Selenized microalgae preparation and application thereof in preparation of immune preparation for treating tumors

Abstract

The invention relates to a selenized microalgae preparation and application thereof in preparation of immune preparations for treating tumors. The microalgae are induced and cultured in a selenium-rich solution for several days, and then are collected and injected to the tumor site to inhibit the growth of the tumor site. The preparation process of the selenized microalgae is simple, and the selenized microalgae can stimulate an organism to generate strong immune response, activate dendritic cells and T cells and regulate an immunosuppressive microenvironment at a tumor through various pathways such as NF-kappa B, so that an efficient and long-term tumor suppression effect is realized; meanwhile, the microalgae can enhance immune checkpoint blocking treatment, and a certain foundation is laid for combined application of microorganism-mediated immunotherapy and immune checkpoint blocking.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms and tumor immunotherapy, and in particular to a selenized microalgae preparation and its application in preparing a tumor immunotherapy preparation. Background Art

[0002] Cancer causes serious damage to human health; its morbidity and mortality rates increase year by year, and it remains one of the major public health issues. The treatment of malignant tumors has undergone continuous development from surgery, radiotherapy, chemotherapy to targeted therapy. Immunotherapy, as an emerging treatment method, mainly aims to improve the patient's own immune system so that it can accurately attack cancer cells. With the phased breakthroughs achieved in this field, immunotherapy has become a hot spot at the forefront of medical research. However, patients receiving immunotherapy, such as immune checkpoint blockade therapy, show large individual differences in response rates; at the same time, immune-related adverse events may occur during treatment, which can be life-threatening in severe cases.

[0003] Microbial therapy has a long history, mainly using foreign microbial substances to stimulate the body to produce an immune response, thereby inhibiting tumor growth. The use of Bacillus Calmette-Guerin Vaccine for the perfusion treatment of human non-muscle invasive bladder cancer is one of the typical clinical options. However, with the deepening of research, some problems have also been exposed in the clinical application of microbial therapy. Taking most new bacterial therapies as an example, although live bacteria can activate a strong immune response, there are still certain risks in clinical use; inactivated bacteria have limited therapeutic effects. How to break through the existing limitations and develop an efficient and safe selenized microalgae treatment strategy is the key to achieving radical cure of tumors. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention aims to develop an efficient and safe selenized microalgae preparation. At a time when health needs are growing, microalgae have shown great development value in the fields of health care products due to their good biological activity and reliable safety, and have become one of the focuses of attention in the biopharmaceutical industry. Inspired by this, the present invention provides a selenized microalgae preparation and its application in the preparation of tumor immunotherapy preparations. In the present invention, the microalgae Synechococcus.sp will form orange selenium particles when exposed to high Na2SeO3 concentrations. After the selenized Synechococcus.sp is injected into the tumor site, the immunosuppressive microenvironment in the tumor can be improved, thereby achieving tumor immunotherapy.

[0005] The present invention is achieved through the following technical solutions:

[0006] The first object of the present invention is to provide a selenized microalgae preparation, comprising selenized microalgae and an immune checkpoint blocker.

[0007] In one embodiment of the present invention, the selenized microalgae are obtained by culturing microalgae in a selenium-rich culture medium.

[0008] In one embodiment of the present invention, the microalgae is a Synechococcus species.

[0009] In one embodiment of the present invention, the amount of selenium source added to the selenium-enriched culture medium is 5 mM-20 mM.

[0010] In one embodiment of the present invention, the culture conditions are: culturing at 20° C.-25° C. for 6 to 8 days.

[0011] Selenized microalgae are cultured in a selenium-rich medium as follows: Synechococcus sp. is cultured in a light incubator until the logarithmic growth phase, and the concentration is adjusted to 0.5×10 7 / mL-2×10 7 / mL, and dispersed in selenium-rich culture medium, and continued to culture for 6-8 days to obtain selenized microalgae (Selenized S.sp).

[0012] In one embodiment of the present invention, the immune checkpoint blocker is selected from one or more of αCTLA-4 antibody, αPD-L1 antibody and αPD-1 antibody.

