Pharmaceutical composition, method for producing the same, and use

Abstract

The present invention discloses a pharmaceutical composition, its manufacturing method, and its use, wherein the pharmaceutical composition comprises a first active ingredient, a second active ingredient, and a pharmaceutically acceptable carrier or excipient, the first active ingredient being a microbial agent containing one or more of Staphylococcus aureus, Bordetella pertussis, diphtheria toxoid, tetanus toxoid, Salmonella typhi, or Salmonella paratyphi, and the second active ingredient comprising polyinosinic acid, polycytidylic acid, and vitamins. The pharmaceutical composition is an artificial active immunotherapy for tumors, which, by "stimulating" the entire immune system, activates the human immune system using bacteria to kill cancer cells, making the treatment extremely stable and reliable, significantly saving and extending the lives of cancer patients, while simultaneously being extremely safe, having very few toxic side effects, and having low manufacturing costs.

Description

[Technical Field]

[0001] This invention relates to the medical field, and more particularly to pharmaceutical compositions, methods for producing them, and their use. [Background technology]

[0002] Cancer is a leading cause of death affecting human life and health. Traditional cancer treatments include surgery, radiation therapy, chemotherapy, and targeted therapy. Over the past decade, tumor immunotherapy, primarily based on passive immunotherapy, has gradually become a treatment option for cancer.

[0003] None of the above therapies can fundamentally and systematically solve the problem. Surgery and radiation therapy can only remove localized lesions. Unless the primary disease is detected early, it is generally impossible to eradicate systemic disease. Chemotherapy as a systemic treatment is currently the main treatment for advanced tumors. While chemotherapy can effectively kill cancer cells, it also indiscriminately attacks and kills normal cells at the same time, causing irreparable damage to the human body. Chemotherapy also damages the immune system of some patients, making recovery and reconstruction impossible, and leading to a near-complete loss of immune surveillance mechanisms, resulting in a significant number of patients dying from tumor spread or disease progression. Targeted therapies have a limited range of action due to their specificity, and drug resistance is an insurmountable obstacle for targeted therapies. Passive immunotherapy, currently the mainstream treatment, is expensive, has a narrow scope of application, does not significantly improve treatment efficacy compared to other treatments, and has relatively significant side effects. In short, at present, surgery, radiation therapy, chemotherapy, targeted therapy, and passive immunotherapy cannot restore or rebuild the body's immune surveillance mechanisms, and the tendency for tumor recurrence and metastasis remains serious.

[0004] Currently approved cancer immunotherapies primarily focus on stimulating adaptive immune responses through T cell activation. However, these T cell immunotherapies have certain limitations; for example, when PD-1 / PD-L1 inhibitors are used as monotherapy, only 10% to 25% of patients respond to treatment in almost all major tumor indications. The response rate to immunotherapies targeting adaptive immune checkpoints is particularly low in "cold tumors" (tumors lacking T cell infiltration) or in non-inflammatory T cell infiltration and immunosuppressive tumor microenvironments. This indicates an urgent need for a new generation of immunotherapies to improve treatment outcomes.

[0005] Currently approved tumor immunotherapies primarily target T-cell immune checkpoints such as PD-1 / PD-L1, CTLA-4, and LAG-3. As shown in the table below, T-cell immune checkpoint inhibitors (such as PD-1 / PD-L1 antibodies) are clinically used in many different types of cancer, including as first-line treatment, but their response rates remain low in almost all major tumor indications.

[0006] [Table 1]

[0007] Note: (1) Response rates are based on the latest standards of the U.S. Food and Drug Administration and the National Food and Drug Administration, but with the exception of colorectal cancer, gastric cancer, small cell lung cancer, ovarian cancer, cholangiocarcinoma and soft tissue sarcoma, these are based on published clinical results. (2) Only monotherapy clinical results are listed. (3) Adjuvant therapy results are not included. Results may differ for different cancer subtypes or clinical trials. (4) All listed clinical results are from the general cancer group (without considering PD-L1 expression status), but with the exception of the overall response rate for cervical cancer, this index is limited to composite positivity score (CPS) ≥ 1 in the PD-L1 positive group.

[0008] Definitions: NSCLC refers to non-small cell lung cancer, SCLC refers to small cell lung cancer, CRC refers to colorectal cancer, GC refers to gastric cancer, HNSCC refers to head and neck squamous cell carcinoma, HCC refers to hepatocellular carcinoma, ESCC refers to esophageal squamous cell carcinoma, BTC refers to cholangiocarcinoma, RCC refers to renal cell carcinoma, OC refers to ovarian cancer, CC refers to cervical cancer, UC refers to urothelial carcinoma, STS refers to soft tissue sarcoma, and DLBCL refers to diffuse large B-cell lymphoma.

[0009] Data source: Frost & Sullivan

[0010] The safety and efficacy of other T-cell immunotherapies also need improvement. Chimeric antigen receptor T-cell (CAR-T) immunotherapy can produce significant and sustained responses in certain B-cell leukemia, lymphoma, and multiple myeloma (MM) subsets, but it faces several limitations, including life-threatening cytokine release syndrome (CRS) and neurotoxicity issues, extremely high costs, and limited efficacy in solid tumors. Similarly, T-cell binding antibodies, such as CD3-based bispecific antibodies, raise safety concerns, including severe cytokine release syndrome and "non-tumor-targeted" toxicity to healthy tissues. To date, clinical trials of numerous candidate drugs worldwide have been discontinued or suspended due to unacceptable toxicity from CAR-T therapy or CD3 bispecific antibodies, including Atara's ATA2271 (autologous mesothelin CAR-T), Amgen's AMG673 (CD3 x CD33), AMG427 (CD3 x FLT3), AMG701 (CD3 x BCMA), REGN's odronextamab (CD3 x CD20), and Pfizer's elranatamab (CD3 x BCMA).

[0011] According to Frost & Sullivan's documentation, tebentafusp is currently the only T-cell linked antibody approved on the market for the treatment of solid tumors, and it is approved for the treatment of uveal melanoma (a rare disease). Furthermore, there are no other CAR-T therapies approved anywhere in the world for the treatment of solid tumors.

[0012] Recent research has shown that by leveraging the synergistic effects between the innate immune system and the innate and adaptive immune systems, the limitations of current immunotherapies can be overcome. To date, there are no approved drugs in the world that activate the innate immune system and then the adaptive immune system to form the synergistic effect between the innate and adaptive immune systems and multi-target anti-cancer agents.

[0013] Tumor active immunotherapy has become an innovative cancer treatment method aimed at eliminating cancer cells by stimulating and activating the patient's own immune system.

[0014] Generally, the human immune system is divided into the innate immune system and the adaptive immune system. The innate immune system is the first line of defense in the human body, which can identify foreign substances and immediately induce non-specific immune responses. The main innate immune cells include macrophages, natural killer cells (NK), and dendritic cells (DC). The adaptive immune system, including T cells and B cells, is the second line of defense, which can more effectively recognize and eliminate specific antigens. The following table compares the major adaptive and innate immune cells in the tumor microenvironment.

Table 2

[0015] Note: The distribution in tumor tissues refers to the proportion of specific immune cells in different tumor tissues. Source: Frost & Sullivan

[0016] Compared to adaptive immune cells, innate immune cells are widely distributed in tumor tissue. Innate immune cells not only function as the first line of defense but also play a crucial role in activating adaptive immune responses, leading to a more complete and effective immune response. For example, activated macrophages and dendritic cells secrete cytokines and chemokines (such as CXCL9 and CXCL10) to recruit T cells into the tumor microenvironment, transforming a "cold tumor" into a "hot tumor" (a tumor infiltrated by T cells and responsive to immunotherapy). Macrophages and dendritic cells can further enhance the T cell response through antigen presentation. When activated, natural killer cells promote T cell differentiation and activation, thereby enhancing the T cell response. Therefore, therapies that target the innate immune system and activate adaptive immunity hold great potential in addressing the limitations of currently approved T cell immunotherapies.

[0017] In recent years, numerous studies have discovered the potential to overcome the limitations of T-cell immunotherapy by targeting innate immunity. Innate immune cells are widely distributed in tumor tissue and, when activated, directly counter cancer cells and enhance the adaptive immune response through interaction with T cells. For example, macrophages can be activated by immunotherapy targeting macrophages, potentially leading to more effective adaptive immune activation. As major antigen-presenting cells, macrophages release cytokines and chemokines that recruit T cells; therefore, activating macrophages can increase the abundance of T cells in the tumor microenvironment, transforming a "cold tumor" into a "hot tumor." Other important innate immune cells, such as natural killer cells and dendritic cells, can also enhance the T-cell immune response through various mechanisms. The synergistic effects of drugs utilizing innate and adaptive immunity have great potential to maximize the effectiveness of immunotherapy and exert potent antitumor activity in "cold tumors."

[0018] Academician Zhenyi Wang, a renowned hematological tumor expert in China, is the inventor who first applied differentiation induction therapy to treat acute promyelocytic leukemia in the world. As early as in the 1980s, he advocated that "new ideas" were needed for cancer control, starting from improving the overall immunity of the body and implementing multi-target immune attacks, that is, advocating the "multi-component multi-target effect". His concept includes the following points: (1) The formation and development of cancer cells are in a balanced state with the body's immunity. As long as the body's immune capacity exists and maintains a sufficient level, cancer cells will not be generated, or will be quickly eliminated (apoptosis), or enter a "dormant" state. Cancer cells do not simply proliferate, but are constantly undergoing apoptosis. As long as the balance between immune cells and cancer cells tilts slightly towards the immune side, a large number of cancer cells will undergo apoptosis, so the growth of tumors will stop. (2) Cells are very complex and have many "targets" (antigens) on their surfaces and inside. More importantly, cancer cells are genetically unstable. Normal cells copy all genetic information to the next generation when they divide, but cancer cells change their genetic information constantly when they divide, generating countless different cells with their own characteristics. The basis of anti-cancer immunity in the body lies in the fact that different immune cells and cytokines act on different cancer cell targets, but a single immune cell or factor (acting alone) may not necessarily be able to eliminate cancer cells. (3) Immunotherapy depends not on "input" but on "activation", and does not activate a single immune component, but activates the "thousands of troops and horses" (the entire immune system). The activated immune components change "flexibly and dynamically" along with the changes in the genetic information of cancer cells, ensuring the effectiveness of the attack on mutant cancer cells.

