) or other highly specific corresponding proteins that prevent the binding of NKG2A-CD94 to HLA-E.

Establishment of mouse model for breast cancer

1. preparation of breast cancer cell suspension: when the breast cancer cells of the 4T1 mouse are in a logarithmic growth phase, removing the culture solution, washing the breast cancer cells of the 4T1 mouse twice by PBS, adding 0.25% protease into a culture dish for digestion for 1 minute, removing the 0.25% protease, adding 3mL of serum-free culture medium, blowing and beating the cells to prepare a suspension, centrifuging for 5 minutes at 1000rpm, removing the supernatant, adding a proper amount of cell culture solution, and preparing the tumor cell suspension.

2. Constructing a breast cancer model mouse: female Bal b/c mice 6 to 8 weeks old were used for tumor cell transplantation. Mice were kept on normal diets and maintained in SPF Animal facilities according to the laboratory Animal Care and Use Committee guidelines of the Animal Care and Use Committee. Counting the tumor cell culture solution by trypan blue staining, and when the ratio of the living cells is more than 90 percent, the cell concentration is 5 multiplied by 10⁷At individual cells/mL, 0.2mL of each mouse can be inoculated subcutaneously into the axillary abdominal wall. A plurality of mice with the weight of 16-22g are selected, and each mouse is inoculated with 4T1 breast cancer cells subcutaneously to construct a breast cancer mouse model.

Fourth, treatment and detection

1. transplanted tumor-bearing mice, which had already developed tumors and had similar tumor volumes, were selected and randomly divided into a control group (saline), and a normal mature DC group (1X 10)⁶individual cells), antigen-loaded Ag-DC vaccine group (1 × 10)⁶Individual cells), an anti-NKG2A antibody group (10. mu.g/kg), a normal mature DC group + anti-NKG2A antibody group, an antigen-loaded Ag-DC vaccine group and an anti-NKG2A antibody composition test group, wherein each group is similarly administered with the same amount of helper cytokines, the IL-2 content is 1000U/ml, the IL-12 content is 1500U/ml, the Poly (I: C) content is 10mg/ml, the TNF-alpha content is 1000U/ml and the like, and the total is six groups, and each group comprises 10 cells.

2. Performing combined immunization therapy by axillary abdominal wall subcutaneous injection on 3 rd, 10 th and 17 th days after tumor inoculation, observing tumor growth condition every day, measuring and recording tumor size every three days during the period, measuring longest diameter and shortest diameter of the tumor, and calculating the tumor volume as 1/2 tumor long diameter x short diameter². The tumor size data measured for six groups of mice in vivo were plotted in FIG. 5.

3. Mouse lymphocyte tumor killing experiment, mice are sacrificed 42 days after tumor inoculation, and spleens of mice of a control group and different treatment groups are taken under a sterile environment. Grinding on sterile 200 mesh sieve to obtain single cell suspension, cracking erythrocytes, adding into lymphocyte separation solution, centrifuging, collecting lymphocyte layer according to the instruction to obtain mouse spleen lymphocyteseffector cells for killing 4T1 cells, 4T1 cells were seeded into each well of 96-well culture plate, 10⁴a certain number of spleen cells and 4T1 cells were co-cultured at the same time per well according to different ratios of effector cells and target cells (effective target ratios of 20:1, 40:1, 80: 1 and 160: 1), and 5 wells were provided with a 4T1 lymphocyte-free control group and a cell-free blank culture solution control group. After 24h, the free effector cells in each well were aspirated, washed 2 times with PBS, 100. mu.l of CCK8 reagent was added to each well, incubation continued for 2h, and absorbance (OD) was measured at 450nm with a microplate reader. Specific lymphocyte killing rate (%) × 100 × (OD-experimental group OD of 4T1 wells)/(OD of 4T1 wells-blank OD). The data were processed using GraphPad Prism software, the data were expressed as mean ± standard deviation, t-test was used for comparisons between 2 groups, and one-way anova was used for statistical analysis between groups, to give fig. 6.

