Application and preparation method of chlorella spores for relieving T cell depletion

Abstract

The invention relates to the technical field of biological medicines, and discloses a preparation method of chlorella spores, which comprises the following steps: taking a chlorella solution, and carrying out primary centrifugation at 4 DEG C to obtain primary supernate; carrying out secondary centrifugation on the primary supernate at 4 DEG C to obtain secondary supernate; centrifuging the secondary supernatant for the third time, and discarding the supernatant to obtain a chlorella spore precipitate; the invention further discloses application of the chlorella spores in relieving T cell depletion. The invention provides an immunotherapy preparation with active chlorella spores, aiming at the problems of chronic infection and T cell depletion in a tumor microenvironment, the immunotherapy preparation relieves ammonia accumulation in the tumor microenvironment through ammonia metabolism, regulates the ammonia metabolism process in the tumor microenvironment, relieves the inhibition effect on T cells, and improves the immunotherapy effect. The T cell depletion state is reversed to a certain extent.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of Chlorella spores in alleviating T cell exhaustion and a preparation method thereof. Background Art

[0002] In chronic viral infection and tumor microenvironment, due to continuous and repeated antigen stimulation, CD8+T cells gradually lose their function and enter a specific differentiation state, which is called T cell exhaustion. Unlike normal effector T cells, these exhausted T cells lose their immune response function, and the expression of inhibitory receptors (such as PD-1, CTLA-4, TIM-3, LAG-3, etc.) increases, and their ability to kill tumor cells is significantly reduced.

[0003] The formation of exhausted T cells is one of the important mechanisms of immune escape, and it is also a major reason why many chronic infections and tumors are difficult to cure. At present, T cell exhaustion is mainly inhibited by using immune checkpoint blockers in clinical practice, but a single therapeutic target has limited therapeutic effect in the face of some specific tumors, especially affected by some metabolites in the tumor microenvironment. These metabolites generally have an inhibitory effect on the function of T cells, leading to T cell dysfunction. Therefore, targeting the formation mechanism of exhausted T cells and finding ways to restore their function is an important research direction in the field of immunotherapy, which is of great significance to tumor immunotherapy.

[0004] Related studies have shown that in some cancer mouse models, ammonia, as a ubiquitous metabolite, accumulates in large quantities in the tumor microenvironment. These high concentrations of ammonia inhibit T cell growth and function, leading to T cell exhaustion and immunotherapy resistance. These findings provide a new idea for reversing T cell exhaustion: whether it is possible to alleviate the process of T cell exhaustion from the perspective of ammonia metabolism, relieve the immunosuppressive state in the tumor microenvironment, and synergize with conventional immunotherapy methods to solve the problem of drug resistance during tumor treatment, restore T cell function, and achieve better tumor treatment effects.

[0005] Therefore, how to remove the accumulated ammonia in the tumor microenvironment is an urgent problem to be solved. Summary of the invention

[0006] The purpose of the present invention is to provide an application and preparation method of Chlorella spores for alleviating T cell exhaustion, so as to solve the technical problems in the prior art.

[0007] In order to solve the above technical problems, the present invention specifically provides the following technical solutions:

[0008] The present invention provides a method for preparing Chlorella spores, comprising the following steps:

[0009] The Chlorella solution was centrifuged for the first time at 4°C to obtain a primary supernatant;

[0010] Centrifuging the primary supernatant for a second time at 4° C. to obtain a secondary supernatant;

[0011] The secondary supernatant is centrifuged for the third time, and the supernatant is discarded to obtain a Chlorella spore pellet.

[0012] As a preferred embodiment of the present invention, the first centrifugation has a rotation speed of 1300-1500 rpm and a centrifugation time of 5-7 min;

[0013] The second centrifugation speed is 4500-5500 rpm, and the centrifugation time is 9-11 min;

[0014] The rotation speed of the third centrifugation is 14000g, and the centrifugation time is 1 hour.

[0015] As a preferred embodiment of the present invention, the Chlorella spore precipitate is dissolved using a PBS solution.

[0016] As a preferred embodiment of the present invention, the diameter of the Chlorella spores is 100-300 nm.

