Anti-tumor combination vaccine

Abstract

Provided is use of a combination of three or more of the following microorganisms in the preparation of an anti-tumor combination vaccine: Bordetella pertussis, Salmonella typhi, Salmonella paratyphi A, Salmonella paratyphi B, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Proteusbacillus vulgaris, Lactic acid bacteria, Bifidobacterium longum, Bordetella pertussis, Corynebacterium diphtheriae and Clostridium tetani, Clostridium acetobutylicum, Salmonella typhimurium, and Streptococcus pyogenes. The anti-tumor combination vaccine is broad-spectrum, safe and non-toxic, and thus has the prospect of clinical applications.

Description

Anti-tumor composite vaccine Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a broad-spectrum anti-tumor compound vaccine. Background Art

[0002] In recent years, with the rapid development of medical research on the tumor microenvironment (TME) and tumor microbiome, and the good anti-tumor prospects shown by immune checkpoint inhibitors (ICI) and chimeric antigen receptor-T cells (CAR-T) in clinical applications, new strategies for treating tumors with bacteria and bacterial components have attracted more and more attention and research.

[0003] As early as the 1980s, my country began exploring the use of bacteria to treat tumors, but found that bacterial vaccines alone were ineffective. Consequently, Academician Wang Zhenyi, a renowned Chinese hematologist and oncologist and the inventor of induced differentiation therapy for acute promyelocytic leukemia, proposed a new approach to combating cancer. He advocated boosting the body's overall immunity through a multi-target immune attack, known as a "multi-component, multi-target approach." Together with doctors Kong Runlian and Xu Kecheng, he pioneered the research of combined bacterial vaccine therapy for tumors. Referring to patent documents CN101569746A, CN101628114B, CN106667907A and CN112618581A, the mechanism of combined microbial vaccines (also known as composite vaccines, combined bacterial vaccines) for treating tumors can be summarized as organically combining the uniqueness and targeting of different microbial vaccines to stimulate the body to produce a variety of non-specific antibodies and comprehensive immunity in an all-round and multi-target manner, thereby "flexibly" resisting the development and even eliminating cancer cells of different types with complex and different physiological properties, generating broad-spectrum immunity, and effectively combating the great heterogeneity, dynamics and volatility of cancer, thereby achieving an overall therapeutic effect of non-specific resistance to tumors. See the literature Therapeutic bacteria to combat cancer; current advances, challenges, and opportunities. Sedighi, Mansour, et al., [J]. Cancer Medicine, 2019, 8(6): 3167-3181. doi: 10.1002 / cam4.2148. and the literature Recent advances in bacteria-mediated cancer therapy. Frontiers in Bioengineering and Biotechnology 2022, 10 https: / / doi.org / 10.3389 / fbioe.2022.1026248. etc. Many clinical research results in the medical community over the years have also confirmed the scientific nature and clinical application prospects of the above-mentioned combined bacterial vaccine for treating tumors. Summary of the Invention

[0004] Since 1990, the inventors have continued Wang Zhenyi's research group's research on anti-tumor compound vaccines, focusing on the practical clinical application of broad-spectrum anti-tumor compound vaccines. They have continuously optimized the composition of compound vaccine formulations. Through research collaborations with the Shanghai Institute of Medical Engineering, the Shanghai Institute of Biochemistry, and the Shanghai Institute of Materia Medica, the Chinese Academy of Sciences, and animal experiments, they have developed a safe, safe, and side-effect-free compound microbial vaccine formulation with significant therapeutic efficacy against various malignant tumors. Based on these research findings, the present invention includes the following technical solutions.

[0005] The first aspect of the present invention is to provide the use of the following microorganisms in the preparation of an anti-tumor composite vaccine, which comprises a combination of three or more microorganisms: Bordetella pertussis, Salmonella typhi, Salmonella paratyphi A, Salmonella paratyphi B, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Proteus, lactic acid bacteria, Bifidobacterium longum, Diphtheria and Tetanus, Clostridium acetobutylicum, Salmonella typhimurium, and Streptococcus pyogenes.

