Use of combination of living plasmodium vivax and antimalarial drug in preparation of product for activating CIK cells

Abstract

The use of the combination of living plasmodium vivax and an antimalarial drug in the preparation of a product for activating CIK cells. It is found in the present application that maintaining the number of plasmodium vivax at a low density level by administering a low-dose antimalarial drug can activate CIK cells in a human body, thereby improving the tumor killing ability of CIK cells. The present invention is expected to be used in the preparation of a product for activating CIK cells and for killing tumor cells, and has the advantages of good safety, low toxicity and side effects, etc.

Description

Application of live Plasmodium vivax and antimalarial drugs in combination to prepare products for activating CIK cells Technical Field

[0001] The present application belongs to the field of biomedicine technology and relates to the use of a combination of live Plasmodium vivax and antimalarial drugs in the preparation of a product for activating CIK cells. Background Art

[0002] There are many types of malarial parasites, with five main species parasitizing humans: Plasmodium vivax, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi. Plasmodium parasites inhabit human liver cells (hepatic stage) and red blood cells (erythrocytic stage) and are the causative agent of malaria. Plasmodium vivax is a relatively benign malarial parasite, predominantly found in tropical and subtropical regions outside of Africa. Inoculation with Plasmodium vivax was used to treat neurosyphilis in the early 20th century.

[0003] Currently, there are many antimalarial drugs for treating malaria, including artesunate. Scientific research and clinical practice have demonstrated that, among all antimalarial drugs, intravenous artesunate can rapidly kill asexual propagules (rings, trophozoites, schizonts, and merozoites) of Plasmodium in the blood, thereby rapidly controlling the red blood cell infection rate of Plasmodium. It also has a strong inhibitory effect on the sexual propagules (gametocytes) of Plasmodium (see: Nacher M, et al. Antimicrob Agents Chemother. 2004), thus preventing the spread of malaria. Furthermore, artesunate is one of the antimalarial drugs with the lowest toxic side effects.

[0004] Cytokine-induced killer cells (CIK cells) refer to CD3 + CD56 + T lymphocytes, because they express two membrane protein molecules, CD3 (T cell marker molecule) and CD56 (NK cell marker molecule), they are also called NK cell (natural killer cell)-like T lymphocytes. They have the powerful anti-tumor activity of T lymphocytes, and at the same time have the non-MHC restricted tumor-killing advantage of NK cells. They have a strong ability to recognize tumor cells and can accurately kill tumor cells without harming normal cells. They are especially effective for patients after surgery or chemotherapy, and can eliminate residual tiny metastatic lesions, prevent the spread and recurrence of cancer cells, and enhance the body's immunity.

[0005] CIK cells currently used for adoptive immunotherapy are actually CD3 +CD56 + A heterogeneous cell population mainly composed of mononuclear cells isolated from peripheral blood, bone marrow or cord blood is cultured and expanded in vitro under the stimulation of multiple cytokines such as IL-2, IFN-γ and certain monoclonal antibodies (such as anti-CD3 mcab), and has a wide range of non-MHC restricted and extremely strong oncolytic activity. For example, CN105925526A discloses a method for enhancing CIK cell activity and CIK cells and their preparation method and application. Peripheral blood mononuclear cells are cultured in the presence of cytokines for 9-11 days and then cultured in the presence of DNA demethylating drugs. The cytokines are at least one of interferon-γ, interleukin-2, CD3 monoclonal antibody, fibronectin and interleukin-1a.

[0006] Currently, there are three main methods known to activate CIK: 1. Food / health product activation method, which mainly uses certain nutrients in food to activate CIK; 2. Drug activation method, including small molecule chemical drugs, large molecule drugs such as antibodies / nucleic acid drugs and certain components of traditional Chinese medicine to activate CIK; 3. Immune cell activation method, which mainly uses immune regulatory cells such as dendritic cells (DC) injected into the body to activate CIK in the body.

[0007] In summary, the current method for preparing CIK cells is mainly through in vitro culture, which has problems such as cumbersome operation and high cost. Therefore, the development of a method for activating CIK cells that is simple to operate and low-cost, and can achieve in situ activation in vivo, is of great significance for the clinical application of CIK cells.

[0008] Summary of the Invention

[0009] This application provides the use of a combination of live Plasmodium vivax and antimalarial drugs in the preparation of a product that activates CIK cells. This application is the first to discover that controlling and maintaining the number of Plasmodium at a low density can activate CIK cells in the human body, thereby enhancing the tumor-killing ability of CIK cells. This application aims to provide a fourth CIK activation method, namely a live Plasmodium / antimalarial drug combination activation method, which utilizes the properties of live Plasmodium and antimalarial drugs to activate CIK activity in the human body. This method is the world's first to successfully activate CIK cells in the human body using a live Plasmodium vivax / antimalarial drug combination. It is a new technical method that is different from the three existing traditional methods. Compared with other methods, it has the advantages of simple operation, low cost, and high success rate.