4. Detection of secretion of interferon γ: a part of the mouse spleen lymphocytes obtained above was used as effector cells, 4T1 cancer cells were used as target cells, and the ratio of effector cells to target cells was 20:1, culturing in a U-shaped bottom 96-well plate for 72h, and detecting the content of IFN gamma in the culture supernatant by using a mouse interferon gamma enzyme linked immunosorbent assay kit according to the instruction flow to obtain a figure 7.

as a result:

As shown in fig. 1: when observed under an optical microscope, the cells are round and clear, are evenly suspended in the culture medium, part of the cells are attached to the wall, and the periphery of the cells has burr-shaped protrusions.

as can be seen from fig. 2, there were few mature dendritic cells in the bone marrow of normal mice, demonstrating that dendritic cells are present mostly in an immature state in vivo under normal conditions; in vitro, immature dendritic cells become mature dendritic cells after being induced by 10ng/ml TNF-alpha, and the cell surface molecule CD11c is highly expressed whether or not the cells are stimulated by related antigens.

as shown in fig. 3: and (3) taking partial immature DC and mature DC cells to carry out flow cytometry to detect the expression of co-stimulatory factors such as CD40/CD80/CD86/CD11c/MHC-II and the like, wherein a red line 1 represents the flow map of corresponding antibody isotype control, a blue line 2 represents the flow map of immature DC surface molecules, and a green line 3 represents the flow map of mature DC surface molecules. As can be seen from the figure, the mature dendritic cells exhibited high expression of molecules such as CD40/CD80/CD86/CD11c/MHC-II on their surface.

As shown in fig. 4: the above results show that the CD11c + cell ratio on the day of bone marrow cell extraction is only 0.66%, and after IL-4, GM-CSF and TNF-alpha factor-induced amplification, the mature DC of the mature dendritic cell Ag-DC vaccine and the control group have CD11c + cell ratios higher than 90%, and from FIG. 3, we can see that CD40 of the mature dendritic cells is not changed greatly, but CD80/CD86/CD11c/MHC-II is expressed positively compared with that of the immature dendritic cells. And more than 80% of cells highly express dendritic cell surface markers such as CD11c, MHC-II, CD40, CD80, CD86 and the like, and the separation and induction are proved to obtain a large amount of high-purity mature dendritic cells.

As shown in fig. 5: along with the increase of time, the tumor growth of the control group mouse is fastest, the tumor growth in the mice of the experimental group of the antigen-loaded Ag-DC vaccine and anti-NKG2A antibody composition is obviously inhibited, and compared with the normal mature DC group, the antigen-loaded Ag-DC vaccine group, the anti-NKG2A antibody group and even the normal mature DC group and anti-NKG2A antibody group, the anti-NKG antibody composition has obvious difference, the tumor volume is obviously smaller than that of the mice adopting other treatment schemes, and the tumor inhibition effect is very obvious; the combination application of the Ag-DC vaccine loaded with the antigen and the anti-NKG2A antibody composition is proved to be capable of effectively inhibiting the growth of the breast cancer tumor of the mouse. After the Ag-DC is used for immunizing a mouse, the tumor cell killing capacity of mouse lymphocytes is obviously enhanced, and the anti-NKG2A antibody can enhance the immune response induced by the BMDC vaccine and improve the tumor treatment effect, which means that the anti-NKG2A antibody plays a synergistic role in the tumor treatment effect of the DC vaccine.

as shown in fig. 6: compared with the simple Ag-DC group, the combination group has significant difference of lymphocyte killing rate (P <0.05) when the effective target ratio is 20:1, 40:1, 80: 1 and 160: 1, and the combination of the DC vaccine and the NKG2A antagonist can induce the organism to generate stronger tumor cell killing capacity.

as shown in fig. 7: the spleen lymphocytes of mice in the experimental group of the DC vaccine and anti-NKG2A antibody composition and 4T1 cells are incubated to secrete interferon gamma which is significantly higher than that of the control group, and the vaccine and antibody are separately treated; the expression levels of the interferon gamma of the Ag-DC vaccine and the anti-NKG2A antibody single treatment group are respectively obviously improved compared with those of a control group, which shows that the DC vaccine and the anti-NKG2A antibody composition promote the formation of the interferon gamma, enhance the anti-immune effect of NK cells and T cells, and are beneficial to the anti-breast cancer immune response of an organism.