[0013] The second object of the present invention is to provide the use of the selenized microalgae preparation in the preparation of tumor treatment immune preparations.

[0014] In one embodiment of the present invention, the frequency of use of the tumor immunotherapy preparation is 1-3 times.

[0015] In one embodiment of the present invention, the concentration of selenized microalgae in the tumor treatment immunotherapy preparation is 1×10 8 / mL-4×10 8 pieces / mL.

[0016] In one embodiment of the present invention, the tumor is selected from colon cancer and / or bladder cancer.

[0017] The present invention injects the selenized S.sp of this concentration into the tumor tissue to activate the body's immune response and achieve immunotherapy. 8 / mL-4×10 8 Selenated S.sp at a concentration of 100mg / mL can enhance immune checkpoint blockade therapy, including αCTLA-4 antibodies.

[0018] The present invention is an immunotherapy mediated by selenized S.sp, which can significantly inhibit the growth of solid tumors. At the same time, its inherent immune active components and selenium nanoparticles synthesized in selenium-rich culture medium can stimulate the maturation of dendritic cells (DCs) through pathways such as nuclear factor kappa-B (NF-κB), upregulate the expression of costimulatory molecules CD80, CD86 and major histocompatibility complex class II (MHC II), and thus activate CD8 + Cytotoxic T lymphocytes (CTLs) and CD4 + It stimulates T helper (Th) cell function and increases the infiltration of immune cells into the tumor, thereby improving the immunosuppressive microenvironment. Furthermore, after receiving the selenized S.sp injection, the tumor's responsiveness to immune checkpoint blockers, such as the αCTLA-4 antibody, is enhanced, further improving the therapeutic effect.

[0019] The above technical solution of the present invention has the following advantages over the prior art:

[0020] (1) The algae species used in the present invention is Synechococcus sp., which can be obtained from nature and has strong survival and reproduction capabilities.

[0021] (2) The preparation process of selenized S.sp by the present invention is simple and can be expected to achieve engineering mass production.

[0022] (3) The selenized S.sp of the present invention has good biocompatibility and has a selective inhibitory effect on the growth of tumor cells.

[0023] (4) The selenized S.sp of the present invention has a good immune activation effect. After intratumoral injection, the selenized S.sp can stimulate DC maturation and antigen presentation through multiple pathways, release pro-inflammatory cytokines, and thus promote T cell differentiation and intratumoral infiltration, thereby achieving efficient immunotherapy.

[0024] (5) The selenized S.sp of the present invention has the effect of enhancing immune checkpoint blockade therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0026] Figure 1Figures 1 and 2 show selenized S.sp and purified selenium nanoparticles after induction culture; A is a scanning electron micrograph of selenized S.sp; and B is a transmission electron micrograph of purified selenium nanoparticles.

[0027] Figure 2 is the cytotoxicity of selenized S.sp in Example 1; wherein A is the toxicity of selenized S.sp on the mouse dendritic cell line DC2.4; B is the toxicity of selenized S.sp on the mouse colon cancer cell line CT26; C is the toxicity of selenized S.sp on the mouse bladder cancer cell line MB49;

[0028] Figure 3 is the maturity of mouse bone marrow-derived dendritic cells (BMDCs) after stimulation with selenized S.sp in Example 1;

[0029] Figure 4 is the growth curve of mouse tumor after intratumoral injection of selenized S.sp in Example 1;

[0030] Figure 5 is the maturity of DCs in the tumor-draining lymph nodes of mice after intratumoral injection of selenized S.sp in Example 1;

[0031] Figure 6 Figure 1 shows the CTLs (A) and Th cell infiltration (B) in mouse tumors after intratumoral injection of selenized S.sp in Example 1.