[0006] To emphasize the immunological effects of these microorganisms and their role as pharmaceutically active ingredients (vaccine active ingredients) in combination vaccine formulations, these microorganisms are sometimes referred to herein as "bacterial vaccines" or "vaccine bacteria."

[0007] The second aspect of the present invention is to provide an anti-tumor composite vaccine, the pharmaceutical active ingredient (vaccine active ingredient) of which comprises an inactivated preparation of the following three microorganisms, more preferably consists of an inactivated preparation of the following three microorganisms: Bordetella pertussis, Salmonella typhi, Staphylococcus aureus; or

[0008] An anti-tumor composite vaccine, wherein its pharmaceutical active ingredients (vaccine active ingredients) include, in addition to Bordetella pertussis, Salmonella typhi, and Staphylococcus aureus, Salmonella paratyphi A and Salmonella paratyphi B, that is, its pharmaceutical active ingredients (vaccine active ingredients) include, more preferably consist of, inactivated preparations of the following five microorganisms: Bordetella pertussis, Salmonella typhi, Salmonella paratyphi A, Salmonella paratyphi B, and Staphylococcus aureus; or

[0009] An anti-tumor composite vaccine, whose pharmaceutical active ingredients (vaccine active ingredients) include inactivated preparations of the following nine microorganisms, and more preferably consist of inactivated preparations of the following nine microorganisms: Listeria, Escherichia coli, Proteus, Lactobacillus, Bifidobacterium longum, Diphtheria and Tetanus, Clostridium acetobutylicum, Salmonella typhimurium, and Streptococcus pyogenes.

[0010] The inactivated preparation is an inactivated product of a microbial suspension, for example, an inactivated product of a microbial suspension in physiological saline or a suspension in PBS buffer (phosphate buffer solution) for injection.

[0011] As known to those skilled in the art, the microbial suspension is preferably inactivated by physical methods, including heat inactivation, for example, at 121° C. for 15-20 minutes, or ultraviolet inactivation, or a combination of heat inactivation and ultraviolet inactivation.

[0012] In one embodiment, suspensions of different microorganisms are inactivated separately; or

[0013] Mix suspensions of different microorganisms and inactivate them together; or

[0014] Suspensions of different microorganisms are mixed with vaccine dressings / adjuvants and inactivated together.

[0015] Optionally, before inactivation, the concentration of the suspension of various microorganisms is 10-100 billion cells / ml (10 10-11 cells / mL), preferably 3-5 billion cells / mL (3×10 10 cells / mL to 5×10 10 pcs / mL). That is:

[0016] The concentration of the suspension of Bordetella pertussis is 1-10 billion cells / ml (10 10-11 cells / mL), preferably 3-5 billion cells / mL (3×10 10 cells / mL to 5×10 10 / mL);

[0017] The concentration of the typhoid suspension is 10-100 billion cells / ml (10 10-11 cells / mL), preferably 3-5 billion cells / mL (3×10 10 cells / mL to 5×10 10 / mL);

[0018] The concentration of paratyphoid A suspension is 10-100 billion cells / ml (10 10-11 cells / mL), preferably 3-5 billion cells / mL (3×10 10 cells / mL to 5×10 10 / mL);

[0019] The concentration of the suspension of Salmonella paratyphi B is 10-100 billion cells / ml (10 10-11 cells / mL), preferably 3-5 billion cells / mL (3×10 10 cells / mL to 5×10 10 / mL);

[0020] The concentration of Staphylococcus aureus suspension is 10-100 billion cells / ml (10 10-11 cells / mL), preferably 3-5 billion cells / mL (3×10 10 cells / mL to 5×10 10 / mL);

[0021] The concentration of Listeria suspension is 1-10 billion cells / ml (10 10-11 cells / mL), preferably 3-5 billion cells / mL (3×10 10 cells / mL to 5×10 10 / mL);