[0019] Bacterial inoculation for malignant tumors is an immunotherapy for malignant tumors developed in the 1890s by American physician William Coley and was primarily used in the United States until it was officially discontinued by the FDA in the 1960s. Historically, it is known as the Coley therapy. A notable characteristic of this treatment is its unstable efficacy. Consistent effectiveness has not been achieved in different patients or different tumor types. This is because the treatment lacks a mechanism for reliably identifying cancer cells. The bacterial immunoactivation therapy currently used in China also suffers from this problem and cannot become the mainstream treatment for tumors.

[0020] Tumor metastasis refers to the process by which primary tumor cells invade or spread to other tissues, establish themselves, and proliferate. Tumor metastasis is the leading cause of cancer-related death, yet the biological basis of this extremely complex metastatic process is still largely unknown in the current academic community, and there are very few drugs that can effectively and broadly inhibit the spread and metastasis of cancer cells. As mentioned above, the first-line drugs currently used in mainstream cancer treatment, such as small molecule targeted drugs, immunosuppressants, and CAR-T therapies, are only somewhat effective against a very small number of primary tumors and are basically powerless against metastatic lesions. [Overview of the Initiative]

[0021] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a pharmaceutical composition, a method for producing the same, and a method for using the same in order to solve the problems of the prior art.

[0022] To achieve the above and other related objectives, the present invention provides a pharmaceutical composition comprising a first active ingredient, a second active ingredient, and a pharmaceutically acceptable carrier or excipient, wherein the first active ingredient is a microbial agent comprising one or more of Staphylococcus aureus, Bordetella pertussis, diphtheria toxoid, tetanus toxoid, Salmonella typhi, or Salmonella paratyphi, and the second active ingredient comprises polyinosinic acid, polycytidylic acid, and vitamins.

[0023] The present invention also provides the use of the pharmaceutical composition in the manufacture of disease treatment products, disease prevention products, or disease diagnostic products.

[0024] The disease is selected from cancer, arteriosclerosis, HPV infection, and atrophic gastritis, and preferably the cancer is selected from liver cancer, lung cancer, melanoma, colorectal cancer, gastric cancer, ovarian cancer, bile duct cancer, cervical cancer, pancreatic cancer, etc.

[0025] As described above, the pharmaceutical composition PGc biological injection of the present invention, its manufacturing method, and its use have the following beneficial effects.

[0026] 1. PGc biological injections are an artificial active immunotherapy for tumors that "stimulates" the entire immune system, thereby activating the human immune system using bacteria and making the treatment method that kills cancer cells extremely stable and reliable. This can significantly save the lives of cancer patients, prolong their lives, and improve their quality of life.

[0027] 2. PGc has extremely high safety and very few toxic side effects. To date, no side effects have been observed in patients who have received PGc injections, other than mild redness and swelling at the injection site, short-term fever, and other immune reactions.

[0028] 3. PGc biological injections have low manufacturing costs, making them a sustainable cancer treatment for patients and truly enabling a cure for all patients.

[0029] 4. According to statistics from the "National Educational Textbook on Prevention of Serious Diseases" published by the China Actuarial Association in 2021, the total cost per person for cancer treatment reaches 220,000 to 800,000 yuan, but PGc biological injections can reduce the patient's treatment cost to under 40,000 yuan.

[0030] 5. The broad applicability of PGC biological injections offers new and effective treatment options for conditions currently difficult to treat, such as atherosclerosis, HPV-positive to HPV-negative conversion, and atrophic gastritis. In short, the widespread use of PGC biological injections in treatment will effectively improve the health level of the Chinese population and effectively extend their lifespan. [Brief explanation of the drawing]

[0031] [Figure 1] Figure 1 shows tumor changes in 10 animals after administration of different samples of the present invention. [Figure 2] Figure 2 shows the results of a supplementary experiment for screening the PGc component in Example 2 of the present invention. [Figure 3] Figure 3 shows the results of the PGc formulation optimization test in Example 3 of the present invention. [Figure 4] Figure 4 shows the typical lung anatomy of the NS group and PGc group in Example 4 of the present invention. [Figure 5] Figure 5 shows the experimental design diagram for Example 5 of the present invention. [Figure 6] Figure 6 shows a schematic diagram of the administration site in Example 5 of the present invention. [Figure 7] Figure 7 shows the experimental results of Example 5 of the present invention. [Figure 8] Figure 8 shows the anatomical results of Example 6 of the present invention. [Figure 9] Figure 9 shows the statistical results for Example 6 of the present invention. [Figure 10] Figure 10 shows the statistical results for Example 7 of the present invention. [Figure 11] Figure 11 shows an anatomical diagram of a tumor in Example 8 of the present invention. [Figure 12] Figure 12 shows an anatomical diagram of a tumor in Example 9 of the present invention. [Figure 13] Figure 13 shows an anatomical diagram of a tumor in Example 10 of the present invention. [Figure 14] Figure 14 shows an anatomical diagram of a tumor in Example 11 of the present invention. [Figure 15] Figure 15 shows an anatomical diagram of a tumor in Example 12 of the present invention. [Figure 16] Figure 16 shows the results of promoting M1 macrophage activation by PGc in Example 14 of the present invention. [Figure 17] Figure 17 shows that in Example 14 of the present invention, PGc promotes the infiltration of M1 macrophages in tumor tissue. [Figure 18] Figure 18 shows that in Example 14 of the present invention, PGc promotes T lymphocyte infiltration in tumor tissue. [Figure 19] Figure 19 shows that in Example 14 of the present invention, PGc inhibits the formation of blood vessels in tumor tissue. [Figure 20] Figure 20 shows the extent to which PGc activates various immune responses, as reflected by the biological process enrichment analysis in Example 14 of the present invention. [Figure 21] Figure 21 shows the KEGG signaling pathway enrichment analysis in Example 14 of the present invention, reflecting the activation level of signaling pathways in the immune system after PGc treatment. [Figure 22] Figure 22 shows the molecular function enrichment analysis of genes in Example 14 of the present invention, reflecting the degree of immune system activation after PGc treatment at the molecular level. [Figure 23] Figure 23 shows the results of flow cytometry analysis of changes in T cells, NK cells, NKT cells, and B cells in tumor tissue of LLC model mice collected after PGc treatment in Example 14 of the present invention. [Modes for carrying out the invention]

[0032] The present invention provides a pharmaceutical composition comprising a first active ingredient, a second active ingredient, and a pharmaceutically acceptable carrier or excipient, wherein the first active ingredient is a microbial agent comprising one or more of Staphylococcus aureus, Bordetella pertussis, diphtheria toxoid, tetanus toxoid, Salmonella typhi, or Salmonella paratyphi, and the second active ingredient comprises polyinosinic acid, polycytidylic acid, and vitamins.

[0033] The microbial agent may be a mixture of Staphylococcus aureus, Bordetella pertussis, diphtheria toxoid, tetanus toxoid, Salmonella typhi, or Salmonella paratyphi, or it may be a single bacterial agent of each of the above bacteria, that is, the microbial agent contains Staphylococcus aureus, Bordetella pertussis, diphtheria toxoid, tetanus toxoid, Salmonella typhi, or Salmonella paratyphi.

[0034] The aforementioned microbial agent is an inactivated formulation.

[0035] The microbial agent is a liquid formulation or a solid formulation. In one embodiment, the microbial agent is a liquid formulation. Each liquid formulation is a fermentation broth of the corresponding microorganism, preferably a culture stock.

[0036] The aforementioned liquid formulation can be obtained from a commercially available product or manufactured independently.

[0037] The Bordetella pertussis, diphtheria toxoid, and tetanus toxoid may be a single triad formulation, a combination of a diad formulation and a single formulation, or a triad formulation.

[0038] In a particular embodiment of the present invention, the Staphylococcus aureus is produced in accordance with the Provisional Rules for the Production and Verification of Ruthorn Vaccine No. 05 in the bacterial solution production process of the 1979 edition of the Biological Products Code.

[0039] In certain embodiments of the present invention, the Bordetella pertussis bacterium and diphtheria toxoid are manufactured in accordance with the provisions of the Chinese Pharmacopoeia, Part III, "Adsorbed Pertussis and Diphtheria Combination Vaccine."

[0040] In certain embodiments of the present invention, the diphtheria toxoid may be manufactured in accordance with the provisions of the Chinese Pharmacopoeia, Part 3, "Adsorbent Diphtheria Vaccine."

[0041] In certain embodiments of the present invention, the tetanus toxoid may be manufactured in accordance with the provisions of the Chinese Pharmacopoeia, Part 3, "Adsorbent Tetanus Vaccine."

[0042] In certain embodiments of the present invention, the Salmonella typhi bacillus may be manufactured in accordance with the provisions of the Chinese Pharmacopoeia, Part 3, "Typhoid Vaccine."

[0043] In certain embodiments of the present invention, the paratyphi bacillus may be manufactured in accordance with the provisions of the "Type A and Type B Paratyphi Combined Vaccine" of the Third Part of the Chinese Pharmacopoeia.

[0044] In a specific embodiment of the present invention, the concentration of Staphylococcus aureus is 2 × 10⁻¹⁰ based on the total volume of the pharmaceutical composition. 7 ~3×10 9 The concentration is cells / mL.

[0045] In a specific embodiment of the present invention, the concentration of Bordetella pertussis based on the total volume of the pharmaceutical composition is 7 × 10 7 ~9×10 9 The concentration is cells / mL.

[0046] In a particular embodiment of the present invention, the concentration of the diphtheria toxoid is 1 LF to 5 LF / mL based on the total volume of the pharmaceutical composition.

[0047] In a particular embodiment of the present invention, the concentration of the tetanus toxoid is 0.1 LF to 5 LF / mL based on the total volume of the pharmaceutical composition.

[0048] In a specific embodiment of the present invention, the concentration of Salmonella typhi is 1.5 × 10⁻¹⁶ based on the total volume of the pharmaceutical composition. 6 ~5×10 8 The concentration is cells / mL.

[0049] In a specific embodiment of the present invention, based on the total volume of the pharmaceutical composition, the concentration of the Salmonella typhi bacteria is 1×10 6 ~3×10 8 CFU / mL.

[0050] The Salmonella typhi bacteria are any one or more selected from Salmonella typhi A bacteria, Salmonella typhi B bacteria or Salmonella typhi C bacteria.

[0051] The Salmonella typhi bacteria or Salmonella paratyphi bacteria may be each a single bacterial agent, or a triple bacterial agent of Salmonella typhi bacteria, Salmonella typhi A bacteria, and Salmonella typhi B bacteria, or may also be a quadruple bacterial agent of Salmonella typhi bacteria, Salmonella typhi A bacteria, Salmonella typhi B bacteria, and Salmonella typhi C bacteria.