[0010] In a first aspect, the present application provides the use of live Plasmodium vivax in the preparation of a product for activating CIK cells.

[0011] In this application, a certain amount of live Plasmodium vivax is intravenously injected into the body of a cancer patient (such as non-small cell lung cancer), while the density of the Plasmodium is controlled at a low level. It was found that maintaining the number of Plasmodium at a low density level can activate CIK cells in the human body, thereby enhancing the tumor-killing ability of CIK cells.

[0012] In a second aspect, the present application provides the use of a combination of live Plasmodium vivax and an antimalarial drug in the preparation of a product for activating CIK cells.

[0013] In this application, a certain amount of live Plasmodium vivax parasites was intravenously injected into patients with cancer (such as small cell lung cancer), while a low dose of an antimalarial drug (such as artesunate) was used to control the parasite density at a low level. It was found that maintaining the parasite density at a low dose of antimalarial drugs can activate CIK cells in the human body, thereby enhancing the tumor-killing ability of CIK cells. This product is expected to be used to prepare products that activate CIK cells for killing non-tumor cells. It has the advantages of good safety and low toxicity and side effects, and provides new ideas for the development of new technologies for killing tumors.

[0014] Preferably, the antimalarial drug comprises any one of artesunate, chloroquine or bromoquine, or a combination of at least two of them.

[0015] In a third aspect, the present application provides a combination for activating CIK cells, wherein the combination comprises live Plasmodium vivax and an antimalarial drug.

[0016] Preferably, the antimalarial drug comprises any one of artesunate, chloroquine or bromoquine, or a combination of at least two of them.

[0017] Preferably, the dosage form of the combination includes any pharmaceutically acceptable dosage form.

[0018] Preferably, the combination is a combination of live Plasmodium vivax and an antimalarial drug preparation.

[0019] In a fourth aspect, the present application provides the use of a combination of live Plasmodium vivax and an antimalarial drug in the preparation of a drug for treating tumors.

[0020] Preferably, the antimalarial drug comprises any one of artesunate, chloroquine or bromoquine, or a combination of at least two of them.

[0021] Preferably, the tumor comprises non-small cell lung cancer.

[0022] This application found that the combination of live Plasmodium vivax and antimalarial drugs can effectively activate CIK cells in vivo, which is of great significance for studying the basic behavior of CIK cells, such as constructing high-content CIK cell animal models, preparing CIK cells, etc.

[0023] In a fifth aspect, the present application provides a method for activating CIK cells, comprising:

[0024] Live Plasmodium vivax is administered to the target subject, and when the Plasmodium vivax infection rate of the red blood cells in the target subject's peripheral blood reaches 0.1%, the target subject is administered with antimalarial drugs to control the Plasmodium vivax infection rate of the red blood cells to be lower than 0.1% for 30 to 90 days, thereby activating CIK cells in the target subject's peripheral blood.

[0025] In the present application, the target object can be a human body or an experimental animal, such as a rat, mouse or rabbit.

[0026] In the present application, controlling the malarial parasite red blood cell infection rate to below 0.1% can effectively prevent malarial parasites from suppressing the immune system and avoid serious side effects and complications.

[0027] Preferably, the amount of the live Plasmodium vivax is 100,000 to 10 million, including but not limited to 110,000, 120,000, 150,000, 200,000, 500,000, 1 million, 5 million, 8 million, 9 million, 9.5 million, 9.6 million, 9.8 million or 9.9 million.

[0028] Preferably, the antimalarial drug comprises any one of artesunate, bromoquine or chloroquine, or a combination of at least two of them.

[0029] In a sixth aspect, the present application provides a method for preparing CIK cells, the method comprising:

[0030] The CIK cells in the peripheral blood of the target subject are activated by the method for activating CIK cells according to the fifth aspect, and the CIK cells are isolated from the peripheral blood of the target subject.

[0031] Compared with the prior art, this application has the following beneficial effects:

[0032] This application discovered that using low-dose antimalarial drugs to maintain the number of malarial parasites at a low density level can activate CIK cells in the human body, thereby enhancing the tumor-killing ability of CIK cells. It is expected to be used to prepare products that activate CIK cells for killing non-tumor cells. It has the advantages of good safety and low toxic side effects, providing new ideas for the development of new technologies for killing tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 shows the analysis results of the proportion of CIK cells secreting IFN-γ in healthy subjects and patients with non-small cell lung cancer.