[0022] The concentration of Escherichia coli suspension is 1-10 billion cells / ml (10 10-11 cells / mL), preferably 3-5 billion cells / mL (3×10 10 cells / mL to 5×10 10 / mL);

[0023] The concentration of the Proteus suspension is 10-100 billion cells / ml (10 10-11 cells / mL), preferably 3-5 billion cells / mL (3×10 10 cells / mL to 5×10 10 / mL);

[0024] The concentration of the lactic acid bacteria suspension is 10-100 billion cells / ml (10 10-11 cells / mL), preferably 3-5 billion cells / mL (3×10 10 cells / mL to 5×10 10 / mL);

[0025] The concentration of the suspension of Bifidobacterium longum is 1-10 billion cells / ml (10 10-11 cells / mL), preferably 3-5 billion cells / mL (3×10 10 cells / mL to 5×10 10 / mL);

[0026] The concentration of the suspension of DPT is 1-10 billion cells / ml (10 10-11 cells / mL), preferably 3-5 billion cells / mL (3×10 10 cells / mL to 5×10 10 / mL);

[0027] The concentration of the suspension of Clostridium acetobutylicum is 1-10 billion cells / ml (10 10-11 cells / mL), preferably 3-5 billion cells / mL (3×10 10 cells / mL to 5×10 10 / mL);

[0028] The concentration of the Salmonella typhimurium suspension is 1-10 billion cells / ml (10 10-11 cells / mL), preferably 3-5 billion cells / mL (3×10 10 cells / mL to 5×10 10 / mL);

[0029] The concentration of Streptococcus pyogenes suspension is 1-10 billion cells / ml (10 10-11cells / mL), preferably 3-5 billion cells / mL (3×10 10 cells / mL to 5×10 10 cells / mL).

[0030] Preferably, the dosage form of the anti-tumor composite vaccine is an injection selected from subcutaneous injection and intramuscular injection. Subcutaneous injection and intramuscular injection (IM injection) are preferred because they are simple to use, quick to operate and easy to handle.

[0031] Furthermore, the above-mentioned anti-tumor composite vaccine is a pharmaceutical composition, which includes, in addition to the microbial inactivation preparation as a pharmaceutical active ingredient (vaccine active ingredient), a pharmaceutically acceptable injectable vaccine dressing / adjuvant, and the dressing / adjuvant includes but is not limited to the following ingredients: polyinosinic acid (polyI:C), dextran, lecithin, injection oil, vitamin A, aluminum stearate, CMC-Na (sodium carboxymethylcellulose, pharmaceutical grade, viscosity 800-1200), injectable fat emulsion, Span-20, etc.

[0032] In a preferred embodiment, the anti-tumor composite vaccine is selected from the following formula:

[0033] Formulation 1: 8-15 v / v% (preferably 10-12 v / v%) of an inactivated preparation of Bordetella pertussis, 5-15 v / v% (preferably 8-10 v / v%) of an inactivated preparation of Salmonella typhi, 5-15 v / v% (preferably 8-10 v / v%) of an inactivated preparation of Staphylococcus aureus, 0.05-0.5 wt% (preferably 0.1-0.2 wt%) of polyinosinic acid (polyI:C), 3-10 wt% (preferably 5-8 wt%) of dextran, and the remainder of normal saline to make up 100% by weight;

[0034] Formulation 2: 6-15 v / v% (preferably 8-10 v / v%) of an inactivated preparation of Bordetella pertussis, 8-15 v / v% (preferably 10-12 v / v%) of an inactivated preparation of Salmonella typhi, 2-8 v / v% (preferably 3-5 v / v%) of an inactivated preparation of Salmonella paratyphi A, 2-8 v / v% (preferably 3-5 v / v%) of an inactivated preparation of Salmonella paratyphi B, 3-10 v / v% (preferably 5-8 v / v%) of an inactivated preparation of Staphylococcus aureus, 0.05-0.5 wt% (preferably 0.1-0.2 wt%) of polyinosinic acid (polyI:C), 5-15 wt% (preferably 8-10 wt%) of dextran, and the remainder of normal saline to make up 100% by weight;

