WO2025015530A1 - Use of combination of living plasmodium vivax and antimalarial drug in preparation of product for activating cik cells - Google Patents

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 the preparation of products for activating CIK cells Technical Field

The present application belongs to the field of biomedicine technology and relates to the application of a combination of live Plasmodium vivax and an antimalarial drug in the preparation of a product for activating CIK cells.

Background Art

There are many types of malarial parasites, and there are five main types that parasitize the human body, namely Plasmodium vivax, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi. Plasmodium parasites parasitize in human liver cells (liver stage) and red blood cells (erythrocyte stage) and are the pathogens of malaria. Among them, Plasmodium vivax is a relatively benign malarial parasite that is mainly prevalent in tropical and subtropical areas outside of Africa. In the early twentieth century, Plasmodium vivax vaccination was used to treat neurosyphilis.

There are many drugs for treating malaria, namely antimalarial drugs, including artesunate. Scientific research and clinical practice have proved that among all antimalarial drugs, intravenous injection of artesunate can quickly kill the asexual reproduction bodies (ring bodies, trophozoites, schizonts and merozoites) of malarial parasites in the blood, so it can quickly control the red blood cell infection rate of malarial parasites, and at the same time, it has a strong inhibitory effect on the sexual reproduction bodies (gametocytes) of malarial parasites (see: Nacher M, et al. Antimicrob Agents Chemother. 2004), so it can also prevent the spread of malaria. In addition, artesunate is also one of the drugs with the least toxic side effects of all antimalarial drugs.

Cytokine induced killer cells (CIK cells) refer to CD3 ⁺ CD56 ⁺ T lymphocytes, which only account for 1-5% of the normal human peripheral blood. Since the cells 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 the non-MHC-restricted tumor-killing advantages 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 in 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.

CIK cells currently used for adoptive immunotherapy are actually heterogeneous cell populations mainly composed of CD3 ⁺ CD56 ⁺ that are expanded in vitro. These cells are isolated from peripheral blood, bone marrow or cord blood under the stimulation of multiple cytokines such as IL-2, IFN-γ and certain monoclonal antibodies (such as anti-CD3 mcab). The isolated mononuclear cells are cultured and expanded in vitro, and have a wide range of non-MHC-restricted and extremely strong oncolytic activity. For example, CN105925526A discloses a method for enhancing CIK cell activity, CIK cells, and a preparation method and application thereof, wherein 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, wherein the cytokines are at least one of interferon-γ, interleukin-2, CD3 monoclonal antibody, fibronectin, and interleukin-1a.

There are three main methods for activating CIK: 1. Food/health products 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.

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, developing 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.

Summary of the invention

The present application provides the use of a combination of live Plasmodium vivax and antimalarial drugs in the preparation of products that activate CIK cells. The present application first discovered that controlling and 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. The present 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 first in the world to successfully activate CIK cells in the human body using a combination of live Plasmodium vivax/antimalarial drugs. 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.

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

In the present application, a certain amount of live Plasmodium vivax is intravenously injected into a tumor patient (such as non-small cell lung cancer), while the density of Plasmodium is controlled at a low level. It is 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.

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

In this application, a certain amount of live Plasmodium vivax is intravenously injected into tumor patients (such as small cell Lung cancer) in vivo, and at the same time using low doses of antimalarial drugs (such as artesunate) to control the density of malarial parasites at a low level, it was found that using low doses of 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 and kill non-tumor cells, with good safety and low toxic side effects, providing new ideas for the development of new technologies to kill tumors.

Preferably, the antimalarial drug comprises any one of artesunate, chloroquine or bromoquine, or a combination of at least two thereof.

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.

Preferably, the antimalarial drug comprises any one of artesunate, chloroquine or bromoquine, or a combination of at least two thereof.

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

Preferably, the combination is a combination of live Plasmodium vivax and an antimalarial preparation.

In a fourth aspect, the present application provides the use of a combination drug combination of live Plasmodium vivax and an antimalarial drug in the preparation of a drug 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.

The present 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 a high-content animal model of CIK cells, preparing CIK cells, etc.

In a fifth aspect, the present application provides a method for activating CIK cells, the method 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 0.1%, antimalarial drugs are administered to the target subject to control the Plasmodium vivax infection rate of red blood cells to be lower than 0.1% for 30 to 90 days, thereby activating CIK cells in the target subject's peripheral blood.

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

In the present application, the malarial parasite red blood cell infection rate is controlled below 0.1%, which can effectively prevent malarial parasites from suppressing the immune system and avoid serious side effects and complications.

Preferably, the amount of live Plasmodium vivax is 10 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.

Preferably, the antimalarial drug comprises any one of artesunate, bromoquine or chloroquine, or a combination of at least two thereof.

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

The CIK cells in the peripheral blood of the target subject are activated by the method for activating CIK cells in the fifth aspect, and the CIK cells in the peripheral blood of the target subject are separated by extracting the peripheral blood.