Example two: combination of Ag-DC vaccine and NKG2A antagonist for treating mouse liver cancer subcutaneous transplantation tumor

Firstly, the method comprises the following steps: extraction, culture and identification of C57BL/6J mouse marrow-derived dendritic cells

1. Soaking a 6-7-week-old C57BL/6J mouse in 75% alcohol by volume fraction immediately after cervical dislocation, taking out femur and tibia under sterile environment after 5min, placing in RPMI-1640 culture medium, removing muscle tissue on a sterile gauze pad, soaking clean bone in a new flat dish containing 70% ethanol for 2min, and finally washing with RPMI-1640 culture medium for 2 times.

2. The bone was cut at both ends (epiphyses) with scissors and then transferred to another petri dish, and the marrow cavity was washed with a syringe sucking 2 mlpmpmmi-1640 medium to obtain bone marrow and washed with 1640 until the marrow cavity became white. The epiphyses were trimmed in another petri dish. Mixing the chopped epiphyses and bone marrow, breaking the mass with a pipette, filtering the suspension through a 200 mesh screen, removing particles and collecting in a 50mL centrifuge tube.

5. mouse recombinant GM-CSF was added to final concentrations of 1000U/mL and 20ug/mL mIL-4 in RPMI-1640 medium with 10% FBS and 1% streptomycin. The cell suspension was seeded at approximately 2 mL/well in six well plates.

6. the medium was changed every 2 days, the old medium was removed each time, and finally 2mL of fresh RPMI-1640 complete medium containing 1000U/mL mGM-CSF and 20ug/mL mIL-4 was added.

7. And (3) adding a proper amount of LPS (lipopolysaccharide) to induce the maturation of the DCs on the 7 th day, and randomly dividing the DCs into two groups, wherein one group is added with the obtained H22 liver cancer cell tumor specific antigen peptide, and the ratio of the antigen peptide to the antigen peptide is 1: and 3, mixing the two solutions according to the proportion, adding the solution into the other control group, continuously culturing for 2h, adding 2ml of PBS (phosphate buffer solution) and washing twice, obtaining a mature dendritic cell vaccine (Ag-DC vaccine) loaded with the antigen from the experimental group, and obtaining a mature DC without the antigen from the control group.

8. Immature and final mature DC cells were separately taken, stained with anti-mouse CD11 c-PE antibody, and cell surface CD11c expression was analyzed by flow cytometry, as shown in FIG. 8.

6. Incubating the cells in the tube for 40 minutes at 4 ℃ in a dark place;

8. Repeating the step (7) once;

10. the tube cells were analyzed using a flow cytometer, 3 independent experiments were performed, and the expression data of the surface marker molecules of the mature dendritic cells were processed using GraphPad Prism software, to obtain fig. 9.

Selection and dosage of anti-NKG2A antibodies

The anti-NKG2A antibody, NKG 2A-blocking antibody (20D5) (Innate Pharma source) is selected and used in a dosage of preferably 40ug/kg and 1ug/kg (preferably 10ug/kg-1mg/kg), and commercially available NKG2A antagonists include NKG2A antibody, antigen-binding fragment immunoadhesin, fusion protein, oligopeptide and other molecules that reduce, block, inhibit, or interfere with the binding interaction of NKG2A/CD94 and HLA-E, and the NKG2A antibody may be NKG2A antibody 20D5 or other highly specific corresponding proteins or polypeptides that prevent the binding of NKG2A-CD94 and HLA-E.

Establishment of mouse model of liver cancer

1. Preparing a liver cancer cell suspension: taking H22 mouse liver cancer cells in logarithmic phase, removing culture solution, washing H22 cells twice with PBS, adding 0.25% protease into a culture dish for digestion for 1 minute, removing the protease, adding 3mL of serum-free culture medium, blowing and beating the cells to prepare suspension, centrifuging for 5 minutes at 1000rpm, removing supernatant, adding a proper amount of physiological saline, and preparing the tumor cell suspension.

2. Constructing a liver cancer model mouse: c57BL/6J mice 6 to 8 weeks old were selected for tumor cell transplantation. Animals were kept on normal diets and maintained in SPF Animal facilities according to the laboratory Animal Care and Use Committee guidelines. Counting the tumor cell culture solution by trypan blue staining, and when the ratio of the living cells is more than 90 percent, the cell concentration is 1 multiplied by 10⁷When each cell is per mL, 0.2mL of cell suspension can be inoculated under the right abdominal wall of each mouse to construct a liver cancer mouse model.