[0035] Formula 3: 3-10 v / v% (preferably 5-7 v / v%) of inactivated preparation for Listeria monocytogenes, 3-10 v / v% (preferably 5-7 v / v%) of inactivated preparation for Escherichia coli, 3-10 v / v% (preferably 5-7 v / v%) of inactivated preparation for Proteus, 3-10 v / v% (preferably 5-7 v / v%) of inactivated preparation for Lactobacillus, 3-10 v / v% (preferably 5-7 v / v%) of inactivated preparation for Bifidobacterium longum, 4-10 v / v% (preferably 6-8 v / v%) of inactivated preparation for Diphtheria and Tetanus, 1-5 v / v% (preferably 2-3 v / v%) of inactivated preparation for Clostridium acetobutylicum, 1-5 v / v% (preferably 2-3 v / v%) of inactivated preparation for Salmonella typhimurium, 0.5-5 v / v% (preferably 1-2 v / v%) of inactivated preparation for Streptococcus pyogenes, and Vitamin A. 0.2-2wt%, preferably 0.5-1wt%, aluminum stearate 1-2wt%, preferably 1-2wt%, CMC-Na carboxymethyl cellulose sodium (pharmaceutical grade, viscosity 800-1200) 0.5-5wt%, preferably 1-2wt%, fat emulsion for injection 3-10v / v%, preferably 5-7v / v%, Span-20 0.5-5wt%, preferably 1-2wt%, and the remainder of normal saline is used to make up 100% by weight.

[0036] The percentage of microorganisms in the formulations described herein refers to the weight percentage (wt%) or volume percentage (v / v%) of the microorganism-inactivated preparation. For example, 10% Bordetella pertussis in the formulation refers to 10 wt% or 10 v / v% of the inactivated Bordetella pertussis preparation, and so on.

[0037] Since the specific gravity of physiological saline and PBS buffer for injection is basically the same as that of water, and the specific gravity of fat emulsion for injection is not much different from that of water, the volume percentage v / v% used in this article can also be replaced by weight percentage wt%.

[0038] It should be understood that the terms "comprises," "includes," or any other variations thereof as used herein are intended to cover non-exclusive inclusion, such that a process, method, material, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, material, or apparatus.

[0039] Formula 1 of the anti-tumor composite vaccine may be: approximately 10% by weight of an inactivated preparation of Bordetella pertussis, approximately 10% by weight of an inactivated preparation of Salmonella typhi, approximately 10% by weight of an inactivated preparation of Staphylococcus aureus, approximately 0.1% by weight of polyinosinic acid (polyI:C), approximately 8% by weight of dextran, and the remainder of normal saline to make up 100% by weight, referred to herein as Formula 1A; or

[0040] About 11 wt % of an inactivated preparation of Bordetella pertussis, about 9 wt % of an inactivated preparation of Salmonella typhi, about 9 wt % of an inactivated preparation of Staphylococcus aureus, about 0.2 wt % of polyinosinic acid (polyI:C), about 6 wt % of dextran, and the remainder of normal saline to make up 100 wt %, referred to herein as Formulation 1-B; or

[0041] The inactivated preparation of Bordetella pertussis is approximately 12 v / v%, the inactivated preparation of Salmonella typhi is approximately 8 v / v%, the inactivated preparation of Staphylococcus aureus is approximately 8 v / v%, polyinosinic acid (polyI:C) is approximately 0.1 wt%, dextran is approximately 7 wt%, and the remainder of normal saline is used to make up 100% by weight, referred to herein as Formulation 1-C.

