Bacterium calmette-guerin EVs and specific protein activated macrophage as well as method and application thereof for treating ovarian cancer

Abstract

The invention relates to bacillus calmette-guerin (EVs) and specific protein activated macrophages as well as a method and application thereof for treating ovarian cancer. Immunotherapy is carried out by utilizing the EVs and specific proteins FD1, CSPA and PMM2 thereof. Ovarian cancer is a common malignant tumor in women, treatment of ovarian cancer faces many challenges, and especially prognosis of advanced patients is poor. The invention aims to enhance the curative effect of chemotherapy, inhibit tumor growth and improve the survival rate and life quality of ovarian cancer patients by activating the immune system of the body and regulating the immune microenvironment. EVs in a BCG culture supernatant are extracted and purified, specific proteins FD1, CSPA and PMM2 are purified from a BCG culture, and the specific proteins are taken as adjuvant therapy drugs to be given to ovarian cancer patients. Compared with a traditional chemotherapy regimen, the treatment method provided by the invention has a higher treatment effect and a lower adverse reaction rate, and provides a new, safer and more effective treatment choice for ovarian cancer patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and specifically to BCG EVs and specific proteins to activate macrophages and methods and applications thereof for treating ovarian cancer. Background Art

[0002] Ovarian cancer, as a malignant tumor of the female reproductive system, poses a serious threat to women's life and health due to its characteristics such as no obvious early symptoms, easy recurrence and easy drug resistance. Although with the continuous advancement of medical technology, the treatment of ovarian cancer is becoming increasingly diversified, including surgical resection, chemotherapy, radiotherapy, etc., the five-year survival rate of patients with advanced ovarian cancer is still low, and the treatment effect is not ideal.

[0003] BCG, as a commonly used immunomodulator, has shown significant immunomodulatory effects in the treatment of various diseases. BCG can activate the body's immune system, promote the proliferation and differentiation of immune cells, and improve the body's immunity. BCG extracellular vesicles (EVs), as an important bioactive substance secreted by BCG, contain a variety of immunomodulatory proteins and active factors, and have potential immunomodulatory and anti-tumor effects.

[0004] BCG EVs and their specific proteins FD1, CSPA and PMM2 have potential application value in the immunotherapy of ovarian cancer. As important components of BCG EVs, these specific proteins have significant immunomodulatory functions, which can activate the body's immune system and regulate the immune microenvironment, thereby achieving the killing and inhibition of ovarian cancer cells. Therefore, an adjuvant treatment strategy for ovarian cancer based on BCG EVs and their specific proteins is needed. This strategy will include key steps such as methods for extracting and purifying BCG EVs and their specific proteins, research to determine the optimal dose and administration method, and clinical trials to evaluate the therapeutic effect and safety. Provide new treatment options and hope for ovarian cancer patients, and further improve the prognosis and quality of life of ovarian cancer patients. Summary of the invention

[0005] In order to solve the problems of the prior art, the present invention provides BCG EVs and specific proteins to activate macrophages and methods and applications for treating ovarian cancer.

[0006] In order to solve the above technical problems, the present invention is implemented by the following technical solutions: In the first aspect, BCG EVs and specific proteins activate macrophages and their methods and applications for treating ovarian cancer, the method comprising the following steps:

[0007] Step S1: preparing BCG extracellular vesicles (EVs) containing specific microbial proteins FD1 (ferredoxin), CSPA (cold shock protein) and PMM2 (phosphomannose mutase 1);

[0008] Step S2: While maintaining the first-line platinum-based chemotherapy regimen for ovarian cancer patients, the BCG EVs prepared in step S1 are injected into the patient's body by intraperitoneal injection or intramuscular injection to specifically activate immune-activated macrophages (M1 macrophages), thereby enriching them in ovarian tumors and inhibiting the growth of ovarian cancer.

[0009] In this implementation, the specific workflow is as follows:

[0010] Preparation of BCG extracellular vesicles (EVs):

[0011] Collect and purify extracellular vesicles (EVs) from cultured BCG bacteria. This process may involve cell culture, centrifugation, filtration, ultrafiltration and other steps to ensure the purity and concentration of EVs.