Fourth, treatment and detection

1. Mice with subcutaneous transplanted tumors that had developed tumors and similar tumor volumes were selected and randomly divided into control group (saline), normal mature DC group (1X 10)⁶Individual cell) Antigen-loaded Ag-DC vaccine group (1X 10)⁶Individual cells), an anti-NKG2A antibody group (10. mu.g/kg), a normal mature DC group + anti-NKG2A antibody group, an antigen-loaded Ag-DC vaccine group and an anti-NKG2A antibody composition test group, wherein each group is similarly administered with the same amount of helper cytokines, the IL-2 content is 1000U/ml, the IL-12 content is 1500U/ml, the Poly (I: C) content is 10mg/ml, the TNF-alpha content is 1000U/ml and the like, and the total is six groups, and each group comprises 10 cells.

2. Performing immunization combination therapy by axillary abdominal wall subcutaneous injection respectively on 3 days, 10 days and 17 days after tumor inoculation, observing tumor growth condition every day, measuring and recording tumor size every three days during the observation, measuring longest diameter and shortest diameter of the tumor, and calculating the tumor volume which is 1/2 tumor long diameter x short diameter². The measured tumor size data for six groups of mice in vivo were plotted in FIG. 10.

3. mouse lymphocyte tumor killing experiment, after 27 days after tumor inoculation, the mice are sacrificed, and spleens of mice of a control group and different treatment groups are taken under a sterile environment. Grinding on a sterile 200-mesh sieve to obtain single cell suspension, cracking erythrocytes, adding into lymphocyte separation solution, centrifuging, taking lymphocyte layer according to the instruction to obtain mouse spleen lymphocytes, using as effector cells for H22 cell killing experiment, inoculating H22 cells into each hole of 96-hole culture plate, and culturing at 10 deg.C⁴On day 2, a certain number of spleen cells and H22 cells were co-cultured at different ratios of effector cells to target cells (5: 1, 10: 1, 20:1 and 40:1 effective target ratios), and 5 wells were provided for both the lymphocyte-free control group and the cell-free control group. After 24h, the free effector cells in each well were aspirated, washed 2 times with PBS, 100. mu.l of CCK8 reagent was added to each well, incubation continued for 2h, and absorbance (OD) was measured at 450nm with a microplate reader. Specific lymphocyte killing rate (%) × (H22 well OD-experimental group OD)/(H22 well OD-blank control group OD) 100 ×. The data were processed using GraphPad Prism software, the data were expressed as mean ± standard deviation, t-test was used for comparisons between 2 groups, and one-way anova was used for statistical analysis between groups, to give fig. 11.

4. Detection of secretion of interferon γ: part of the mouse spleen lymphocytes obtained above was used as effector cells, and H22 cancer cells were used as target cells, and the ratio of effector cells to target cells was 20:1, culturing in a U-shaped bottom 96-well plate for 72h, detecting the content of IFN gamma in the culture supernatant by using a mouse interferon gamma enzyme linked immunosorbent assay kit according to the instruction flow, and obtaining a figure 12.

As a result:

as shown in fig. 8-9: the above results show that CD11c was obtained on the day of bone marrow cell extraction⁺The cell ratio is only 1.66%, after the mature DC of the mature dendritic cell Ag-DC vaccine and the control group are induced and amplified by IL-4, GM-CSF and LPS factors, the mature DC of the mature dendritic cell Ag-DC vaccine and the control group highly express CD11c, and CD80, CD86 and MHC-II are positively expressed compared with the immature dendritic cells. The separation induction is proved to obtain a large amount of mature dendritic cells with high purity.

As shown in fig. 10: along with the increase of time, the tumor growth of the mice in the control group is fastest, the tumor growth in the mice of the experimental group of the antigen-loaded Ag-DC vaccine and anti-NKG2A antibody composition is obviously inhibited, and compared with the normal mature DC group, the antigen-loaded Ag-DC vaccine group, the anti-NKG2A antibody group and even the normal mature DC group and anti-NKG2A antibody group, the anti-NKG antibody composition has obvious difference, the tumor volume is obviously smaller than that of the mice of other treatment groups, and the tumor inhibition effect is very obvious; proves that the immunotherapy which combines the Ag-DC vaccine loaded with the antigen and the anti-NKG2A antibody can effectively inhibit the growth of the liver cancer tumor of the mouse. After the Ag-DC is used for immunizing a mouse, the tumor cell killing capacity of mouse lymphocytes is obviously enhanced, the anti-NKG2A antibody can enhance the immune response induced by the BMDC vaccine, and the tumor treatment effect is improved, which means that the anti-NKG2A antibody and DC vaccine composition can obviously enhance the anti-tumor capacity of an organism.