[0042] Formula 2 of the anti-tumor composite vaccine may include: approximately 10% by weight of an inactivated preparation of Bordetella pertussis, approximately 10% by weight of an inactivated preparation of Salmonella typhi, approximately 3% by weight of an inactivated preparation of Salmonella paratyphi A, approximately 5% by weight of an inactivated preparation of Salmonella paratyphi B, approximately 8% by weight of an inactivated preparation of Staphylococcus aureus, approximately 0.1% by weight of polyinosinic acid (polyI:C), approximately 8% by weight of dextran, and the remainder of normal saline to make up 100% by weight, referred to herein as Formula 2A; or

[0043] An inactivated preparation of Bordetella pertussis, approximately 8% by weight, an inactivated preparation of Salmonella typhi, approximately 12% by weight, an inactivated preparation of Salmonella paratyphi A, approximately 5% by weight, an inactivated preparation of Salmonella paratyphi B, approximately 5% by weight, an inactivated preparation of Staphylococcus aureus, approximately 0.2% by weight of polyinosinic acid (polyI:C), approximately 10% by weight of dextran, and the remainder of normal saline to make up 100% by weight, referred to herein as Formulation 2B; or

[0044] An inactivated preparation of Bordetella pertussis, approximately 9 v / v%, an inactivated preparation of Salmonella typhi, approximately 11 v / v%, an inactivated preparation of Salmonella paratyphi A, approximately 4 v / v%, an inactivated preparation of Salmonella paratyphi B, approximately 7 v / v%, an inactivated preparation of Staphylococcus aureus, approximately 0.1 wt % of polyinosinic acid (polyI:C), approximately 9 wt % of dextran, and the remainder of normal saline to make up 100% by weight, referred to herein as Formulation II C; or

[0045] The inactivated preparation of Bordetella pertussis is approximately 10 v / v%, the inactivated preparation of Salmonella typhi is approximately 11 v / v%, the inactivated preparation of Salmonella paratyphi A is approximately 5 v / v%, the inactivated preparation of Salmonella paratyphi B is approximately 3 v / v%, the inactivated preparation of Staphylococcus aureus is approximately 6 v / v%, polyinosinic acid (polyI:C) is approximately 0.1 wt%, dextran is approximately 10 wt%, and the remainder of normal saline is used to make up 100% by weight, which is referred to herein as Formulation 2D.

[0046] Preferably, Formulation 1 and Formulation 2 are in the form of subcutaneous injection or intramuscular injection (IM).

[0047] Surprisingly, despite containing only three bacterial vaccines (B. pertussis, Salmonella typhi, and Staphylococcus aureus) and two excipients (polyinosinic-polycytidylic acid and β-glucan), Formula 1 demonstrates broad-spectrum anti-tumor activity. Compared to existing multi-component vaccines, this reduction in raw material usage facilitates standardized production management and quality control, and is economically viable.

[0048] Formula III of the anti-tumor composite vaccine may include: approximately 7% by weight of an inactivated preparation of Listeria monocytogenes, approximately 5% by weight of an inactivated preparation of Escherichia coli, approximately 7% by weight of an inactivated preparation of Proteus, approximately 5% by weight of an inactivated preparation of Lactobacillus, approximately 5% by weight of an inactivated preparation of Bifidobacterium longum, approximately 8% by weight of an inactivated preparation of Diphtheria and Tetanus, approximately 2.5% by weight of an inactivated preparation of Clostridium acetobutylicum, approximately 2% by weight of an inactivated preparation of Salmonella typhimurium, approximately 1% by weight of an inactivated preparation of Streptococcus pyogenes, approximately 0.5% by weight of Vitamin A, approximately 1% by weight of aluminum stearate, approximately 1% by weight of CMC-Na (sodium carboxymethylcellulose, pharmaceutical grade, viscosity 800-1200), approximately 5% by weight of injectable fat emulsion, approximately 1% by weight of Span-20, and the remainder of normal saline to make up 100% by weight, referred to herein as Formula III A; or