[0032] Figure 7 These are the growth curves of the proximal (A) and distal tumors (B) of mice after intratumoral injection of selenized S.sp and immune checkpoint blocker αCTLA-4 antibody in Example 1. DETAILED DESCRIPTION

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0034] While live bacteria can stimulate a strong immune response, they still present certain clinical risks. Inactivated bacteria have limited therapeutic efficacy. Therefore, to overcome existing limitations and develop an efficient and safe microbial immunotherapy strategy, the present invention provides a selenized microalgae preparation and its use in the preparation of a drug for treating tumors. The induced cultured selenized microalgae exhibits a certain immunostimulatory effect. Intratumoral injection can improve the immunosuppressive tumor microenvironment, thereby achieving microalgae-mediated immunotherapy.

[0035] In the present invention, the microalgae is Synechococcus sp., which can produce selenium nanoparticles during the culture process; the culture cell concentration is 0.5×107 / mL-2×10 7 pieces / mL.

[0036] In the present invention, the selenium source in the selenium-enriched BG11 culture medium of the microalgae Synechococcus.Sp is Na2SeO3, and the concentration is 5μM-20μM.

[0037] In the present invention, the microalgae Synechococcus.Sp is cultured for 6-8 days, with a light-dark ratio of 12:12.

[0038] In the present invention, selenized Synechococcus sp has a certain immune stimulation function, and the concentration of microalgae injected into the tumor is 1×10 8 / mL-4×10 8 pieces / mL.

[0039] In the present invention, the selenized Synechococcus.sp has a certain immune stimulation function, and the number of intratumoral injections is 1-3 times.

[0040] In the present invention, selenized Synechococcus.sp has a certain immune checkpoint blocking sensitization function, and the immune checkpoint blocker is an αCTLA-4 antibody; the number of intratumoral injections is 1-3 times.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are all commercially available unless otherwise specified.

[0042] The Synechococcus sp. (FACHB-805) was purchased from the Freshwater Algae Seed Bank of the National Aquatic Biological Germplasm Resources Bank.

[0043] BG11 culture medium (HB8793) was purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0044] αCTLA-4 antibody (BE0164) was purchased from BioXCell.

[0045] Example 1: Inducing selenization of Synechococcus.sp

[0046] The Synechococcus sp. (FACHB-805) was placed in a conical flask and cultured to the logarithmic growth phase, and the concentration was adjusted to 1×10 7 After culturing in BG11 medium containing 10mM Na2SeO3 for 6 days at room temperature, selenized S.sp was obtained. After 30kHz ultrasonic disruption, selenium nanoparticles were purified by centrifugation. Figure 1As shown, it can be seen that S.sp and the selenium nanoparticles it generates have a particle size of about 50nm-100nm.

[0047] Example 2: Cytotoxicity of Selenated S.sp

[0048] Selenized S.sp was co-incubated with mouse dendritic cell line DC2.4, mouse colon cancer cell line CT26, and mouse bladder cancer cell line MB49 for 12 hours. Figure 2 As shown. Figure 2 It can be seen that selenized S.sp has no significant cytotoxicity to DC2.4, but has a certain inhibitory effect on the proliferation of CT26 and MB49.

[0049] Example 3: Selenated S.sp stimulates maturation of mouse bone marrow-derived DCs

[0050] Selenized S.sp was co-incubated with bone marrow-derived dendritic cells (BMDCs) for 12 h, and the expression changes of CD80 and CD86 were detected by flow cytometry. Figure 3 As shown. Figure 3 It can be seen that after stimulation of selenium S.sp, CD80 + CD86 + The proportion of BMDCs was significantly increased, suggesting that selenized S.sp has certain immune activation function.

[0051] Example 4: Treatment of tumors with selenized S.sp

[0052] Two experimental groups were set up, namely the control group (untreated) and the selenized S.sp group. When the volume of the subcutaneous CT26 tumor of mice reached 50-100mm 3 Treatment started when the tumor volume reached 1000mm 3 When the mice were considered dead, the tumor volume was stopped. 8 Selenized S.sp was uniformly injected into the CT26 tumor at a dose of 50 μL. The entire process was repeated three times. The tumor volume of mice in different experimental groups was measured using a vernier caliper at different time points. The tumor growth curve of the mice is shown in Figure 2. Figure 4 As shown, it can be seen that compared with the control group, selenized S.sp can significantly inhibit tumor growth and increase the survival rate of mice to 4 / 5.