[0052] In a specific embodiment of the present invention, the Salmonella paratyphi bacteria are selected from Salmonella typhi A bacteria and Salmonella typhi B bacteria. Based on the total volume of the pharmaceutical composition, the concentration of the Salmonella typhi A bacteria is 0.1×0 7 ~8×10 7 CFU / mL, and / or the concentration of the Salmonella typhi B bacteria is 0.1×10 7 ~8×10 7 CFU / mL.

[0053] The polyinosinic acid and polycytidylic acid are selected from a polymer formed by inosinic acid alone and a polymer formed by cytidylic acid alone, or are selected from poly(I:C) which is a copolymer of inosinic acid and cytidylic acid. Poly(I:C) is an analog of double-stranded RNA, one strand is poly(I), and the other strand is poly(C). Correspondingly, the second active ingredient is obtained after mixing the three of polyinosinic acid, polycytidylic acid and vitamin together, or is obtained after mixing poly(I:C) and vitamin.

[0054] In certain embodiments of the present invention, based on the total volume of the pharmaceutical composition, the concentrations of polyinosinic acid and polycytidylic acid are both 0.5 g / 100 mL or less, for example, 0.01 g / 100 mL to 0.5 g / 100 mL. Alternatively, for example, the concentrations of polyinosinic acid and polycytidylic acid are both selected from any of the following concentration ranges: 0.01 g / 100 mL to 0.05 g / 100 mL, 0.05 g / 100 mL to 0.1 g / 100 mL, 0.1 g / 100 mL to 0.2 g / 100 mL, 0.2 g / 100 mL to 0.3 g / 100 mL, 0.3 g / 100 mL to 0.4 g / 100 mL, and 0.4 g / 100 mL to 0.5 g / 100 mL.

[0055] In certain embodiments of the present invention, when the polyinosinic acid and polycytidylic acid are selected from polymers formed by inosinic acid alone and polymers formed by cytidylic acid alone, the mass ratio of the polyinosinic acid and polycytidylic acid is 1:0.1 to 1:10. For example, the mass ratio of the polyinosinic acid and polycytidylic acid is 1:0.1 to 1:0.5, 1:0.5 to 1:1, 1:1 to 1:2, 1:2 to 1:4, 1:4 to 1:6, 1:6 to 1:8, and 1:8 to 1:10. In preferred embodiments, the mass ratio of the polyinosinic acid and polycytidylic acid is 1:1. Specifically, when the mass ratio of the polyinosinic acid and polycytidylic acid is 1:1, the parts by mass of the polyinosinic acid and polycytidylic acid may be 2, 4, 6, 8, or 10 parts.

[0056] The aforementioned polyinosinic acid and polycytidylic acid only need to meet the standards of the National Pharmacopoeia, and there are no specific requirements regarding their degree of polymerization.

[0057] The vitamin in question is vitamin A. The vitamin A is selected from vitamin A1 and / or vitamin A2.

[0058] In certain embodiments of the present invention, the concentration of vitamin A is 1 g / 100 mL or less based on the total volume of the pharmaceutical composition. For example, the concentration of the vitamin is selected from any of the following concentration ranges: 0.1 g / 100 mL to 0.2 g / 100 mL, 0.2 g / 100 mL to 0.3 g / 100 mL, 0.3 g / 100 mL to 0.4 g / 100 mL, 0.4 g / 100 mL to 0.5 g / 100 mL, 0.5 g / 100 mL to 0.8 g / 100 mL, and 0.8 g / 100 mL to 1.0 g / 100 mL.

[0059] The synergistic effect of polyinosinic acid, polycytidylic acid, and vitamin A can effectively identify abnormal cells in the human body (e.g., tumor cells) and induce changes in their cell membranes.

[0060] In the aforementioned pharmaceutically acceptable carrier or excipient, "pharmaceutically acceptable" means that when the drug is appropriately administered to an animal or human, no adverse reaction, allergic reaction, or other adverse reaction occurs.

[0061] A "pharmaceutically acceptable carrier or excipient" must be compatible with the active ingredient, that is, it must be able to be mixed with the active ingredient under normal circumstances without significantly reducing the drug's effect. Specific examples of substances that can be used as pharmaceutically acceptable carriers or excipients include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; solid lubricants such as tragacanth powder, malt, gelatin, talc, stearic acid, and magnesium stearate; vegetable oils such as calcium sulfate, peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; emulsifiers such as alginic acid and Tween; wetting agents such as sodium lauryl sulfate; colorants, fragrances, tableting agents, stabilizers, antioxidants, preservatives, water without a pyrogenic source, isotonic salt solutions, or phosphate buffer solutions. These substances are used as needed to enhance the stability of the formulation, to increase its activity or bioavailability, or to produce a taste or smell that is acceptable when taken orally.

[0062] In certain embodiments of the present invention, the pharmaceutically acceptable carrier or excipient comprises sodium carboxymethylcellulose, aluminum stearate, tween 80, lecithin, soybean oil, dextran, fat emulsion, and water.

[0063] The aluminum stearate is selected from aluminum monostearate and aluminum distearate.

[0064] In certain embodiments of the present invention, the concentration of sodium carboxymethylcellulose in the pharmaceutically acceptable carrier or excipient is 2 g / 100 mL or less, based on the total volume of the pharmaceutical composition. Based on the total volume of the pharmaceutical composition, the concentration of sodium carboxymethylcellulose is 0.2 to 2 g / 100 mL. For example, the concentration of sodium carboxymethylcellulose is selected from any of the following concentration ranges: 0.2 g / 100 mL to 0.5 g / 100 mL, 0.5 g / 100 mL to 1.0 g / 100 mL, 1.0 g / 100 mL to 1.5 g / 100 mL, or 1.5 g / 100 mL to 2 g / 100 mL.

[0065] Based on the total volume of the pharmaceutical composition, the concentration of aluminum stearate in the pharmaceutically acceptable carrier or excipient is 3 g / 100 mL or less. Based on the total volume of the pharmaceutical composition, the concentration of aluminum stearate is 0.3 to 3 g / 100 mL. For example, the concentration of aluminum stearate is selected from any of the following concentration ranges: 0.3 g / 100 mL to 0.5 g / 100 mL, 0.5 g / 100 mL to 1.0 g / 100 mL, 1.0 g / 100 mL to 1.5 g / 100 mL, 1.5 g / 100 mL to 2.0 g / 100 mL, 2.0 g / 100 mL to 2.5 g / 100 mL, and 2.5 g / 100 mL to 3.0 g / 100 mL.

[0066] Based on the total volume of the pharmaceutical composition, the concentration of tween80 in the pharmaceutically acceptable carrier or excipient is 1 ml / 100 mL or less. Based on the total volume of the pharmaceutical composition, the concentration of tween80 is 0.1 to 1 mL / 100 mL. For example, the concentration of tween80 is selected from any of the following concentration ranges: 0.1 mL / 100 mL to 0.3 mL / 100 mL, 0.3 mL / 100 mL to 0.5 mL / 100 mL, 0.5 mL / 100 mL to 0.7 mL / 100 mL, and 0.7 mL / 100 mL to 1.0 mL / 100 mL.

[0067] Based on the total volume of the pharmaceutical composition, the concentration of lecithin in the pharmaceutically acceptable carrier or excipient is 50 mg / 100 mL or higher. Based on the total volume of the pharmaceutical composition, the concentration of lecithin is 50 to 2000 mg / 100 mL. For example, the concentration of lecithin is selected from any of the following concentration ranges: 50 mg / 100 mL to 100 mg / 100 mL, 100 mg / 100 mL to 300 mg / 100 mL, 300 mg / 100 mL to 500 mg / 100 mL, 500 mg / 100 mL to 1500 mg / 100 mL, and 1500 mL to 2000 mg / 100 mL.

[0068] Based on the total volume of the pharmaceutical composition, the concentration of soybean oil in the pharmaceutically acceptable carrier or excipient is 15 ml / 100 mL or less. Based on the total volume of the pharmaceutical composition, the concentration of soybean oil is 1 to 15 mL / 100 mL. For example, the concentration of soybean oil is selected from any of the following concentration ranges: 1 mL / 100 mL to 3 mL / 100 mL, 3 mL / 100 mL to 6 mL / 100 mL, 6 mL / 100 mL to 9 mL / 100 mL, 9 mL / 100 mL to 12 mL / 100 mL, and 12 mL / 100 mL to 15 mL / 100 mL.

[0069] Based on the total volume of the pharmaceutical composition, the concentration of dextran in the pharmaceutically acceptable carrier or excipient is 10 g / 100 mL or less. Based on the total volume of the pharmaceutical composition, the concentration of dextran is 1 to 10 g / 100 mL. For example, the concentration of dextran is selected from any of the following concentration ranges: 1 g / 100 mL to 2 g / 100 mL, 2 g / 100 mL to 4 g / 100 mL, 4 g / 100 mL to 6 g / 100 mL, 6 g / 100 mL to 8 g / 100 mL, 8 g / 100 mL to 10 g / 100 mL.

[0070] Based on the total volume of the pharmaceutical composition, the concentration of the fat emulsion in the pharmaceutically acceptable carrier or excipient is 10 ml / 100 mL or higher. Based on the total volume of the pharmaceutical composition, the concentration of the fat emulsion is 10 to 80 mL / 100 mL. For example, the concentration of the fat emulsion is selected from any of the following concentration ranges: 10 mL / 100 mL to 20 mL / 100 mL, 20 mL / 100 mL to 30 mL / 100 mL, 30 mL / 100 mL to 40 mL / 100 mL, 40 mL / 100 mL to 50 mL / 100 mL, 50 mL / 100 mL to 60 mL / 100 mL, 60 mL / 100 mL to 70 mL / 100 mL, and 70 mL / 100 mL to 80 mL / 100 mL.

[0071] The synergistic effect of aluminum stearate and lecithin can effectively solve the challenge of enabling effector macrophages and T cells to smoothly enter the tumor microenvironment.

[0072] The form of the pharmaceutical composition is not particularly limited and may be in the form of various substances such as solids, liquids, gels, semi-fluids, or aerosols.

[0073] In a particular embodiment of the present invention, the pharmaceutical composition is an injectable preparation.

[0074] The present invention also provides a method for producing a pharmaceutical composition, which includes mixing the first active ingredient, the second active ingredient, and a pharmaceutically acceptable carrier or excipient to obtain the pharmaceutical composition.

[0075] The present invention also provides the use of pharmaceutical compositions in the manufacture of disease treatment products, disease prevention products, or disease diagnostic products.

[0076] In this invention, the term "prevention" includes preventive treatments that produce desired pharmacological and / or physiological effects. These superior effects refer to the ability to medically prevent or delay the onset of a disease and / or reduce the risk of disease progression or worsening.