[0049] An inactivated preparation of Listeria monocytogenes is about 5 v / v%, an inactivated preparation of Escherichia coli is about 7 v / v%, an inactivated preparation of Proteus is about 5 v / v%, an inactivated preparation of lactic acid bacteria is about 7 v / v%, an inactivated preparation of Bifidobacterium longum is about 7 v / v%, an inactivated preparation of diphtheria and pertussis is about 6 v / v%, an inactivated preparation of Clostridium acetobutylicum is about 3 v / v%, an inactivated preparation of Salmonella typhimurium is about 3 v / v%, an inactivated preparation of Streptococcus pyogenes is about 2 v / v%, about 0.8 wt % of vitamin A, about 1.7 wt % of aluminum stearate, about 2 wt % of CMC-Na (sodium carboxymethyl cellulose, pharmaceutical grade, viscosity 800-1200), about 7 v / v of fat emulsion for injection, about 2 wt % of Span-20, and the remainder of normal saline is used to make up 100% by weight, referred to herein as Formulation III B; or

[0050] The inactivated preparation of Listeria monocytogenes is approximately 6 v / v%, the inactivated preparation of Escherichia coli is approximately 6 v / v%, the inactivated preparation of Proteus is approximately 6 v / v%, the inactivated preparation of lactic acid bacteria is approximately 6 v / v%, the inactivated preparation of Bifidobacterium longum is approximately 6 v / v%, the inactivated preparation of diphtheria and pertussis is approximately 7 v / v%, the inactivated preparation of Clostridium acetobutylicum is approximately 2 v / v%, the inactivated preparation of Salmonella typhimurium is approximately 3 v / v%, the inactivated preparation of Streptococcus pyogenes is approximately 2 v / v%, vitamin A is approximately 0.9 wt%, aluminum stearate is approximately 1.5 wt%, CMC-Na (sodium carboxymethyl cellulose, pharmaceutical grade, viscosity 800-1200) is approximately 1.5 wt%, fat emulsion for injection is approximately 6 v / v%, Span-20 is approximately 1.5 wt%, and the remainder of normal saline is used to make up 100% by weight, which is referred to as Formulation III C herein.

[0051] Formulation three can be a subcutaneous injection dosage form or an intramuscular injection (IM) dosage form.

[0052] In order to avoid possible infections such as sepsis and other diseases, the anti-tumor composite vaccine of the present invention, including formula one, formula two and formula three, when in the form of an injection, no longer adopts the intravenous injection form reported in the prior art such as patent documents CN101569746A, CN101628114B, CN106667907A and CN112618581A, but instead adopts a safe subcutaneous injection form or intramuscular injection form to prevent any medical accidents.

[0053] It should be understood that when expressing numerical characteristics in this article, the terms "about", "approximately" or "around" mean that the number represented may have an error range or floating range of ±10%, ±9%, ±8%, ±6% or ±5%.

[0054] In an optional embodiment, the above-mentioned anti-tumor composite vaccine can be prepared into a lyophilized agent to facilitate storage and transportation, and can be dissolved in sterile double-distilled water or physiological saline to a specified volume or concentration when used.

[0055] In one embodiment, the tumor described in the above-mentioned anti-tumor composite vaccine is one or a combination of two or more selected from the following cancers / tumors: malignant epithelial tumors, lymphomas, blastomas, sarcomas, leukemias, basal cell carcinomas, bile duct cancer; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); liver cancer; hepatoma; intraepithelial neoplasia; kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma); lymphoma, including Hodgkin lymphoma and non-Hodgkin lymphoma Lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urological cancer; vulvar cancer; and other carcinomas and sarcomas; and B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-dividing cell NHL; bulky EHL (bulky disease NHL); mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs syndrome.

[0056] In an exemplary embodiment, the cancer / tumor is one or a combination of two or more of the following: liver cancer, sarcoma, bladder cancer, prostate cancer, colon cancer, rectal cancer, ovarian cancer, kidney cancer, breast cancer, glioblastoma, melanoma, malignant melanoma or lung cancer.