[0017] As an application of Chlorella spores in alleviating T cell exhaustion of the present invention, the Chlorella spores alleviate T cell exhaustion by metabolizing ammonia.

[0018] As a preferred embodiment of the present invention, it also includes a product prepared from the Chlorella spores;

[0019] The product is an immunotherapeutic preparation targeting ammonia metabolism;

[0020] The immunotherapeutic preparation contains a pharmaceutically acceptable carrier and the Chlorella spores.

[0021] As a preferred embodiment of the present invention, the administration route of the immunotherapy preparation includes intravenous administration, subcutaneous administration or local administration.

[0022] As a preferred embodiment of the present invention, the Chlorella spores are used in combination with PD-1 monoclonal antibody to reduce the ammonia concentration in tumor interstitial fluid.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides an immunotherapy preparation with active Chlorella spores by exploring the ammonia-degrading ability of Chlorella. This immunotherapy preparation targets the problems of chronic infection and T cell exhaustion in the tumor microenvironment, alleviates the accumulation of ammonia in the tumor microenvironment through ammonia metabolism, regulates the ammonia metabolism process in the tumor microenvironment, reduces the inhibitory effect on T cells, and reverses the T cell exhaustion state to a certain extent.

[0025] The immunotherapy preparation disclosed in the present invention has the advantages of good biocompatibility and low immunogenicity, and can be used in combination with general anti-tumor drugs. After being used in combination with PD-1, its ability to deaminize is further improved, promoting CD8+T cell infiltration, reducing exhaustion-related indicators, and promoting CD8+T cell proliferation and the enhancement of TNF-α expression, and has good clinical application prospects in the field of tumor treatment immunity.

[0026] The invention obtains the chlorella spores by multiple centrifugation, and the preparation method is simple and easy to implement. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0028] Figure 1 The present invention provides a schematic flow chart of a method for preparing Chlorella spores;

[0029] Figure 2 This is a transmission electron microscope characterization of the morphology of Chlorella spores;

[0030] Figure 3 Particle size characterization of Chlorella spores for nanoparticle tracking analysis;

[0031] Figure 4 A table showing the ability of Chlorella spores to deammonify in vitro environment for ammonia content detection;

[0032] Figure 5 This is a graph showing the effect of Chlorella spores on T cell exhaustion indicators detected by fluorescent real-time quantitative PCR;

[0033] Figure 6 This is a photo of the image analysis of the tumor size in the breast cancer mouse model to determine the therapeutic effect of Chlorella spores on breast cancer;

[0034] Figure 7This is a photo of the image analysis of the tumor size in the breast cancer mouse model to determine the therapeutic effect of the combination of Chlorella spores and PD-1 monoclonal antibody on breast cancer;

[0035] Figure 8 The results of the investigation of the ammonia content detection of Chlorella spores on the ammonia removal ability in the tumor microenvironment of the breast cancer mouse model;

[0036] Fig. 9 This is the result of flow cytometry detection of the effect of Chlorella spores on CD3+CD8+ cell infiltration in breast cancer mouse model;

[0037] Fig.10 This is a flow cytometry test showing the effect of Chlorella spores on CD3+CD8+ cell-related exhaustion indicators in a breast cancer mouse model;

[0038] Fig.11 This is the result of flow cytometry detection of the effect of Chlorella spores on CD3+CD8+ cell proliferation and tumor killing ability in a breast cancer mouse model. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] like Figure 1 As shown, the present invention provides a method for preparing biological spores, which are quasi-spores derived from Chlorella, specifically named as microalgae coccospores, and the microalgae coccospores can be used in the preparation of products for alleviating T cell exhaustion. Figure 2 as well as Figure 3 The morphology and particle size of Chlorella spores were characterized by transmission electron microscopy and nanoparticle tracking analysis.

[0041] Algae can effectively remove ammonia nitrogen and nitrite nitrogen from water bodies, and are widely used in life to improve water quality and reduce eutrophication of water bodies. Inorganic nitrogen sources in the environment are actively or passively transported into cells through specific transport proteins on the cell membrane of algae plants, and converted into their own components to participate in the metabolic process in the body.

[0042] Utilizing the biological characteristics of algae, the present invention provides a novel immunotherapy preparation targeting ammonia metabolism to alleviate the accumulation of ammonia in the tumor microenvironment and reverse the state of T cell exhaustion.