As shown in fig. 11: compared with the simple Ag-DC group, the lymphocyte killing rate difference is significant (P is less than 0.05) when the effect-target ratio is 5: 1, 10: 1, 20:1 and 40:1, and the lymphocyte killing rate is very similar when the effect-target ratio is 20:1 and 40:1, which indicates that the optimal effect-target ratio is 20:1 in the experiment, and proves that the composition formed by combining the DC vaccine with NKG2A can induce the organism to generate stronger tumor cell killing capacity.

as shown in fig. 5: the interferon gamma secreted by splenic lymphocytes of the experimental group of the DC vaccine and anti-NKG2A antibody composition in incubation with H22 cells is significantly higher than that of the control group, and the vaccine and antibody are separately treated; the expression levels of the Ag-DC vaccine and the anti-NKG2A antibody single treatment group interferon gamma are respectively obviously improved compared with a control group, which shows that the combined application of the DC vaccine and the anti-NKG2A antibody composition promotes the formation of the interferon gamma, enhances the anti-immune effect of NK cells and T cells, and is beneficial to the anti-liver cancer immune response of organisms.

Claims

1. A composition of a DC vaccine and an NKG2A antagonist, wherein: the composition comprises a DC vaccine loaded with tumor specific antigen and an NKG2A antagonist, wherein the DC vaccine is stored in a non-animal derived culture medium; the DC vaccine contains dendritic cells with the number of 5 x 10⁶-5*10⁸The content of the NKG2A antagonist is 10ug/ml to 10 mg/ml; the DC vaccine comprises a first adjuvant or other therapeutic-aiding cytokine together with adjuvants IL-2, IL-12, Poly (I: C) and TNF-alpha.

2. the composition of DC vaccine and NKG2A antagonist according to claim 1, wherein: the NKG2A antagonists include anti-NKG2A antibodies and small molecule antagonists; the anti-NKG2A antibody is a recombinant monoclonal or polyclonal antibody, the anti-NKG2A binding antibody is an IgA, IgG, genetically modified IgG isotype, or an antigen binding fragment thereof, or is a Fab ', F (ab ')2, F (ab ')3, monovalent scFv, divalent scFv, bispecific, nanobody, or single domain antibody; or human, humanized or deimmunized antibodies, and the small molecule antagonist is an inhibitory nucleic acid, RNA, or other chemically synthesized small molecule inhibitor.

3. The composition of DC vaccine and NKG2A antagonist according to claim 2, wherein: the anti-NKG2A binding antibody is humanized anti-NKG2A antibody, monatizumab or murine anti-NKG2A antibody 20D 5.

4. Use of a combination of a DC vaccine according to claims 1 to 3 and an NKG2A antagonist against breast or liver cancer, wherein: extracting and inducing dendritic cells, loading specific breast cancer or liver cancer antigen to dendritic cells extracted from a breast cancer or liver cancer patient after in vitro culture and maturation, enabling the dendritic cells to carry the specific cancer antigen on the surface, forming a composition with an NKG2A antagonist, and returning the composition to the patient body to stimulate natural immunity and induce NK cells and T lymphocytes to generate acquired immune response, and strengthening the immune effect of the NK cells and the T lymphocytes; the NKG2A antagonist and DC vaccine loaded with breast cancer or liver cancer specific antigen are used to form composition for enhancing the immunity of body against tumor.

5. The use of a combination of a DC vaccine according to claim 4 and an NKG2A antagonist against breast or liver cancer, wherein: the composition treats breast cancer or liver cancer by means of intravenous, intradermal, intratumoral, intramuscular, intraperitoneal, subcutaneous or local administration of a composition of the NKG2A antagonist and the DC vaccine, wherein the composition of the DC vaccine and the NKG2A antagonist is administered three times with one or two weeks between each time.