[0057] The newly formulated compound vaccine developed by the present invention is highly safe. Acute toxicity experiments conducted on Wistar rats, SD mice, and Kunming mice showed that after subcutaneous injection of a compound vaccine equivalent to 900-15,000 times the adult dose, all animals survived, their renal function was unaffected, and no toxic side effects were observed. Moreover, the newly formulated compound vaccine of the present invention can effectively inhibit the growth and metastasis of tumors such as liver cancer, sarcoma, bladder cancer, melanoma, etc., and significantly reduce the weight and volume of solid tumors, demonstrating its broad-spectrum anti-tumor effect and showing good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 shows photos of the study on the tumor inhibition effect of composite vaccine formula 3A on Lewis lung cancer in C57 mice.

[0059] FIG2 shows photos of the study on the tumor inhibition effect of composite vaccine formula 1A on Lewis lung cancer in C57 mice.

[0060] FIG3 shows photos of the study on the tumor inhibition effect of composite vaccine formula II A on Lewis lung cancer in C57 mice.

[0061] Figure 4 shows photos of the study on the tumor inhibition effect of seven composite vaccine formulations on B16 melanoma in BL / 6 mice. DETAILED DESCRIPTION

[0062] The new formula compound vaccine developed by the present invention has a broad-spectrum anti-tumor activity. Multiple mouse tumor models have confirmed that the anti-tumor compound vaccine (including formula one and formula two) containing Bordetella pertussis, Staphylococcus aureus, Salmonella typhi and / or Salmonella paratyphi A and / or Salmonella paratyphi B can be used to treat solid tumors such as liver cancer, sarcoma, bladder cancer and / or melanoma.

[0063] As used herein, the term "or" sometimes means "and / or," and the term "either" sometimes means "and / or." The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone).

[0064] As used herein, the terms "anti-tumor combination vaccine," "anti-tumor vaccine," "tumor vaccine," "combined bacterial vaccine," "combined microbial," "combined bacterial vaccine," and "combined vaccine" have the same meaning and may be used interchangeably, particularly to refer to Formulations 1, 2, and 3. For ease of description, the term "vaccine" may be referred to herein simply as "vaccine."

[0065] The composite vaccine of the present invention is preferably administered to a subject by injection, including but not limited to intravenous (IV), intramuscular (IM), subcutaneous (IH / SC), intradermal (IC), intraperitoneal (IP), intravenous drip, or intratissue injection. Subcutaneous (IH / SC) and intramuscular (IM) injections are preferred because they are simple to use, quick to administer, and easy to handle.

[0066] The term "subject" refers to a human or animal. Typically, the animal is a vertebrate, such as a primate, rodent, livestock, or game animal. Primates include chimpanzees, crab-eating macaques, spider monkeys, and macaques, such as rhesus macaques. Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species (e.g., house cats), and canine species (e.g., dogs, foxes, wolves). In some embodiments, the subject is a mammal, such as a primate such as a human. The terms "individual," "patient," and "subject" are used interchangeably herein. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cattle, but is not limited to these examples. The subject can be male or female.

[0067] Those skilled in the art will readily appreciate that the aforementioned composite vaccines are generally pharmaceutical compositions that, in addition to the primary ingredients of various inactivated bacteria, may also include a pharmaceutically acceptable carrier. Preferably, the carrier imparts functions such as providing a suitable dosage of the active ingredient for administration to a subject, and being suitable for pharmaceutical storage and transportation, while maintaining freshness, i.e., preserving biological activity.

[0068] For example, when the composite vaccine is an injectable pharmaceutical composition, pharmaceutically acceptable injectable vaccine dressings / adjuvants include but are not limited to the following ingredients: polyinosinic-acid (polyI:C), dextran, lecithin, oil for injection, vitamin A, aluminum stearate, CMC-Na (sodium carboxymethylcellulose, pharmaceutical grade, viscosity 800-1200), injectable fat emulsion, Span-20, etc.