[0034] FIG2 is a graph showing the statistical analysis results of CIK cells secreting IFN-γ in healthy subjects and patients with non-small cell lung cancer. * indicates P < 0.05.

[0035] FIG3 is a graph showing the analysis results of the ratio of CIK killing tumor cells in healthy individuals and patients with non-small cell lung cancer.

[0036] FIG4 is a graph showing the statistical analysis of CIK killing tumor cells in healthy subjects and patients with non-small cell lung cancer, where * indicates P < 0.05. DETAILED DESCRIPTION

[0037] To further illustrate the technical means and effects of this application, the following further describes this application in conjunction with examples and drawings. It should be understood that the specific implementation methods described herein are only used to explain this application, rather than to limit this application.

[0038] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0039] Example 1

[0040] This example performs a comparative analysis of the activation of CIK cells in patients with non-small cell lung cancer using live Plasmodium vivax and artesunate, and their functions.

[0041] 1. Intravenous injection of live Plasmodium vivax and low concentrations of artesunate into patients with non-small cell lung cancer

[0042] Three patients with non-small cell lung cancer (NSCLC) were intravenously injected with 5 million live Plasmodium vivax red blood cells (1 ml volume) into their bodies. The parasite density (parasitemia density) in the patients' peripheral blood was then monitored daily. When the parasitemia density exceeded 0.1%, 6-12 mg of artesunate was administered intravenously each time to control the parasitemia density to below 0.1%. This low parasitemia was maintained for 90 days. Afterward, sufficient amounts of effective antimalarial drugs were administered to completely eliminate the parasites in the patients (consisting of one course of treatment). During this period, peripheral blood samples were collected from the patients one day before and 30 days after inoculation to culture CIK cells and test their activity. Peripheral blood samples from healthy controls were used as controls.

[0043] 2. Pretreatment of peripheral blood from patients with non-small cell lung cancer / healthy subjects

[0044] 1. Take one drop (100 μL) of venous peripheral blood from patients with non-small cell lung cancer and healthy adults, and perform anticoagulation.

[0045] 2. Peripheral whole blood was mixed with 2 ml of 1× red blood cell lysis buffer (Biolegend), vortexed for 10 seconds, and then allowed to stand at 25°C in the dark for 15 minutes;

[0046] 3. Centrifuge (350 × g, 5 minutes), pour off the supernatant, and then suspend the precipitated cells in 2 ml of cell staining solution (PBS solution containing 2.5% fetal bovine serum);

[0047] 4. Add leukocyte stimulating factor (BD) at a concentration of 0.1% and incubate the cells at 37°C for 6 hours.

[0048] 3. Analysis of the development and differentiation degree of CIK cells in the peripheral blood of patients with non-small cell lung cancer and healthy subjects

[0049] The reserved blood cells were centrifuged (350×g) for 5 minutes, the supernatant discarded, and the cells were suspended in 100 μL of cell staining solution. 2 μL of anti-human CD3 and CD56 antibodies (Biolegend) were then added, and the cells were incubated at 25°C for 30 minutes. Then, 2 mL of cell staining solution was added and the cells were centrifuged twice (350×g) for 5 minutes each. After discarding the supernatant, the cells were fixed with 2 mL of 2% formalin solution and incubated at 25°C in the dark for 20 minutes before use.

[0050] 4. Functional analysis of CIK cells in peripheral blood of patients with non-small cell lung cancer and healthy subjects

[0051] 1. Resuspend the fixed cells in 2 ml of cell permeabilization buffer (Biolegend) and centrifuge (350 × g) for 10 min twice.

[0052] 2. Resuspend the pelleted cells after centrifugation in 100 μL of cell permeabilization buffer, then add 2 μL of IFN-γ (Biolegend) antibody and incubate at 25°C in the dark for 30 minutes;

[0053] 3. Resuspend the incubated cells in 2 ml of cell permeabilization buffer and repeat centrifugation (350 × g) for 5 minutes twice;

[0054] 4. Finally, pour off the supernatant and resuspend the precipitated cells in 0.5 ml of cell staining solution and analyze them using flow cytometry.

[0055] 5. Determination of tumor cell apoptosis test ratio

[0056] The tumor cells were stained with apoptosis reagent 7AAD, and the positive ratio of 7AAD was determined by flow cytometry as the apoptosis ratio of tumor cells.