[0043] Chlorella is a single-cell eukaryotic green algae with a diameter of 2 to 12um. It has a short growth cycle, strong reproductive capacity, good tolerance, and a wide ecological distribution. Chlorella does not need to go through the nitrification process but can directly use the assimilation of ammonia, converting and absorbing ammonia in the environment into the cell body for use, and has a strong potential for ammonia removal.

[0044] The present invention utilizes Chlorella spores as a novel drug carrier, giving full play to its own ammonia-degrading effect and advantages such as good biocompatibility and low immunogenicity, and is used in combination with general anti-tumor drugs to regulate the ammonia metabolism process in the tumor microenvironment and reduce the inhibitory effect on T cells, which has good clinical application prospects in the field of tumor treatment immunity.

[0045] The immunotherapy preparation disclosed in the present invention, wherein the active ingredient is Chlorella spores, may also provide other pharmaceutically acceptable carriers for use in combination with the active ingredient, such as PBS solution, physiological saline, etc.

[0046] The immunotherapy preparation of the present invention can act directly on an organism, and the administration route includes intravenous administration, subcutaneous administration or local administration. After administration, the Chlorella spores act in the organism to alleviate the exhaustion of T cells.

[0047] Specifically, the steps for preparing Chlorella spores are as follows:

[0048] The Chlorella culture solution is cultured at room temperature and under light to a suitable density to obtain a Chlorella solution, the Chlorella solution is centrifuged for the first time at 4°C to obtain a primary supernatant, the primary supernatant is centrifuged for the second time at 4°C to obtain a secondary supernatant, the secondary supernatant is centrifuged for the third time, the supernatant is discarded, and a Chlorella spore precipitate is obtained.

[0049] The invention obtains chlorella spores with a diameter of 100-300nm by centrifuging the chlorella solution multiple times. The preparation method is simple and easy to implement.

[0050] Preferably, the first centrifugation speed is 1300-1500 rpm, the centrifugation time is 5-7 min, the second centrifugation speed is 4500-5500 rpm, the centrifugation time is 9-11 min, and the third centrifugation speed is 14000 g, and the centrifugation time is 1 h.

[0051] After obtaining the precipitate, the precipitate can be dissolved using a PBS solution to obtain an immunotherapy preparation.

[0052] The following examples further illustrate the chlorella spores:

[0053] Embodiment 1:

[0054] (1) Preparation of Chlorella Spore Solution:

[0055] The chlorella used was a freshwater species, Latin species name: Chlorella sp, purchased from the freshwater algae species bank of the Institute of Hydrobiology, Chinese Academy of Sciences, with a specification of 500 ml.

[0056] Take 200mL of Chlorella solution with good growth status and moderate density, divide the solution into ordinary 50ml centrifuge tubes, centrifuge at 1500rpm for 6min, transfer the supernatant to a new centrifuge tube after centrifugation, centrifuge at 5000rpm for 10min, and continue to transfer the supernatant to an ultracentrifuge tube; centrifuge at 14000g, 4℃ for 1h, discard the supernatant, dissolve the precipitate with 1mL PBS to obtain 1mL Chlorella spore solution.

[0057] The particle size and concentration of Chlorella spores were detected by nanoparticle tracking analyzer (NTA).

[0058] Experimental results: The spore concentration was 2×10 10 / mL, diameter is 100-300nm, see Figure 3 .

[0059] The function of the Chlorella spore solution prepared in Example 1 is explained by verification below.

[0060] (2) After incubation with NH4Cl, the concentration of ammonia can be reduced

[0061] Chlorella spores (Example 1), Scenedesmus spores, and Oocystis spores were added to NH4Cl solutions of the same concentration, respectively, and the concentration change of ammonia in the solution was detected after incubation for 16 hours.

[0062] Chlorella spores and H22 tumor cell-derived vesicles were added to NH4Cl solutions of the same concentration, and the concentration of ammonia in the solution was detected after incubation for 16 hours. The concentration of NH4Cl was 10 mM, and the concentrations of spores and vesicles used were the same, 2×10 10 Pieces / mL.