[0077] In this invention, the term "diagnosis" refers to the ability to determine whether or not a particular disease is present.

[0078] In this invention, the term "treatment" includes therapeutic or palliative treatments that produce desired pharmacological and / or physiological effects. This superior effect refers to medically reducing one or more symptoms of a disease or completely eliminating the disease.

[0079] The aforementioned diseases are selected from cancer, arteriosclerosis, HPV infection, atrophic gastritis, and others.

[0080] In this application, "cancer" refers to any medical condition that is mediated by the growth, proliferation, or metastasis of tumors or malignant cells, causing solid tumors and non-solid tumors such as leukemia. In this invention, "tumor" refers to the physical substance of a tumor and / or malignant cells.

[0081] The aforementioned cancers are selected from liver cancer, lung cancer, melanoma, colorectal cancer, stomach cancer, ovarian cancer, bile duct cancer, cervical cancer, pancreatic cancer, etc.

[0082] The arteriosclerosis is selected from atherosclerosis, medial arterial sclerosis, or arteriolar sclerosis.

[0083] The HPV infections are selected from high-risk, intermediate-risk, or low-risk types. High-risk types are selected from HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. Intermediate-risk types are selected from HPV26, 53, 66, 73, and 82. Low-risk types are selected from HPV6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, and 89.

[0084] The present invention also provides a method for treating or preventing a disease, comprising administering a therapeutically effective amount of the pharmaceutical composition to a subject.

[0085] "Subjects" include, but are not limited to, animals (preferably mammals), and the mammals are preferably rodents, artiodactyls, odd-toed ungulates, lagomorphs, primates, etc. The mammals include, for example, humans, non-human primates (e.g., monkeys), mice, pigs, cattle, goats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep, or other non-human mammals, and the non-mammals include, for example, non-mammalian vertebrates, such as birds (e.g., chickens or ducks) or fish, and non-mammalian invertebrates. Subjects may be humans, for example, immunocompromised patients or cancer patients.

[0086] "Treatment" or "therapy" of a condition includes prevention or reduction of a condition, a decrease in the onset or progression rate of a condition, a reduction in the risk of developing a condition, prevention or delay of the onset of symptoms associated with a condition, reduction or cessation of symptoms associated with a condition, achievement of complete or partial reversal of a condition, a cure of a condition, or a combination thereof. With respect to cancer, "treatment" or "therapy" may mean inhibiting or delaying the growth, proliferation, metastasis, or combination thereof of a tumor or malignant cells. With respect to tumors, "treatment" or "therapy" includes removal of all or part of a tumor, inhibition or delay of tumor growth and metastasis, prevention or delay of tumor development, or a combination thereof.

[0087] Examples of the aforementioned cancers include lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycoses fungoids, Merkel cell carcinoma, and other hematological malignancies such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and negative PTLD, EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, nasopharyngeal cancer, and HHV8-associated primary exudative lymphoma.

[0088] In the present invention, the terms “therapeutic effective dose” or “effective dose” refer to a dose or concentration of a drug that is effective in treating a disease or condition associated with a chimeric antigen receptor antigen. For example, in the use of an antibody or its antigen-binding fragment disclosed in the present invention, the therapeutic effective dose is a dose or concentration at which the antibody or antigen-binding compound can eliminate all or part of a tumor, inhibit or slow the growth of a tumor, inhibit the growth or proliferation of cells mediating a cancerous condition, inhibit the metastasis of tumor cells, alleviate symptoms or markers associated with a tumor or cancerous condition, prevent or slow the progression of a tumor or cancerous condition, or a combination thereof.

[0089] Specifically, when administering to subjects, the dosage will vary depending on the patient's age and weight, the characteristics and severity of the disease, and the route of administration. The results of animal experiments and various circumstances can be taken into consideration, and the total dosage cannot exceed a certain range.

[0090] In some embodiments, the methods described herein may further include combination administration with other compounds known in the art or other cancer treatment regimens.

[0091] Other cancer treatments include, but are not limited to, surgery, radiation therapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy, cancer vaccines (e.g., HPV vaccine, hepatitis B vaccine, Oncophage, Provenge), and gene therapy, and any combination thereof. Immunotherapy includes, but is not limited to, adoptive cell therapy, induction of stem cells and / or dendritic cells, blood transfusion, lavage and / or other therapies, and cryo-tumors.

[0092] The embodiments of the present invention will be described below by specific examples, but those skilled in the art will readily understand other advantages and effects of the present invention from the disclosure herein. The present invention may also be carried out or applied by other different specific embodiments, and each detail herein may be modified or altered in various ways based on different viewpoints and applications without departing from the spirit of the invention.

[0093] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below, and that the terms used in the embodiments of the present invention are for the purpose of describing specific embodiments and do not limit the scope of protection of the present invention. In the specification and claims of the present invention, unless otherwise clearly specified in the context, the singular forms "one," "1," and "this" include the plural forms.

[0094] Where the examples indicate numerical ranges, it should be understood that any numerical values ​​at both endpoints of each range and between those endpoints can be adopted, unless otherwise specified in the present invention. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as generally understood by those skilled in the art. Except for the specific methods, apparatus, and materials used in these examples, it is also possible for those skilled in the art to implement the present invention using any of the prior art methods, apparatus, and materials that are similar or equivalent to those described in the examples of the present invention, based on prior art knowledge and the description of the present invention.

[0095] Example 1: PG component screening test 1. Test Protocol PGa does not contain PI+PC, and the other ingredients are a blend of PGb, PGc, and PGd. PGb does not contain lecithin, and the other ingredients are a blend of PGA, PGc, and PGd. PGd does not contain Va, while the other ingredients are a blend of PGa, PGb, and PGc. The table below shows the detailed formulations of PGa, PGb, PGc, and PGd. [Table 3] [Table 4]

[0096] 2. Main Steps 2.1 LLC cell culture Cells were subcultured in DMEM medium containing 10% high-quality fetal bovine serum in a cell incubator at 37°C with 5% carbon dioxide. 2.2 Subcutaneous Tumor Formation Actively proliferating LLC tumor cells were collected, and 0.1 mL of the cell suspension was inoculated into the subcutaneous tissue of the axilla of corresponding C57 / BL / 6 mice. The cell count was approximately 1 × 10⁶. 6 That was the case. 2.3 Random group assignment All mice were randomly assigned to groups before administration on day 1 after tumor cell inoculation. 2.4 Administration to animals The drug was administered according to the efficacy test design method. 2.5 Tracking and Measurement of Tumor Volume On days 7 and 9 after tumor cell inoculation, the length (a) and width (b) of the tumors in each group of animals were measured on the surface, and the formula V = 0.52 × a × b 2 The tumor volume was estimated according to the following formula. 2.6 End of Exam The experiment was concluded 10 days after tumor cell inoculation, and the tumors from each group of animals were removed and weighed.

[0097] 3. Test Results and Analysis The anatomical diagram of the tumor is shown in Figure 1. The statistical analysis and calculation of the tumor inhibition rate are as follows. Statistical analysis of the data is performed using GraphPad Prism software, and the tumor inhibition rate is calculated based on tumor size or tumor weight using the following formula: Tumor inhibition rate % = [(Average tumor volume or tumor weight of the control group - Average tumor volume or tumor weight of the treatment group) / Average tumor volume or tumor weight of the control group] × 100%. [Table 5] [Table 6] [Table 7] Test conclusion: 1. In the predetermined effective formulation PGc, the new formulation PGa, formed by removing the raw materials PI and PC, did not exhibit a significant tumor-inhibiting effect. 2. In the predetermined effective PGc formulation, the new PGd formulation formed by removing the raw material Va did not exhibit tumor-inhibiting effects. 3. The new PGb formulation, formed by removing the excipient lecithin from the predetermined effective PGc formulation, did not exhibit any tumor-inhibiting effect. 4. Overall conclusion: Only when the specified inactivated bacterial stock solution is used as prescribed can the harmony of the active pharmaceutical ingredient and auxiliary agents in the PGc formulation, as well as the systemic action, be effectively achieved to form the optimal therapeutic effect.

[0098] Example 2: Supplemental experiment for PGC component screening 1. Test Objectives Evaluation of the antitumor effect of PGC-containing formulations lacking both pI+PC and vitamin A.

[0099] 2. Test sample 2.1 Name: PGc, PGc-PW Here, the PGc formulation is the same as the experimental formulation in Example 1, and the PGc-PW formulation is the same as the other components of the PGc formulation except that vitamin A, polyinosinic acid, and polycytidylic acid have been removed. 2.2 Provided by: CMC

[0100] 3. Test materials: 3.1 Negative control and solvent: Sterile physiological saline

[0101] 4. Tumor source: 4.1 We used the H22 mouse hepatocellular carcinoma cell line provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0102] 5. Test animals: 5.1 Origin: C57 / BL / 6 mice, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. 5.2 weeks old: 9 weeks old 5.3 Biology: Female 5.4 Number of animals: PGc sample group (2 groups), negative control group (1 group), 6 mice in each group.

[0103] 6. Method of administration and dosage setting:

[0104] 7. Main examination steps: 7.1 Randomly assign to groups 7.2 Actively growing tumors were collected and subcutaneously inoculated into the axilla of the corresponding host (0.1 mL / mouse, approximately 2 × 10⁻⁶). 6 (Individual cells). This was performed at the Beijing laboratory of Beijing Vital River Laboratory Animal Technology Co., Ltd., and the cells were sent to the Hangzhou laboratory after tumor formation. [Table 8] 7.3 Random group assignment again 7.4 The drug was administered according to the experimental design, and the size of the tumor was measured and calculated using calipers before each administration.

[0105] 8. Analysis of test results: The tumor inhibition rate was calculated from the change in tumor size, or the animals in each group were euthanized at the end of the experiment, the tumors were removed and weighed, and the tumor inhibition rate was calculated according to the following formula: Tumor inhibition rate % = [(Average tumor weight of control group - Average tumor weight of treatment group) / Average tumor weight of control group] × 100%.

[0106] 9. Experimental results: The anatomical diagram of the tumor is shown in Figure 2. [Table 9] Experimental conclusion: Under conditions in which the specified PGC formulation is effective, the new formulation, formed by removing the active ingredients PI+PC and vitamin A, showed almost no tumor-inhibiting effect.