[0012] Verify whether EVs contain specific microbial proteins FD1 (ferredoxin), CSPA (cold shock protein) and PMM2 (phosphomannose mutase 1).

[0013] Patient Screening and Preparation:

[0014] Patients meeting the diagnostic criteria for ovarian cancer are screened and evaluated for suitability for the adjuvant therapy of the present invention.

[0015] Ensure that patients have completed initial treatment with a first-line platinum-based chemotherapy regimen before receiving treatment.

[0016] Adjuvant therapy implementation:

[0017] The prepared BCG EVs or purified specific proteins FD1, CSPA and PMM2 are injected into the patient through intraperitoneal injection or intramuscular injection.

[0018] The injection dose and frequency need to be adjusted according to the patient's specific condition and the doctor's advice.

[0019] Monitoring and Evaluation:

[0020] Patients' tumor growth, immune response levels, and chemotherapy efficacy were regularly monitored.

[0021] The therapeutic effect is evaluated through imaging examinations (such as CT, MRI), laboratory tests (such as blood routine, biochemical indicators) and immunological tests (such as cytokine levels, immune cell numbers, etc.).

[0022] Function and principle

[0023] effect:

[0024] Activation of macrophages: BCG EVs and their specific proteins FD1, CSPA and PMM2 can specifically activate immune-activated macrophages (M1 macrophages). After activation, these cells can release a variety of cytokines and chemokines, thereby triggering a stronger immune response.

[0025] Inhibit tumor growth: Activated M1 macrophages can migrate into ovarian tumors and inhibit tumor growth and spread by phagocytizing tumor cells, releasing cytotoxic substances, and regulating the immune microenvironment.

[0026] Enhance the efficacy of chemotherapy: By activating macrophages and regulating the immune microenvironment, the method of the present invention can enhance the body's sensitivity to platinum-based chemotherapy drugs, thereby improving the efficacy of chemotherapy.

[0027] Broad-spectrum immune activation: In addition to adjuvant treatment of ovarian cancer, the method of the present invention may also have a broad-spectrum immune activation effect, providing new ideas for the treatment of other non-tumor diseases.

[0028] principle:

[0029] Immune activation mechanism: BCG EVs and their specific proteins, as immunogenic substances, can stimulate the body's immune system to respond. These proteins bind to receptors on the surface of immune cells (such as macrophages), triggering signal transduction pathways, leading to the activation and differentiation of immune cells.

[0030] Regulation of tumor immune microenvironment: Activated macrophages can regulate the tumor immune microenvironment by reducing the number and function of immunosuppressive cells (such as regulatory T cells, myeloid-derived suppressor cells, etc.) while increasing the number and function of immune-activating cells (such as M1 macrophages, dendritic cells, etc.), thereby creating a microenvironment that is conducive to anti-tumor immune response.

[0031] Chemotherapy enhancement mechanism: By activating macrophages and regulating the immune microenvironment, the method of the present invention can enhance the body's sensitivity to platinum-based chemotherapy drugs. Macrophages can release a variety of cytokines and chemokines, which can promote the absorption and distribution of chemotherapy drugs, while reducing the drug resistance and side effects of chemotherapy drugs.

[0032] In summary, the present invention uses BCG extracellular vesicles and their specific proteins to specifically activate macrophages to assist in the treatment of ovarian cancer, which provides a new treatment option for ovarian cancer patients through multiple mechanisms such as activating immune responses, regulating the immune microenvironment and enhancing the efficacy of chemotherapy.

[0033] In a specific embodiment of the first aspect, BCG EVs are obtained by culturing BCG in vitro and collecting and purifying its extracellular vesicles.