[0063] Experimental results:

[0064] 1. After Chlorella spores were incubated with NH4Cl for 16 hours, the concentration of ammonia in the solution was detected to be reduced ( Figure 3 ).

[0065] 2. Compared with Scenedesmus spores, Oocystis spores, and H22 tumor cell vesicles, Chlorella spores had a more obvious reduction effect.

[0066] (3) After 24 hours of co-culture with NH4Cl, the expression of T cell exhaustion indicators PD-1, LAG3, TIM3, and CTLA4 genes decreased

[0067] T cells were extracted from the spleen of 4-week-old C57BL / 6 mice using a mouse CD8+T cell magnetic bead sorting kit, and IL-2 (10 ng / mL) and anti-CD3 / 28 antibodies were added to activate T cells. The cells were cultured for three days and seeded into six-well plates. 2×10 5 T cells.

[0068] The cells were divided into four groups: blank control group, group with NH4Cl, group with NH4Cl and 2×10 9 spores of Chlorella vulgaris, adding NH4Cl and 2×10 10 / mL Chlorella spores.

[0069] Incubate for 24 hours in a 37°C, 5% CO2 incubator. Collect RNA from the four groups of T cells, and use qPCR to detect the gene expression of T cell exhaustion indicators PD-1, LAG3, TIM3, and CTLA4 in each group. The concentration of NH4Cl was 10mM.

[0070] Experimental results: Compared with the blank control group, the T cell exhaustion index increased in the group adding NH4Cl, and decreased in the group adding Chlorella spores. 10 Compared with 2×10 9 The level of decrease in the group with Chlorella spores / mL was even greater ( Figure 5 ).

[0071] It can be seen that Chlorella spores are the effective ingredients that consume NH4Cl.

[0072] (4) Chlorella spores have a good therapeutic effect on mouse breast cancer model

[0073] Four-week-old BALB / c mice were used to construct a breast cancer mouse model. Each mouse was subcutaneously injected with 2×10 5 4T1 cells (mouse breast cancer cell line).

[0074] The mice were divided into PBS group, Chlorella spore group, and Oocystis spore group, with 6 mice in each group. The mice were injected with drugs through the tail vein every other day from the 7th day of tumor bearing, for a total of five injections. The injection volume of the PBS group was 0.1 mL, and the injection concentration of the Oocystis spore group and the Chlorella spore group was 2×10 10 / mL, injection volume 0.1mL. On the 21st day, the tumor site of the mouse was removed to observe the size and morphological changes.

[0075] Experimental results: Chlorella spores have a good therapeutic effect on mouse breast cancer model ( Figure 6 ).

[0076] (5) Combined with PD-1 monoclonal antibody, it has a good therapeutic effect on mouse breast cancer

[0077] Four-week-old BALB / c mice were used to construct a breast cancer mouse model. Each mouse was subcutaneously injected with 2×10 5 4T1 cells.

[0078] The mice were divided into PBS group, Chlorella spores alone group, PD-1 monoclonal antibody alone group, and Chlorella spores and PD-1 monoclonal antibody combined group, with 6 mice in each group. The mice were injected once every other day through the tail vein from the 7th day of tumor bearing, for a total of five injections. The injection volume was 0.1 mL, the PD-1 monoclonal antibody injection concentration was 10 ug / mouse, and the injection concentration of the Chlorella spore group was 2×10 10 pcs / mL.

[0079] On the 21st day, the mouse tumor sites were removed to observe changes in size and morphology. The tumor tissue was then chopped up and centrifuged at 2000rpm for 10 minutes to extract the tumor interstitial fluid. The kit was used to detect the concentration of ammonia in the tumor tissue interstitial fluid. The chopped tumor tissue was then placed in 3mL of the prepared digestion solution and digested at 37°C for 1.5 hours, then ground and filtered through gauze. The filtrate was centrifuged at 800rpm for 5 minutes, the supernatant was transferred to a new centrifuge tube, and centrifuged at 400g for 5 minutes. 3mL of ELS solution was added to the resulting precipitate for red blood cell lysis. After standing for 5 minutes, 3mL of serum-containing culture medium was added for neutralization, and centrifuged at 400g for 5 minutes.