[0107] Example 3: PGc formulation optimization test in a H22 mouse liver cancer drug efficacy model. 1. Test Objectives: a) Efficacy evaluation of PI+PC substitution with Poly(I:C) in PGC formulations; b) Evaluation of the efficacy of different PI+PC dosages in PGC formulations;

[0108] 2. Test sample 2.1 Sample composition: In this test, the only change in the mass ratio of polyinosinic acid and polycytidylic acid is the same as in Example 1's PGc formulation; all other components and their ratios remain identical. The specific groupings are shown below. B6:PI+PC dosage 8:8, C6:PI+PC dosage 4:4, D6:PI+PC dosage 8:1, E6:PI+PC dosage 4:1, F6: Use only Poly(I:C). G6:PI + PC dosage 8:8, no lipid emulsion added. 2.2 Provided by: CMC

[0109] 3. Test materials: 3.1 Negative control and solvent: Sterile physiological saline

[0110] 4. Tumor source: 4.1 We used the H22 mouse hepatocellular carcinoma cell line provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0111] 5. Test animals: 5.1 Origin: C57 / BL / 6 mice, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. 5.2 weeks old: 9 weeks old 5.3 Sex: Female 5.4 Number of animals: PGc sample group (6 groups), negative control group (1 group), 6 mice in each group.

[0112] 6. Method of administration and dosage setting: This is the same as in Example 2.

[0113] 7. Main steps of the exam: 7.1 Randomly assign to groups 7.2 Actively growing tumors were collected and subcutaneously inoculated into the axilla of the corresponding host (0.1 mL / mouse, approximately 2 × 10⁻⁶). 6 (Individual cells). This was performed at the Beijing laboratory of Beijing Vital River Laboratory Animal Technology Co., Ltd., and the cells were sent to the Hangzhou laboratory after tumor formation. 7.3 Randomly divide into groups again 7.4 The drug was administered according to the experimental design, and the size of the tumor was measured and calculated using calipers before each administration.

[0114] 8. Analysis of test results: The tumor inhibition rate was calculated from the change in tumor size, or the animals in each group were euthanized at the end of the experiment, the tumors were removed and weighed, and the tumor inhibition rate was calculated according to the following formula: Tumor inhibition rate % = [(Average tumor weight of control group - Average tumor weight of treatment group) / Average tumor weight of control group] × 100%.

[0115] 9. Test Results The anatomical details of the tumor are shown in Figure 3. [Table 10] Conclusion: 1. PGc biological injection has a significant inhibitory effect on the proliferation of liver cancer, and the efficacy of equal or unequal doses of PI+PC is superior to Poly(I:C), while the efficacy of equal doses of PI+PC is significantly superior to Poly(I:C). 2. The optimal combination is to combine PI and PC in an equal 4:4 ratio.

[0116] Example 4: Efficacy evaluation study of PGc formulation in a B16 melanoma mouse metastasis model. 1. Test Protocol [Table 11]

[0117] 2. Main Steps 2.1 B16 cell culture Cells were subcultured in DMEM medium containing 10% high-quality fetal bovine serum in a cell incubator at 37°C with 5% carbon dioxide. 2.2 Tail vein vaccination Actively proliferating B16 tumor cells were collected, and 0.1 mL of the cell suspension was inoculated into the tail vein of corresponding C57 / BL / 6 mice. The cell count was approximately 2 × 10⁶. 5 That was the case. 2.3 Random group assignment Three days after tumor cell inoculation, prior to administration, all mice were randomly assigned to groups. 2.4 Administration to animals The drug was administered according to the efficacy trial design scheme. 2.5 End of Exam The experiment was concluded 20 days after tumor cell inoculation. The lungs were dissected and removed for observation, and the presence or absence of B16 lung metastases and the number of metastases with a diameter of ≥2 mm were counted.

[0118] 3. Test Results and Analysis Typical lung anatomical diagrams for the NS and PGc groups are shown in Figure 4. Statistical analysis is as follows: GraphPad Prism software was used for statistical analysis of the data. [Table 12] Conclusion: PGc biological injection has a statistically significant effect in inhibiting the spread and metastasis of malignant melanoma. This represents a very important advance in nonclinical research in the development of tumor therapies.

[0119] Example 5: Efficacy test in PBMC model mice This study was commissioned to Shanghai Biaodu Biotechnology Co., Ltd.

[0120] 1. Experimental Objective This study aimed to evaluate the antitumor activity of the test substance PGc administered subcutaneously in a PBMC humanized mouse non-small cell lung cancer model and to compare it with the antitumor activity of an anti-hPD-1 antibody administered by intraperitoneal injection. PGc was provided by Pugong Biotechnology (Hangzhou) Co., Ltd. (hereinafter referred to as Pugong Biotechnology), and the anti-hPD-1 antibody was purchased commercially.

[0121] 2. Experimental materials 2.1 Test substance [Table 13] 2.2 Solvent [Table 14] 2.3 Other Reagent Materials [Table 15] 2.4 Equipment [Table 16]

[0122] 3. Laboratory animals 3.1 Animal information [Table 17] 3.2 Animal acceptance, health assessment, and acclimatization period Upon arrival at the breeding facility, the animals were received by the facility staff and quarantine was performed. The quarantine adaptation period for mice was 7 days. Before inoculation with PBMC cells, the experimenter conducted an examination, which included the external appearance, limbs, and cavities. The examination also checked for any abnormalities in the animals at rest or during movement, and animals in poor physical condition were excluded. 3.3 Cages and Animal Identification Each mouse was assigned a unique number. Before grouping the animals, cage tags were labeled with the project number, species / strain, sex, cage number, and animal number. After grouping the animals, the cage tags were replaced, and group information was added to the tags based on the above information. The grouping status was recorded in the experimental log.

[0123] 4. Experimental Method and Steps 4.1 Experimental Design As shown in Figure 5, 7 days prior, PBMC was 5 × 10 6 Intraperitoneal inoculation was performed using the cell / mouse dose, and 5 × 10⁶ HCC827 (ATCC:CRL-2868) cells were administered one day prior to the inoculation. 6 Subcutaneous inoculation was performed at the cell / mouse dose. Physiological saline, anti-hPD-1, and PGc were administered on days 0, 4, 8, and 12, respectively, and blood was collected on day 14. The group assignments and administration schedule are shown below. Here, 3+3 or 4+4 are derived from only two donors, representing 3 animals / donor or 4 animals / donor. Administration method for the saline group: subcutaneous administration (same administration method as the PGc group), administered a total of 5 times during the experimental period. Administration method for the anti-hPD-1 group: intraperitoneal injection (ip), administered a total of 5 times during the experimental period. Administration method for the PGc (same formulation as Example 1) group: subcutaneous administration, administered a total of 5 times during the experimental period. The administration locations are shown in Figure 6, with (1), (2), (3), (4), and (5) indicating the administration order and administration location. [Table 18] 4.2 Cell Culture and Inoculation Prepare two PBMC cell suspensions (two donor cells): Remove the cell cryotubes from liquid nitrogen, thaw them in a 37°C water bath, centrifuge the cells and remove the supernatant, resuspend the PBMC cells in DPBS, and adjust the cell concentration after resuspending to 5 × 10⁻⁶. 7 The concentration was adjusted to cells / mL. Subsequently, on October 11, 2022, mice were inoculated by intraperitoneal administration (ip), with each mouse receiving an inoculation volume of 100 μL, and 10 mice were inoculated with PBMCs from each donor. The human lung cancer HCC827 cell line used in this experiment was obtained from ATCC (catalog number: CRL-2868), and culture conditions were RPMI-1640 + 10% FBS + 0.1% P / S, culture environment: 5% CO2, 37°C. The experiment was performed before the cells were cultured continuously for 10 generations. The cells were centrifuged and the supernatant was removed, and the HCC827 cells were resuspended in DPBS. The HCC827 cell concentration after resuspending was set to 5 × 10⁶. 7 The solution was adjusted to cells / mL. It was administered subcutaneously (sc) into the right axilla of mice on October 17, 2022, with each mouse receiving a dose of 100 μL. 4.3 Classification of Animals 1) PBMCs were derived from two donors and inoculated into 20 NCG mice, with 10 mice inoculated from each donor, i.e., Donor Group 1 and Donor Group 2. The 20 mice were randomly assigned to these two groups and inoculated with PBMCs. 2) Ten mice from each of the donor groups (group 1 and group 2) were inoculated with HCC827 cells. 3) Before administration, 10 mice each were randomly divided into three groups from Donor Group 1 and Donor Group 2, with each group consisting of 3 or 4 mice (the PGc group had 4 mice), resulting in a total of 6 groups. These were Donor 1-1, Donor 1-2, Donor 1-3, Donor 2-1, Donor 2-2, and Donor 2-3. 4) Administration: Administration was carried out according to the administration methods for the physiological saline group (Donor 1-1 group and Donor 2-1 group), anti-hPD-1 (Donor 1-2 group and Donor 2-2 group), and PGc (Donor 1-3 group and Donor 2-3 group). 4.4 Data Collection Cage-side observations: From the start of inoculation to the end of the experiment, the appearance and behavior of each mouse were observed daily during working hours. In the PBMC donor 1 model, the three mice with ear tags 724, 725, and 727 in the saline group (i.e., donor 1-1 group) developed kyphosis on days 24, 20, and 20, respectively. In the anti-hPD-1 group (donor 1-2 group), the mouse with ear tag 730 developed kyphosis on day 20 and died on day 24. In the PGc group (donor 1-3 group), the mice with ear tags 721, 723, and 726 developed kyphosis on day 20, and the mouse with ear tag 731 died on day 14. Furthermore, in the PBMC donor 2 model, two mice with ear tags 732 and 741 in the saline group (i.e., donor 2-1 group) developed kyphosis on days 20 and 22, respectively. In the anti-hPD-1 group (i.e., donor 2-2 group), mouse with ear tag 736 died on day 2, and two mice with ear tags 733 and 735 developed kyphosis on days 20 and 24, respectively. In the PGc group (i.e., donor 2-3 group), mice with ear tags 737, 738, 739, and 740 developed kyphosis on day 20. Tumor volume: How to calculate tumor volume (TV): Volume = (Length × Width) 2 ) / 2. From day 6 onwards, measurements were taken three times a week (Monday, Wednesday, and Friday), and the same experimenter performed the measurements (to reduce measurement errors). Animal weight: After grouping, the weight of the mice was recorded simultaneously with the measurement of tumor volume. 4.5 Sample Collection Blood collection: 21 days after PBMC inoculation, approximately 100 μL of peripheral blood was collected from the orbit of mice and placed in a 1.5 mL EDTA anticoagulant tube. This was then sent at room temperature to the Institute of Biochemistry, Chinese Academy of Sciences for detection. 4.6 Criteria for Euthanasia If any one or more of the following situations occur during the experiment, the animal must be euthanized. 1) The tumor volume of one animal in the group is 3000 mm 3 If it exceeds that. 2) Ulceration, necrosis, or infection of the tumor. 3) The animal develops behavioral abnormalities or paralysis. 4) The animal's body weight decreases by more than 20% compared to its body weight at the time of the first administration.