[0053] Example 5: Selenized S.sp improves the tumor immunosuppressive microenvironment

[0054] On the 3rd and 7th day, the tumor-draining lymph nodes and tumor samples of each group of mice in Example 4 were collected to prepare single cell suspensions. The maturity of DCs in the lymph nodes and tumors, CTLs and Th cell infiltration were analyzed by flow cytometry. Figure 5 As shown in the figure, compared with the control group, the maturity of DCs in the mouse lymph nodes and tumors was significantly improved on the 3rd day after intratumoral injection of selenized S.sp, indicating that selenized S.sp can activate the body's innate immunity. On the 7th day, the CTLs infiltration of mouse tumors was as follows: Figure 6 As shown, the proportions of CTLs and Th cells increased in the selenized S.sp group, suggesting the occurrence of a later adaptive immune response. These results demonstrate that the selenized S.sp-based therapeutic strategy can improve the immunosuppressive microenvironment within the tumor, thereby killing the tumor.

[0055] Example 6: Selenated S.sp enhances immune checkpoint blockade therapy:

[0056] Three experimental groups were set up, namely control group (untreated), αCTLA-4 group and selenized S.sp+αCTLA-4 group. When the volume of the subcutaneous CT26 proximal tumor of mice reached 50-100mm 3 Treatment was started when the proximal or distal tumor volume reached 1000 mm 3 When the tumor volume was ≥ 100 μg / kg, the mice were considered dead and the tumor volume was stopped. 8 Selenized S.sp was uniformly injected into the CT26 tumor at a dose of 50 μL, followed by intraperitoneal injection of αCTLA-4 antibody at 1 mg / kg into the mouse. The entire process was repeated three times. The tumor volume of mice in different experimental groups was measured using a vernier caliper at different time points. The proximal and distal tumor growth curves of mice are shown in Figure 2. Figure 7 As shown, there was no significant difference in tumor volume between the αCTLA-4 group and the control group, suggesting a low response rate to the immune checkpoint blocker in mice. However, after injection of selenized S.sp, the mice's response to the αCTLA-4 antibody treatment was significantly enhanced. This combined treatment even activated the systemic immune response, successfully achieving complete regression of proximal and distal tumors in 5 mice in the group, with no signs of recurrence. This discovery provides a new approach to overcoming the limitations of single-agent immune checkpoint blockers, and also lays an experimental foundation for the combined use of microbial-mediated immunotherapy and immune checkpoint blockers.

[0057] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A selenized microalgae preparation, characterized in that: Including selenized microalgae and immune checkpoint blockers.

2. The selenized microalgae preparation according to claim 1, characterized in that The selenized microalgae are obtained by culturing the microalgae in a selenium-rich culture medium.

3. The selenized microalgae preparation according to claim 2, characterized in that: The microalgae is a Synechococcus species.

4. The selenized microalgae preparation according to claim 2, characterized in that The amount of selenium source added to the selenium-enriched culture medium is 5mM-20mM.

5. The selenized microalgae preparation according to claim 2, characterized in that: The culture conditions are: 20° C.-25° C. for 6 to 8 days.

6. The selenized microalgae preparation according to claim 1, characterized in that The immune checkpoint blocker is selected from one or more of αCTLA-4 antibody, αPD-L1 antibody and αPD-1 antibody.

7. Use of the selenized microalgae preparation according to any one of claims 1 to 6 in the preparation of a tumor treatment immune preparation.

8. The use according to claim 7, characterized in that The frequency of using the tumor treatment immune preparation is 1-3 times.

9. The use according to claim 7, characterized in that The concentration of selenized microalgae in the tumor treatment immune preparation is 1×10 8 / mL-4×10 8 pieces / mL.

10. The use according to claim 7, characterized in that The tumor is selected from colon cancer and / or bladder cancer.

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