[0080] The supernatant was discarded to obtain a cell pellet containing immune cells. The cell suspension was adjusted to an appropriate density with a certain amount of PBS, and then stained with flow cytometry antibodies CD3, CD8, CD45, PD-1, CTLA4, ki67, and TNF-α, and analyzed using a flow cytometer.

[0081] Digestion solution: DNase 5μg / μl, hyaluronidase 100mg / μl, collagenase 1mg / μl, DMEM medium. ELS solution: NH4Cl4.14g, NaHCO3 0.51g, EDTA 0.0186g, add water to make up to 500mL.

[0082] Experimental results: Compared with the PBS control group, the group of single administration of Chlorella spores, the group of single administration of PD-1 monoclonal antibody, and the group of combined administration of Chlorella spores and PD-1 monoclonal antibody all showed certain therapeutic effects, among which the combined administration of Chlorella spores and PD-1 monoclonal antibody had the best effect on tumor treatment ( Figure 6 ), the concentration of ammonia in the tumor interstitial fluid decreased more ( Figure 7 ), promoting CD8+T cell infiltration, reducing exhaustion-related indicators, and promoting CD8+T cell proliferation and TNF-α expression ( Figure 8, 9 , 10).

[0083] Through this embodiment, it can be explained that the biological spores provided by the present invention have a strong ammonia-degrading ability, and can be used in the preparation of products for alleviating T cell exhaustion. The Chlorella spores are used as a new type of drug carrier to give full play to their own ammonia-degrading effect and good biocompatibility and low immunogenicity. At the same time, they are combined with general anti-tumor drugs to regulate the ammonia metabolism process in the tumor microenvironment and reduce the inhibitory effect on T cells. It has a good clinical application prospect in the field of tumor treatment immunity.

[0084] The present invention provides a new method for improving ammonia accumulation in the tumor microenvironment through the ammonia-degrading effect of Chlorella spores, thereby reversing the T cell exhaustion state. The technical scheme includes preparing Chlorella spores, verifying their ammonia-degrading ability through in vitro experiments, and evaluating their effects on restoring T cell function and inhibiting tumor growth in animal models.

[0085] The main use of the present invention is to serve as a potential immunotherapy to improve the efficiency of tumor immunotherapy and provide a new strategy for cancer treatment.

[0086] The above embodiments are only exemplary embodiments of the present application and are not intended to limit the present application. The protection scope of the present application is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present application within the essence and protection scope of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present application.

Claims

1. A use of Chlorella spores in alleviating T cell exhaustion, characterized in that: The Chlorella spores alleviate T cell exhaustion by metabolizing ammonia.

2. The use of Chlorella spores in alleviating T cell exhaustion according to claim 1, characterized in that: Including products prepared from the Chlorella spores; The product is an immunotherapeutic preparation targeting ammonia metabolism; The immunotherapeutic preparation contains a pharmaceutically acceptable carrier and the Chlorella spores.

3. The use of Chlorella spores in alleviating T cell exhaustion according to claim 2, characterized in that: The administration routes of the immunotherapeutic preparation include intravenous, subcutaneous or local administration.

4. The use of Chlorella spores in alleviating T cell exhaustion according to claim 1, characterized in that: The Chlorella spores are used in combination with PD-1 monoclonal antibody to reduce the ammonia concentration in tumor interstitial fluid.

5. A method for preparing the Chlorella spores according to any one of claims 1 to 4, characterized in that: The steps include: The Chlorella solution was centrifuged for the first time at 4°C to obtain a primary supernatant; Centrifuging the primary supernatant for a second time at 4° C. to obtain a secondary supernatant; The secondary supernatant is centrifuged for the third time, and the supernatant is discarded to obtain a Chlorella spore pellet.

6. The method according to claim 5, characterized in that The first centrifugation has a rotation speed of 1300-1500 rpm and a centrifugation time of 5-7 min; The second centrifugation speed is 4500-5500 rpm, and the centrifugation time is 9-11 min; The rotation speed of the third centrifugation is 14000g, and the centrifugation time is 1 hour.

7. The method according to claim 5, characterized in that The Chlorella spore precipitate is dissolved by using PBS solution.

8. The method according to claim 5, characterized in that The diameter of the Chlorella spore is 100-300 nm.

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