[0124] 5.Statistical analysis method Efficacy results were presented as mean ± SEM. Comparisons between different groups were detected using ANOVA or other analytical methods, and the difference was considered statistically significant if p < 0.05.

[0125] 6. Abbreviations [Table 19]

[0126] 7. Experimental Results Evaluation of the antitumor activity of PGc in the HCC827PBMC model As shown in Figure 7 and the table below, in an HCC827PBMC model using two donors, the PGc group and the anti-hPD-1 group significantly inhibited tumor growth compared to the negative control group (saline solution). Specifically, the tumor volume in the PGc group was significantly smaller than that of the negative control group from day 6 to day 17, and significantly smaller than that of the anti-hPD-1 group from day 6 to day 15. The tumor inhibition effect was similar between the PGc group and the anti-hPD-1 group, and no significant difference in tumor volume was observed. [Table 20] TIFF2026516846000021.tif211170 Experimental conclusion: In the humanized mouse PBMC HCC827 lung cancer mouse model of the present invention, the overall tumor inhibition rate of PGc biological injection reached 68.515%.

[0127] Example 6: Antitumor Efficacy Test of PGc-containing Samples 1. Test Objectives Antitumor efficacy testing of PGc-containing samples, and preliminary tumor inhibition experiments in small-scale samples using combination therapy with anti-mouse PD-1 antibody.

[0128] 2. Test materials and equipment 2.1 Test specimen: 2.1.1 Name: PGc (same formulation as Example 1), 2.1.2 Provider: Pugong Biotechnology (Hangzhou) Co.,Ltd. 2.2 Reagent materials: 2.2.1 Cell medium: DMEM, Gibco, Catalog number: C11995500BT, Batch number: 8122010, Serum: Yeasen, Catalog number: 40130ES76, Batch number: 504102121, 2.2.2 Negative and positive control solvents: Sterile physiological saline (NS), Beyotime, product number: ST341-500mL 2.2.3 Positive control: Cyclophosphamide for injection (CTX), Baxter, Batch number: 0G391A 2.2.4 Concomitant medication: Anti-mouse PD-1, bioxcell, catalog number: BE0146, batch number: 695318A1, 599016M2C; Dilution buffer: bioxcell, catalog number: IP0070, batch number: 710920M1. 2.3 Tumor source: Mouse Lewis lung cancer cells (LLC) were obtained from the Chinese Academy of Sciences Cell Bank. 2.4 Test animals: 2.4.1 Origin: C57 / BL / 6 mice were provided by Shanghai SLAC Laboratory Animal Co., Ltd., Certificate of Conformity Number: 20170005068458, CEMCS Test Animal Use License Number: SYXK(Shanghai)2018-0007; 2.4.2 Weight: average 19g, 2.4.3 Sex: Female, 2.4.4 Number of animals: 10 animals in the negative control group, 10 animals in the positive control group, 10 animals in the PG-B sample group, 10 animals in the PGc sample group, 5 animals in the anti-PD-1 control group, and 5 animals in the PGc and anti-PD-1 combination group. 2.5 Equipment and facilities: FENYE inverted biological microscope, model: ZLD200-37T; Heal Force biosafety cabinet, model: II, A2; Thermo CO2 cell incubator, model: BB15.

[0129] 3. Efficacy Test Protocol [Table 21]

[0130] 4. Main Examination Steps 4.1 LLC cell culture Cells were subcultured in DMEM medium containing 10% high-quality fetal bovine serum in a cell incubator at 37°C with 5% carbon dioxide. 4.2 Subcutaneous Tumor Formation Actively proliferating LLC tumor cells were collected, and 0.1 mL of the cell suspension was inoculated into the subcutaneous tissue of the axilla of corresponding mice. The cell count was approximately 1 × 10⁶. 6 That was the case. 4.3 Random group assignment All mice were randomly assigned to groups before administration on day 1 after tumor cell inoculation. 4.4 Administration to animals The drug was administered according to the efficacy trial design scheme. 4.5 End of Exam The experiment was concluded 10 days after tumor cell inoculation, and the tumors from each group of animals were removed and weighed.

[0131] 5. Test Results and Analysis The anatomical diagram of the tumor is shown in Figure 8. Statistical analysis and calculation of tumor inhibition rate: Statistical analysis of the data was performed using GraphPad Prism software, and the tumor inhibition rate was calculated based on tumor size or tumor weight using the following formula: Tumor inhibition rate % = [(Average tumor weight of control group - Average tumor weight of treatment group) / Average tumor weight of control group] × 100%. The results are shown in the table and Figure 9 below. [Table 22] The results showed that when C57 / BL / 6 mice were inoculated with an LLC lung cancer cell model, the tumor inhibition rate of PGc biological injection as a monotherapy reached 60.11%, and when used in combination with PD-1, the tumor inhibition rate reached 82.27%.

[0132] Example 7 Flow cytometry analysis of antitumor immune cell activation using PGc-containing samples 1. Test Objectives This study provides ideas for further investigating the antitumor mechanism of PGc-containing samples by performing flow cytometry analysis on the activation of antitumor immune cells by PGc-containing samples.

[0133] 2. Test materials and equipment 2.1 Test specimen: 2.1.1 Name: PGc (same formulation as Example 1), 2.1.2 Provider: Pugong Biotechnology (Hangzhou) Co., Ltd. 2.2 Reagent materials: 2.2.1 Cell medium: DMEM, Gibco, Catalog number: C11995500BT, Batch number: 8122010, Serum: Genimi, Catalog number: 900-123, Batch number: A24F02G, 2.2.2 Negative and positive control solvents: Sterile physiological saline (NS), Beyotime, product number: ST341-500mL 2.2.3 Positive control: Cyclophosphamide for injection (CTX), Baxter, Batch number: 0G391A 2.2.4 Isolation of lymphocytes from tumor tissue: Collagenase IV, Sigma, catalog number: C-5138; DNase I, Roche, catalog number: 143582; Percoll, GE Healthcare, catalog number: 17-0891-01. 2.2.5 Flow cytometry antibodies: Anti-CD3εFITC, eBioscience, catalog number: 11-0031-82; Anti-CD4 PerCP-Cy5.5, eBioscience, catalog number: 45-0042-82; Anti-CD8aAPC, eBioscience, catalog number: 17-0081-83; Anti-B220FITC, eBioscience, catalog number: 11-0452-82; Anti-CD45 PerCP-Cy5.5, BD, catalog number: 561047; Anti-NK1.1PE, eBioscience, catalog number: 12-5941-81. 2.3 Tumor source: Mouse Lewis lung cancer cells (LLC) were obtained from the Chinese Academy of Sciences Cell Bank. 2.4 Test animals: 2.4.1 Origin: C57 / BL / 6 mice were provided by Shanghai SLAC Laboratory Animal Co., Ltd., Certificate of Conformity Number: 20170005064721, CEMCS Test Animal Use License Number: SYXK (Shanghai) 2018-0007. 2.4.2 Age in weeks: 8 weeks old, 2.4.3 Sex: Female, 2.4.4 Number of animals: PGc-containing sample group (1 group), negative control group (1 group), positive control group (1 group), 10 mice in each group. 2.5 Instruments and Equipment: Flow cytometer: CytoFLEX, Backman.

[0134] 3. Flow Cytometry Test Protocol Administration was discontinued 10 days after tumor cell inoculation. In the NS control group and the PGc group, tumors were removed after autopsy, and immunocytometry analysis was performed. [Table 23]

[0135] 4. Main steps of the exam 4.1 LLC cell culture: Cells were subcultured in DMEM medium containing 10% high-quality fetal bovine serum in a cell incubator at 37°C with 5% carbon dioxide. 4.2 Subcutaneous Tumor Formation Actively proliferating LLC tumor cells were collected, and 0.1 mL of the cell suspension was inoculated into the subcutaneous tissue of the axilla of corresponding mice. The cell count was approximately 1 × 10⁶. 6 That was the case. 4.3 Random group assignment All mice were randomly assigned to groups before administration on day 1 after tumor cell inoculation. 4.4 Administration to animals and termination The drug was administered according to the experimental design, and the experiment was completed 10 days after tumor cell inoculation. Tumors were then removed from both the NS control group and the PGc group. 4.5 Flow Cytometry Analysis 4.5.1 Isolation of lymphocytes from tumor tissue: The mouse tumor was excised along the tumor margin and placed in a 6 cm petri dish filled with PBS. A portion of the tumor was treated with 1 mg / mL of collagenase type IV and 1 mg / mL of DNase, in an amount approximately 5-6 times the tumor volume. Add I, seal with Parafilm, digest in a 37°C shaker for 0.5-1 hour, place a 70 μm filter in a 6 cm petri dish, add the cell suspension colrogenase solution to the filter, and grind with a grinding rod until the single cell suspension flows into the petri dish, transfer the single cell suspension to a 15 ml centrifuge tube, centrifuge at 1200 rcf for 5 minutes, discard the supernatant, add 3 ml of 35% Percoll and resuspend to precipitate, add 3 ml of 70% Percoll to a 15 ml centrifuge tube, slowly add the 3 ml obtained in the previous step on top so as not to disturb the layer, centrifuge at 2500 rpm for 20 minutes, aspirate the intermediate lymphocyte layer, wash once with 15 ml of PBS, centrifuge at 2000 rpm for 5 minutes, discard the supernatant and resuspend in PBS. 4.5.2 Flow cytometry staining: Resuspended cells were added to a centrifuge tube containing flow cytometry antibody (flow cytometry antibody: 1 μL / sample) and uniformly sprayed using a spray gun. Reaction system volume: 50 μL. The cells were incubated in the dark at 4°C for 45-60 minutes. 1 mL of PBS was added to the centrifuge tube, mixed uniformly with a pipette, and centrifuged in a horizontal centrifuge (1000 rpm, 5 minutes). The supernatant was removed, the cells were resuspended in 200 μL of PBS, transferred to a flow cytometry tube, and flow cytometry detection was performed in the dark.

[0136] 5. Test Results and Analysis Flow cytometry analysis of cells in tumor tissue from the NS group and the PGC group revealed that CD45 was different between these two groups. + immune cells, CD3 + T cells and CD4 + Statistically significant differences were observed in T cells, with P=0.0027, P<0.0001, and P=0.0107 respectively, but CD8 + No statistically significant differences were observed in T cells, NK cells, NKT cells, and B cells. (Figure 10)

[0137] Example 8: Verification of the efficacy of PGc-B36 against B16f0 melanoma. Tumor source: B16f0 was purchased from Zhejiang Meisen Industrial Co., Ltd. and transplanted subcutaneously into B / C mice. Tumor culture and mouse tumor retention were completed by Hangzhou Medical College.