[0057] 6. Results Analysis

[0058] 1. Flow cytometry was used to detect the expression of the molecule CD56 on human peripheral blood CIK cells. This data was used to evaluate the differentiation and maturation of human peripheral blood CIK cells. The results are shown in Figure 1. Healthy subjects represent healthy controls; NSCLC patients represent non-small cell lung cancer patients before inoculation with Plasmodium vivax (Pv); and NSCLC patients + Pv represent non-small cell lung cancer patients after inoculation with Plasmodium vivax.

[0059] 2. Functional analysis of human peripheral blood CIK cells: flow cytometry analysis of cytokine IFN-γ in CD3 + CD56 + The secretion expression in the cells (expressed as %) is shown in Figures 1 and 2. The expression level of IFN-γ in CIK cells of patients with non-small cell lung cancer after low-density Plasmodium infection was significantly higher than that in healthy controls and patients with non-small cell lung cancer. It can be seen that low-density Plasmodium infection effectively increased the activity of CIK cells in patients with non-small cell lung cancer, indicating that the combination of live Plasmodium vivax and artesunate effectively overcomes the inhibitory effect of non-small cell lung cancer on the activity function of CIK cells.

[0060] Example 2

[0061] This example verifies that low-density Plasmodium infection effectively enhances the tumor-killing ability of CIK cells in patients with non-small cell lung cancer.

[0062] Peripheral blood was collected from non-small cell lung cancer patients and healthy volunteers before and after inoculation with Plasmodium in Example 1. The blood was then mixed with H-1299 non-small cell lung cancer cells at a ratio of 10:1 in complete culture medium and cultured at 37°C for 5 hours. After culture, the H-1299 non-small cell lung cancer cells were stained with eFluor 670 dye and then with 7-AAD and FITC-annexin. The apoptosis rate of tumor cells was tested. The results are shown in Figures 3 and 4. Lysis (%) represents the tumor cell lysis rate / apoptosis rate. These results indicate that low-density Plasmodium infection effectively enhances the tumor-killing ability of CIK cells in non-small cell lung cancer patients.

[0063] In summary, this application discovered that using low-dose antimalarial drugs to maintain the number of Plasmodium at a low density level can activate CIK cells in the human body, thereby enhancing the tumor-killing ability of CIK cells. This product is expected to be used to prepare products that activate CIK cells for killing non-tumor cells. It has the advantages of good safety and low toxic side effects, providing new ideas for the development of new technologies for killing tumors.

[0064] The applicant declares that while the above-mentioned embodiments are used to illustrate the detailed methods of the present application, the present application is not limited to the above-mentioned detailed methods, which does not mean that the present application must rely on the above-mentioned detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present application, equivalent replacements for the raw materials of the present application's products, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present application.

Claims

1. Application of live Plasmodium vivax in the preparation of products for activating CIK cells.

2. The use of a combination of live Plasmodium vivax and antimalarial drugs in the preparation of products for activating CIK cells.

3. The application according to claim 2, wherein: The antimalarial drug comprises any one of artesunate, chloroquine or bromoquine, or a combination of at least two of them.

4. A combination for activating CIK cells, comprising live Plasmodium vivax and an antimalarial drug.

5. The combination for activating CIK cells according to claim 4, wherein: The antimalarial drug includes any one of artesunate, chloroquine or benquine, or a combination of at least two of them; Preferably, the dosage form of the combination comprises any pharmaceutically acceptable dosage form; Preferably, the combination is a combination of live Plasmodium vivax and an antimalarial drug preparation.

6. Application of a combination of live Plasmodium vivax and antimalarial drugs in the preparation of drugs for treating tumors; Preferably, the antimalarial drug comprises any one of artesunate, chloroquine or bromoquine, or a combination of at least two thereof; Preferably, the tumor comprises non-small cell lung cancer.

7. A method for activating CIK cells, comprising: Live Plasmodium vivax is administered to the target subject, and when the Plasmodium vivax infection rate of red blood cells in the target subject's peripheral blood is 0.01-0.1%, antimalarial drugs are administered to the target subject to control the Plasmodium vivax infection rate of red blood cells to be lower than 0.01-0.1% for 30 to 90 days, thereby activating CIK cells in the target subject's peripheral blood.

8. The method for activating CIK cells according to claim 7, wherein: The dosage of the living Plasmodium vivax is 10 to 10 million.

9. The method for activating CIK cells according to claim 7, wherein: The antimalarial drug comprises any one of artesunate, chloroquine or bromoquine, or a combination of at least two of them.

10. A method for preparing CIK cells, comprising: The CIK cells in the peripheral blood of the target subject are activated by the method for activating CIK cells according to any one of claims 7 to 9, and the CIK cells in the peripheral blood of the target subject are isolated by extracting the peripheral blood.

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