[0034] In a second aspect, a method for adjuvant treatment of ovarian cancer is characterized in that the method comprises the following steps:

[0035] Step 1: preparing purified BCG strain-specific proteins FD1, CSPA and PMM2;

[0036] Step 2: While maintaining the first-line platinum chemotherapy regimen for ovarian cancer patients, the purified specific proteins FD1, CSPA and PMM2 prepared in step 1 are injected into the patient's body by intraperitoneal injection or intramuscular injection to specifically activate immune-activated macrophages (M1 macrophages), thereby enriching them in ovarian tumors and inhibiting the growth of ovarian cancer.

[0037] In a specific embodiment of the second aspect, the method can enhance the body's sensitivity to platinum-based chemotherapy drugs, thereby improving the efficacy of chemotherapy.

[0038] In a specific embodiment of the second aspect, the method is also applicable to increasing the number of immune-activated macrophages in non-tumor diseases, and has a potential broad-spectrum immune activation effect.

[0039] In a third aspect, a composition for the adjuvant treatment of ovarian cancer comprises BCG extracellular vesicles (EVs), wherein the EVs comprise specific microbial proteins FD1 (ferredoxin), CSPA (cold shock protein) and PMM2 (phosphomannose mutase 1).

[0040] In a specific embodiment of the third aspect, the composition comprises purified BCG species-specific proteins FD1, CSPA and PMM2.

[0041] The beneficial effects of the present invention are:

[0042] 1. The present invention uses BCG extracellular vesicles (EVs) and its specific proteins FD1, CSPA and PMM2 to assist in the treatment of ovarian cancer, providing a new immunomodulatory-based strategy for the treatment of ovarian cancer. This method not only enriches the treatment methods for ovarian cancer, but also provides new inspiration for the treatment of other tumors. Through clinical verification, we found that patients who received BCG EVs or specific protein adjuvant therapy showed significant effects in tumor growth control, immune function enhancement, and chemotherapy efficacy enhancement. This treatment strategy can more effectively inhibit tumor growth and spread, prolong patient survival, and improve quality of life;

[0043] 2. Compared with traditional chemotherapy regimens, the treatment method proposed in the present invention can significantly reduce the side effects of chemotherapy. By activating the immune system and regulating the immune microenvironment, this method can enhance the body's sensitivity to chemotherapy drugs, thereby reducing the dose and frequency of chemotherapy drugs, alleviating the patient's pain and discomfort, and the treatment method is highly personalized. Doctors can flexibly adjust the dose and injection method of EVs or specific proteins according to the patient's specific conditions (such as tumor type, stage, immune function status, etc.) to develop the most suitable treatment plan for the patient. DETAILED DESCRIPTION

[0044] The following will be combined with 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 making creative work are within the scope of protection of the present invention.

[0045] Example 1: Preparation of BCG extracellular vesicles (EVs) and their application in adjuvant therapy of ovarian cancer.

[0046] Materials and Methods

[0047] BCG culture: Select a standard BCG strain and culture it in a suitable culture medium until it reaches the logarithmic growth phase.

[0048] EVs collection and purification: BCG extracellular vesicles (EVs) were collected and purified from the culture supernatant by differential centrifugation, ultrafiltration, and sucrose density gradient centrifugation. Western blot and other methods were used to verify whether EVs contained specific microbial proteins FD1, CSPA, and PMM2.

[0049] Patient screening: Patients with pathologically confirmed ovarian cancer who had received at least two cycles of first-line platinum-based chemotherapy were selected.

[0050] Adjuvant therapy: Purified BCG EVs are given to patients by intraperitoneal injection, with each injection dose of 5 mg / kg (calculated based on the patient's body weight), once a week for a total of 6 injections.

[0051] Monitoring and evaluation: Monitor changes in tumor size through CT scanning, and detect patients' immune function indicators (such as cytokine levels, number of immune cells, etc.) and chemotherapy efficacy.

[0052] result

[0053] EVs preparation: EVs containing specific microbial proteins FD1, CSPA and PMM2 were successfully collected and purified from the BCG culture supernatant.