[0138] Animal information: Age: 8 weeks old, Sex: Female Number of animals: PGc sample group (1 group), negative control group (1 group), 11 mice in each group.

[0139] The administration method and dosage settings are as shown in the table below: In the table below, A64 is the negative control, specifically sodium chloride injection, and the formulation of PGc-1208 in B64 is the same as in Example 1. Different codes represent only different manufacturing batches.

[0140] The units for injection doses in the table are mL. PGc0.1+0.1 indicates that 0.1 mL of PGc is injected at two different locations, and the same applies to the other examples. [Table 24]

[0141] The anatomical diagram of the tumor is shown in Figure 11. The tumor weight data (in g) and tumor inhibition rates are shown in the table below: the total represents the total tumor weight of 11 mice. [Table 25]

[0142] The results showed that PGc biological injections can effectively inhibit the proliferation of melanoma.

[0143] Example 9: Verification of the efficacy of PGc-F31 / B36 for RM-1 prostate cancer. Tumor source: In this study, the RM-1 mouse prostate cancer cell line was used and transplanted subcutaneously into C57 mice. Tumor culture and mouse tumor retention were completed by Hangzhou Medical College.

[0144] Animal information: Age: 8 weeks old, Sex: Male Number of animals: PGc sample group (2 groups), negative control group (1 group), 5-6 mice in each group.

[0145] The negative control group was sodium chloride injection, and the formulation of PGc-B36 was the same as in Example 1.

[0146] Administration method and dosage setting: [Table 26]

[0147] The unit of injection volume in the table is ml.

[0148] The anatomical diagram of the tumor is shown in Figure 12. The tumor weight data (in g) and tumor inhibition rate are shown in the table below. The first row of the table, numbers 1-6, each represent a different mouse, and the total represents the total tumor weight of 6 mice. [Table 27]

[0149] The results indicate that the biological injection of PGc can significantly inhibit the growth of prostate tumors.

[0150] Example 10: Experiment to verify the efficacy of PGc against HT1080 human sarcoma. Tumor source: Human HT1080 sarcoma transplanted subcutaneously into nude B / C mice. Tumor culture and mouse tumor retention were completed by Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0151] Animal information: Age: 8 weeks old, Sex: Male.

[0152] The negative control group was sodium chloride injection, with the PGc-C16 formulation being the same as in Example 1; different codes represent only different manufacturing batches. Dosage method and setting: [Table 28]

[0153] The anatomical diagram of the tumor is shown in Figure 13. The tumor weight data (in g) and tumor inhibition rate are shown in the table below. Rows 1-6 in the first row of the table represent different mice, and the total represents the total tumor weight of 6 mice. [Table 29]

[0154] The results showed that PGc biological injections can effectively inhibit sarcoma growth.

[0155] Example 11 Experiment to Verify the Effectiveness of PGcc-B32N against 4T1 Breast Cancer Tumor source: In this study, the 4T1 mouse breast cancer cell line was used and transplanted subcutaneously into B / C mice. Tumor culture and mouse tumor bearing were completed by Hangzhou Medical College.

[0156] Animal information: Age: 8 weeks old, Gender: Female.

[0157] The negative control group was sodium chloride injection. The formulation of PGc-B32N was the same as in Example 1, and the different codes only represent different production batches.

[0158] Administration method and dosage setting:

Table 30

[0159] The anatomical diagram of the tumor is as shown in Figure 14. The tumor weight data (unit: g) and tumor inhibition rate are as shown in the following table. The 1 - 6 in the first row of the table represent different mice, and the total represents the total tumor weight of 6 mice.

Table 31

[0160] The results showed that the PGc biological injection could effectively inhibit the growth of breast cancer.

[0161] Example 12 Test to Verify the Drug Effect of PGc-C16 against Pan02 Pancreatic Cancer Tumor source: Pan02 cell line. Tumors were transplanted subcutaneously into B / C mice. Tumor culture and mouse tumor bearing were completed by Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0162] Animal information: Age: 8 weeks old, Gender: Female.

[0163] The negative control group was sodium chloride injection, and the formulation of PGc-C16 was the same as in Example 1. Different codes only represent different production batches. Administration method and dosage setting:

Table 32

[0164] The anatomical diagram of the tumor is as shown in Figure 15. The tumor weight data (unit: g) and tumor inhibition rate are as shown in the following table. The numbers 1 to 6 in the first row of the table represent different mice, and the total represents the total tumor weight of 6 mice.

Table 33

[0165] The results showed that the PGc biological injection could effectively inhibit the growth of pancreatic cancer.

[0166] Example 13 Evaluation of the potential toxic reaction of the PGc broad-spectrum anti-tumor preparation on the living body The purpose of this study was to observe the potential toxic reaction when the PGc broad-spectrum anti-tumor injection was repeatedly administered subcutaneously to cynomolgus monkeys for 3 months (administered once every 3 days in the first week, a total of 3 times, and then once a week, a total of 12 times), and to identify the possible toxic target organs. The formulation of the PGc broad-spectrum anti-tumor injection was the same as in Example 1.

[0167] Two male cynomolgus monkeys were subcutaneously injected with high and low doses of a broad-spectrum antitumor agent (PGc) (4.0 mL / milli or 2.0 mL / milli) on days 1, 4, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, and 91, respectively. Dissection was performed at the end of the administration period (day 93). During the study period, various indicators were evaluated, including clinical observation, body weight, food intake, body temperature (rectal temperature), electrocardiogram, surface temperature, hematology, blood coagulation, plasma biochemistry, immunoglobulins and complement, lymphocyte immunophenotype, urine, cytokines, and macroscopic morphological observation. Since the evaluation methods for each indicator are all extremely common techniques in the art, they will not be described in detail in this invention.

[0168] During the study period, the main changes caused by the test compound were observed through clinical observation, body weight, surface temperature, clinicopathology (hematology, blood coagulation, plasma biochemistry, immunoglobulins, lymphocyte immunophenotype), cytokines, and macroscopic autopsy observation. The main changes are shown in the table and explanation below.

[0169] Clinical observations: In animals in both the high-dose and low-dose groups, multiple raised areas (hard to the touch) were observed at the injection site after repeated administration, with significantly more in the high-dose group than in the low-dose group. Furthermore, reversible erythema developed at the injection site in two animals after the initial administration.

[0170] Body weight: In the high-dose group, slight weight loss was observed several times during the mid- and late-stages of drug administration. Specifically, animals in the PGc 4.0 mL / animal group showed slight weight loss (up to approximately 7%) on multiple occasions during the mid- and late-stages after administration (days 21, 49, 56, 63, 70, 77, and 91) compared to their pre-administration body weight (1 day prior). No significant abnormalities in body weight were observed in the PGc 2.0 mL / animal group during the experiment.

[0171] Body surface temperature: Animals in the high-dose group showed a slight increase in body surface temperature on day 3 and day 5 (8 and 16 hours after administration).

[0172] Hematology: Elevated WBC and NEUT were observed in animals in both dose groups on day 2 after the 3rd, 6th, 10th, and 15th doses, but no clear correlation was found between the elevation and the dose.

[0173] Blood coagulation: In the two administration groups, FIB (days 8, 29, 57, and 92), APTT (days 8, 57, and 92), and PT (day 8) increased two days after the 3rd, 6th, 10th, and 15th doses. In the PGc 2.0 mL / animal group, FIB (days 8, 29, 57, and 92) and APTT (day 57) also increased at the same time points.

[0174] Plasma biochemistry, immunoglobulins: In two dose groups of animals, decreased ALB and increased GLOB (decreased A / G ratio) were observed two days after 3, 6, 10, and 15 doses (days 8, 29, 57, and 92). In the high-dose group, a slight increase in IgM and / or IgG was also observed at the above time points.

[0175] Lymphocyte immunophenotype: In animals in the two dose groups, a decrease in CD3+CD8+, an increase in CD4+ / CD8+, and a slight increase in CD3-CD16+ (observed only after the third dose) were observed two days after the 3rd, 6th, 10th, and 15th doses. Animals in the high-dose group also showed a significant increase in CD3-CD11b+ after the 15th dose.

[0176] Cytokines: In animals in the two dose groups, elevated IL-6 levels were observed 2 hours and 24 hours after administration on the day of the 1st, 6th, 10th, and 15th doses (days 7, 28, 56, and 91).

[0177] Autopsy and macroscopic observation: During autopsy, multifocal elevations were not observed only at the last administration site (back of the neck) in the animals from the two dose groups.

[0178] No changes related to the test compound were observed in other evaluation indicators (feed intake, rectal temperature, electrocardiogram, and complement).

Table 34

[0179] From the above, under the conditions of this test, when cynomolgus monkeys were repeatedly subcutaneously administered 2.0 and 4.0 mL / kg of the PGc broad-spectrum anti-tumor injection for 3 months, all animals in each dose group were able to tolerate it. The main changes were the injection site irritation reaction (mainly manifested as swelling at the injection site) and mild immune enhancement (mainly manifested as increases in IgM, IgG, WBC count, NEUT count, and IL-6). Therefore, it was shown that the PGc broad-spectrum anti-tumor injection of the present invention has safety.