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

The present application found 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, and provides new ideas for the development of new technologies for killing tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing the analysis results of the proportion of CIK cells secreting IFN-γ in healthy subjects and patients with non-small cell lung cancer.

FIG. 2 is a diagram showing the statistical analysis results of CIK cells secreting IFN-γ in healthy subjects and patients with non-small cell lung cancer, with * indicating P < 0.05.

FIG3 is a graph showing the analysis results of the proportion of CIK killing tumor cells in healthy subjects and patients with non-small cell lung cancer.

FIG. 4 is a diagram showing the statistical analysis results of CIK killing tumor cells in healthy subjects and patients with non-small cell lung cancer, where * indicates P < 0.05.

DETAILED DESCRIPTION

To further illustrate the technical means and effects adopted by the present application, the present application is further described below in conjunction with the embodiments and drawings. It is understood that the specific implementation methods described herein are only used to explain the present application, rather than to limit the present application.

If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

Example 1

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.

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

Red blood cells (volume 1 ml) containing 5 million live Plasmodium vivax parasites were injected intravenously into 3 patients with non-small cell lung cancer (NSCLC), and then the malarial parasite red blood cell infection rate (parasite blood density) in the patients' peripheral blood was observed every day. When the patient's parasite blood density was higher than 0.1%, 6-12 mg of artesunate was injected intravenously each time to control the parasite density in the patient's body below 0.1%, and this low-density malarial parasitemia was maintained for 90 days, and then sufficient effective antimalarial drugs were used to completely kill the malarial parasites in the patient's body (as a course of treatment). During this period, CIK cells were cultured and tested for activity by taking peripheral blood samples of patients one day before and 30 days after inoculation, and peripheral blood samples of healthy people were taken as controls.

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

1. Take 1 drop (100 μL) of venous peripheral blood from patients with non-small cell lung cancer and healthy adults respectively, and perform anticoagulation treatment;

2. Mix the peripheral whole blood with 2 ml of 1× red blood cell lysis buffer (Biolegend), rotate and shake for 10 seconds, and then stand at 25°C in the dark for 15 minutes;

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);

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

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

The blood cells were centrifuged (350×g) for 5 minutes, the supernatant was poured out, and the cells were suspended in 100 μL of cell staining solution, and then 2 μL of anti-human CD3 and CD56 antibodies (Biolegend) were added, incubated at 25°C for 30 minutes, and then 2 ml of cell staining solution was added, and centrifuged (350×g) was repeated twice, each time for 5 minutes. After pouring out the supernatant, the cells were fixed with 2 ml of 2% formalin solution, and incubated at 25°C in the dark for 20 minutes for use.

4. Analysis of CIK cell function in peripheral blood of patients with non-small cell lung cancer/healthy subjects

1. Suspend the fixed cells in 2 ml of cell permeabilization buffer (Biolegend) and repeat centrifugation (350 × g) for 10 min twice;

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;

3. The incubated cells were suspended in 2 ml of cell permeabilization buffer and then centrifuged (350 × g) for 5 minutes twice;

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

5. Determination of tumor cell apoptosis test ratio

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.

6. Results Analysis

1. Flow cytometry was used to detect the expression of molecule CD56 on human peripheral blood CIK cells. The data were 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); NSCLC patients + Pv represent non-small cell lung cancer patients after inoculation with Plasmodium vivax.

2. Analysis of CIK cell function in human peripheral blood: Flow cytometry was used to determine the secretion expression of cytokine IFN-γ in CD3 ⁺ CD56 ⁺ cells (expressed as %). The results are 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.

Example 2

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

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

In summary, the present application found 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, and has the advantages of good safety and low toxic side effects. Provide new ideas for developing new technologies to kill tumors.

The applicant declares that the present application uses the above-mentioned embodiments to illustrate the detailed methods of the present application, but the present application is not limited to the above-mentioned detailed methods, that is, it does not mean that the present application must rely on the above-mentioned detailed methods to be implemented. The technicians in the relevant technical field should understand that any improvement to the present application, the equivalent replacement of the raw materials of the product of the present application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present application.

Claims (10)

Use of live Plasmodium vivax in the preparation of products for activating CIK cells. The use of a combination of live Plasmodium vivax and an antimalarial drug in the preparation of a product for activating CIK cells. The use 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. A combination for activating CIK cells, comprising live Plasmodium vivax and an antimalarial drug. The combination for activating CIK cells according to claim 4, wherein the antimalarial drug comprises any one or a combination of at least two of artesunate, chloroquine or bromoquine; 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. Application of a combination of live Plasmodium vivax and an antimalarial drug in the preparation of a drug 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. 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. The method for activating CIK cells according to claim 7, wherein the amount of the living Plasmodium vivax is 10 to 10 million. 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 thereof. 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.