[0054] Patient response: In patients receiving BCG EVs-assisted therapy, a significant slowdown in tumor growth was observed, and the tumor volume of some patients even shrank. At the same time, the patients' immune function indicators improved, such as increased cytokine levels and increased numbers of immune cells.

[0055] Enhanced chemotherapy efficacy: Compared with patients who received only first-line platinum-based chemotherapy, patients who received BCG EVs adjuvant therapy showed more significant improvements in chemotherapy efficacy, such as enhanced chemotherapy sensitivity and reduced chemotherapy side effects.

[0056] Example 2: Adjuvant therapy application of purified BCG-specific proteins FD1, CSPA and PMM2.

[0057] Materials and Methods

[0058] Protein purification: Specific microbial proteins FD1, CSPA and PMM2 were purified from BCG culture using affinity chromatography, ion exchange chromatography and other methods.

[0059] Patient screening: As in Example 1, patients who were pathologically diagnosed with ovarian cancer and had received first-line platinum chemotherapy were selected.

[0060] Adjuvant therapy: The purified specific proteins FD1, CSPA and PMM2 are given to patients by intramuscular injection, with each injection dose of 100 μg / kg (calculated according to the patient's body weight), once a week, for a total of 6 injections.

[0061] Monitoring and evaluation: Similar to Example 1, CT scanning is used to monitor changes in tumor size and detect the patient's immune function indicators and chemotherapy efficacy.

[0062] result

[0063] Protein purification: Specific microbial proteins FD1, CSPA and PMM2 were successfully purified from BCG culture.

[0064] Patient response: In patients receiving purified protein adjuvant therapy, the same effects were observed, such as slowed tumor growth, improved immune function indicators, and enhanced chemotherapy efficacy. Compared with Example 1, although the injection method and protein source are different, the two have similar effects in the adjuvant treatment of ovarian cancer.

[0065] in conclusion

[0066] Through the research of Example 1 and Example 2, we have demonstrated that BCG extracellular vesicles and their specific proteins FD1, CSPA and PMM2 have significant effects in the adjuvant treatment of ovarian cancer. These results not only provide new treatment options for patients with ovarian cancer, but also provide new ideas for further exploring the application of immunotherapy in tumor treatment. It should be noted that in actual application, the appropriate adjuvant treatment plan should be selected according to the specific situation of the patient and the doctor's advice, and the patient's treatment effect and adverse reactions should be closely monitored.

[0067] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. BCG EVs and specific proteins activate macrophages, characterized by: The composition comprises BCG extracellular vesicles (EVs), wherein the BCG extracellular vesicles (EVs) comprise specific microbial proteins FD1 (ferredoxin), CSPA (cold shock protein) and PMM2 (phosphomannose mutase 1).

2. The BCG EVs and specific protein activated macrophages and the method and use thereof for treating ovarian cancer according to claim 1, characterized in that: The BCG EVs are obtained by culturing BCG in vitro and collecting and purifying its extracellular vesicles.

3. A method and use for adjuvant treatment of ovarian cancer, characterized in that: The method comprises the following steps: Step 1: preparing purified BCG strain-specific proteins FD1, CSPA and PMM2; Step 2: While maintaining the first-line platinum chemotherapy regimen for ovarian cancer patients, inject the purified specific proteins FD1, CSPA and PMM2 prepared in step 1 into the patient's body by intraperitoneal injection or intramuscular injection to specifically activate immune-activated macrophages (M1 macrophages), thereby enriching them in ovarian tumors and inhibiting the growth of ovarian cancer.

4. The method for adjuvant treatment of ovarian cancer according to claim 3, characterized in that: The method can enhance the body's sensitivity to platinum-based chemotherapy drugs, thereby improving the efficacy of chemotherapy.

5. The method for adjuvant treatment of ovarian cancer according to claim 3, characterized in that: The method is also suitable for increasing the number of immune-activated macrophages in non-tumor diseases and has a potential broad-spectrum immune activation effect.

6. A composition for adjuvant treatment of ovarian cancer, characterized in that: The composition comprises purified BCG species-specific proteins FD1, CSPA and PMM2.