[0180] Example 14 Mechanism of action of the PGc broad-spectrum anti-tumor preparation 1. The experimental method for macrophage stimulation by PGc is as follows: 1) Induction, isolation, and culture of primary peritoneal macrophages in mice: Mice aged 8 weeks or older were intraperitoneally injected with an aqueous solution of 3% thioglycolate, 3 mL was injected into each mouse. Four days later, the mice were euthanized by cervical dislocation or carbon dioxide, immersed in ethanol for disinfection for 2 minutes, placed flat with the ventral side up, and a small incision was made on the outside of the abdomen while taking care not to damage the peritoneum. 5 mL of DMEM medium was injected into the mouse peritoneal cavity with a 5 mL syringe, the syringe was removed, the mouse abdomen was massaged for about 3 minutes, the syringe needle was inserted into the mouse peritoneal cavity again, and as much liquid as possible was aspirated, transferred to a centrifuge tube, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and resuspended with an appropriate amount of DMEM complete medium. After counting, seeded in a 12-well plate at 1×10 6 cells / well and cultured overnight, and then the adhered cells were used as peritoneal macrophages for subsequent experiments. 2) Macrophage stimulation by PGc PGc (the same formulation as in Example 1) was diluted in DMEM complete medium at a ratio of 1:100. The medium of the cells cultured overnight above was discarded, and the cells were cultured for 6 hours with a new medium containing PGc. 3) RNA extraction, reverse transcription, real-time quantitative RT-qPCR RNA extraction: After cell processing, discard the culture medium, wash once with PBS, dissolve in 1 ml of Trizol, let stand at room temperature for 5 minutes, add 200 μl of chloroform, vortex vigorously, let stand at room temperature for 3 minutes, centrifuge at 12000 rpm at 4°C for 15 minutes, take the upper aqueous phase (upper liquid, approximately 500 μl), add an equal volume of isopropanol, let stand at room temperature for 10 minutes, centrifuge at 12000 rpm at 4°C for 10 minutes, wash once with 1 mL of 75% ethanol, and centrifuge at 12000 rpm at 4°C for 5 minutes. Remove the supernatant, aspirate the liquid using a small tip, air dry the precipitate for 5-10 minutes, dissolve and mix with 20 μl of ddH2O (DEPC treated), and measure the RNA concentration with Nanodrop. The RNA was stored at -80°C. Reverse transcription: First, 1 μg of RNA was mixed with 100 ng of oligonucleotide (dN6) and adjusted to 14 μl with DEPC water. After incubation at 70°C for 10 minutes and rapid cooling in an ice bath for 2 minutes, 4 μl of reverse transcriptase buffer (5X), 1 μl of M-MLV reverse transcriptase, and 1 μl of dNTP 10 mM were added and adjusted to 20 μl with DEPC water. The mixture was incubated at 37°C for 1 hour and then treated at 72°C for 10 minutes to inactivate the reverse transcriptase activity. 180 μl of deionized water was added to the reverse transcript product and used as a cDNA template for the qPCR reaction. Real-time quantitative RT-qPCR: 20 μl RT-qPCR reaction system: 3.2 μl deionized water, 10 μl SYBR-Green Master Mix, 6 μl diluted cDNA template, 0.8 μl forward primer and reverse primer (10 μM each). Reaction program: 3 minutes at 95°C, 10 seconds at 95°C, 30 seconds at 60°C, 30 seconds at 72°C, fluorescence readings for a total of 40 cycles, 1 minute at 95°C, 1 minute at 55°C, thawing at 55°C to 98°C, increasing temperature by 5°C every 5 seconds, and simultaneously reading fluorescence values. The results are shown in Figures 16-17. The PGc-treated group showed a clear increase in each mRNA compared to the control group. These results indicate that PGc promotes the activation of M1 macrophages.

[0181] 2. Construction of an animal model The formulation of the PGc preparation used in the animal model was the same as in Example 1. The steps for constructing the LLC tumor animal model were the same as in Example 1. The steps for constructing the H22 tumor animal model were the same as in Example 2. Cell staining is a conventional experimental method in this field, and further explanation will be omitted in this example. The RNA-seq experiment was outsourced to a third-party organization. The results are shown in Figures 16-23. Based on pathological autopsy and RNA sequencing analysis, the results indicate that the mechanism of action of PGc has the following directions: firstly, it promotes the activation of M1 macrophages (shown in Figures 16-17); secondly, it promotes the activation of T cells (shown in Figures 18 and 23); thirdly, it inhibits angiogenesis within tumor tissue (shown in Figure 19); and fourthly, it enables the comprehensive activation and effective coordination of the innate and adaptive immune systems (shown in Figures 20-22). Note: The conclusion that there was no significant difference in CD8 in Example 7 was derived based on the results of the initial single experiment. The multiple replicate experiments conducted later, as well as the experiment in this example, all showed a significant change in CD8.

[0182] The present invention demonstrated that the HCC827 lung cancer mouse model of humanized mouse PBMCs achieved an overall tumor inhibition rate of 68.515% with PGc biological injection, the monotherapy tumor inhibition rate of PGc biological injection reached 89.8% in a model of C57 / BL / 6 mice inoculated with H22 liver cancer cells, the melanoma metastasis model showed that PGc biological injection exhibited excellent inhibitory effects against melanoma metastasis, the atherosclerosis mouse model showed that PGc had a significant effect on improving atherosclerosis, and the HPV mouse model showed that PGc had a significant therapeutic effect on conversion from HPV positive to HPV negative.

[0183] The above examples are for illustrative purposes only and should not be construed as limitations on the present invention. Furthermore, the various modifications and methodological changes in the invention enumerated herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been specifically described in combination with several specific preferred embodiments of the invention, it should be understood that the present invention is not limited to these specific embodiments. In fact, obtaining the invention through the various modifications described above, which will be apparent to those skilled in the art, should all be included within the scope of the present invention.

Claims

1. A pharmaceutical composition characterized by comprising a first active ingredient, a second active ingredient, and a pharmaceutically acceptable carrier or excipient, wherein the first active ingredient is a microbial agent comprising one or more of Staphylococcus aureus, Bordetella pertussis, diphtheria toxoid, tetanus toxoid, Salmonella typhi, or Salmonella paratyphi, and the second active ingredient comprises polyinosinic acid, polycytidylic acid, and vitamin A.

2. The pharmaceutical composition according to claim 1, characterized in that the microbial agent is an inactivated formulation and / or the microbial agent is a liquid formulation or a solid formulation.

3. The pharmaceutical composition according to claim 1, further characterized by including one or more of the following features. (1) Based on the total volume of the pharmaceutical composition, the concentration of Staphylococcus aureus is 2 × 10 ７ ~3 x 10 ９ The characteristic is that it is particles / mL. (2) Based on the total volume of the pharmaceutical composition, the concentration of Bordetella pertussis is 7 × 10 ７ ~9 x 10 ９ The characteristic is that it is particles / mL. (3) Based on the total volume of the pharmaceutical composition, the concentration of the diphtheria toxoid is 1 LF to 5 LF particles / mL. (4) Based on the total volume of the pharmaceutical composition, the concentration of the tetanus toxoid is 0.1 LF to 5 LF particles / mL. (5) Based on the total volume of the pharmaceutical composition, the concentration of the typhoid bacillus is 1.5 × 10 ６ ~5 x 10 ８ The characteristic is that it is particles / mL. (6) Based on the total volume of the pharmaceutical composition, the concentration of the paratyphi bacterium is 1 × 10 ６ ~3 x 10 ８ A characteristic feature is that it contains particles per mL.

4. The paratyphoid bacterium is any one or more selected from Salmonella paratyphi A, Salmonella paratyphi B or Salmonella paratyphi C. Preferably, the paratyphoid bacterium is selected from Salmonella paratyphi A and Salmonella paratyphi B. More preferably, based on the total volume of the pharmaceutical composition, the concentration of Salmonella paratyphi A is 0.1×10 ７ to 8×10 ７ CFU / mL, and / or the concentration of Salmonella paratyphi B is 0.1×10 ７ to 8×10 ７ CFU / mL. The pharmaceutical composition according to claim 1, characterized by this.

5. The pharmaceutical composition according to claim 1, characterized in that the polyinosinic acid and polycytidylic acid are selected from polymers formed by inosinic acid alone and polymers formed by cytidylic acid alone, or from copolymers of inosinic acid and cytidylic acid, preferably, when the polyinosinic acid and polycytidylic acid are selected from polymers formed by inosinic acid alone and polymers formed by cytidylic acid alone, the mass ratio of the polyinosinic acid to the polycytidylic acid is 1:0.1 to 1:10, more preferably the mass ratio of the polyinosinic acid to the polycytidylic acid is 1:1, and more preferably, based on the total volume of the pharmaceutical composition, the concentrations of the polyinosinic acid and polycytidylic acid are both 0.5 g / 100 mL or less, more preferably 0.01 g / 100 mL to 0.5 g / 100 mL.

6. The pharmaceutical composition according to claim 1, wherein the vitamin A is selected from vitamin A1 and / or vitamin A2, and more preferably, the concentration of the vitamin A is 1 g / 100 mL or less based on the total volume of the pharmaceutical composition.

7. The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable carrier or excipient comprises sodium carboxymethylcellulose, aluminum stearate, tween 80, lecithin, soybean oil, dextran, fat emulsion, and water, and preferably the aluminum stearate is selected from aluminum monostearate or aluminum distearate.

8. The pharmaceutical composition according to claim 1, further characterized by including one or more of the following features. (1) Based on the total volume of the pharmaceutical composition, the concentration of sodium carboxymethylcellulose in the pharmaceutically acceptable carrier or excipient is 2 g / 100 mL or less, and the preferred concentration is 0.2 to 2 g / 100 mL. (2) Based on the total volume of the pharmaceutical composition, the concentration of aluminum stearate in the pharmaceutically acceptable carrier or excipient is 3 g / 100 mL or less, and the preferred concentration is 0.3 to 3 g / 100 mL. (3) Based on the total volume of the pharmaceutical composition, the concentration of tween80 in the pharmaceutically acceptable carrier or excipient is 1 ml / 100 mL or less, and the preferred concentration is 0.1 to 1 mL / 100 mL. (4) Based on the total volume of the pharmaceutical composition, the concentration of lecithin in the pharmaceutically acceptable carrier or excipient is 50 mg / 100 mL or more, and the preferred concentration is 50 to 2000 mg / 100 mL. (5) Based on the total volume of the pharmaceutical composition, the concentration of soybean oil in the pharmaceutically acceptable carrier or excipient is 15 ml / 100 mL or less, and the preferred concentration is 1 to 15 mL / 100 mL. (6) Based on the total volume of the pharmaceutical composition, the concentration of dextran in the pharmaceutically acceptable carrier or excipient is 10 g / 100 mL or less, and the preferred concentration is 1 to 10 g / 100 mL. (7) Based on the total volume of the pharmaceutical composition, the concentration of the fat emulsion in the pharmaceutically acceptable carrier or excipient is 10 ml / 100 mL or more, and a preferred concentration is 10 to 80 mL / 100 mL.

9. The pharmaceutical composition according to claim 1, characterized in that the pharmaceutical composition is an injectable preparation.

10. Use of the pharmaceutical composition according to any one of claims 1 to 9 in the manufacture of a disease treatment product, a disease prevention product, or a disease diagnostic product.

11. The use according to claim 10, wherein the disease is selected from cancer, arteriosclerosis, HPV infection, and atrophic gastritis, and preferably the cancer is selected from liver cancer, lung cancer, melanoma, colorectal cancer, gastric cancer, ovarian cancer, bile duct cancer, cervical cancer, pancreatic cancer, prostate cancer, sarcoma, and breast cancer.

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