[0069] More preferably, the pharmaceutically acceptable carrier can produce a synergistic effect with various inactivated bacteria as the main active ingredients of the drug, thereby enhancing the anti-tumor effect. For example, for combination vaccine formulations 1 and 2, comparative experiments have shown that the combination of polyinosinic-acid (polyI:C) + glucan dressing with Bordetella pertussis, Staphylococcus aureus, Salmonella typhi and / or Salmonella paratyphi A and / or Salmonella paratyphi B can produce a synergistic effect.

[0070] Polyinosinic-polycytidylic acid (PICP), also known as poly (I)-polycytidylic acid (PC), is a synthetic double-stranded RNA (dsRNA) consisting of poly(I) and poly(C). PIP is an interferon inducer, inducing the production of interferon in cells within the body. It exhibits similar antiviral and immunomodulatory properties to interferon. It is used to treat chronic hepatitis B, hemorrhagic fever, Japanese encephalitis, viral keratitis, herpes zoster, various warts, and respiratory infections. It possesses broad-spectrum antiviral and immunomodulatory properties and can be used as an adjunctive therapy for viral infections and tumors.

[0071] β-glucan, also known as glucan, is a natural polysaccharide found in a variety of plants and fungi, such as mushrooms, yeast, and algae. β-glucan possesses numerous important biological and pharmacological activities, earning it the nickname "immune gold," playing a crucial role in the immune system. It can enhance the activity of immune cells, promote their proliferation and differentiation, and improve their ability to recognize and eliminate pathogens. β-glucan also exhibits anti-inflammatory, anti-tumor, and antioxidant properties, modulating immune system function and maintaining immune balance. It is widely used in medicine, health supplements, and food additives.

[0072] In an optional embodiment, the pharmaceutical composition may also include another type of anti-tumor drug such as certain nucleic acid molecules (RNA or DNA) drugs or polypeptide drugs without side effects, provided that the biological activity of the bacterial vaccine is not compromised. As used herein, the term "nucleic acid" or "nucleic acid molecule" refers to any molecule, preferably a polymeric molecule, comprising units of ribonucleic acid, deoxyribonucleic acid, or their analogs. Nucleic acids may be single-stranded or double-stranded. A single-stranded nucleic acid may be a nucleic acid chain of a denatured double-stranded DNA. Alternatively, it may be a single-stranded nucleic acid that is not derived from any double-stranded DNA. On the one hand, the nucleic acid may be DNA. On the other hand, the nucleic acid may be RNA.

[0073] The phrase "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. Pharmaceutically acceptable carriers are well known in the art and include liquid or solid fillers, diluents, excipients, solvents or encapsulating materials. Each carrier must be "acceptable" in the sense that it is compatible with the other ingredients of the formulation and harmless to the patient, including, for example, aqueous solutions (such as water or physiologically buffered saline) or other solvents or vehicles (such as glycols, glycerol, oils (such as olive oil) or injectable organic esters). Excipients can be selected, for example, to achieve delayed release of the agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form, such as tablets, capsules (including dispersible capsules and gelatin capsules), granules, powders, solutions, syrups, suppositories, injections, etc. The composition can also be present in a transdermal delivery system (e.g., a skin patch). The composition may also be presented in a solution suitable for topical administration (eg, a lotion, cream, or ointment).

[0074] For example, the pharmaceutically acceptable carrier includes a protein stabilizer, which can be selected from stabilizers commonly used in protein drugs and / or living cell drugs to maintain protein and / or cell activity, for example, including but not limited to at least one of the following groups, or a combination of two or more: ① buffer: such as sodium citrate-citric acid buffer; ② surfactant: such as non-ionic surfactant polysorbates; ③ sugars and polyols: such as sucrose, glucose, trehalose, maltose, glycerol, mannitol, sorbitol, PEG and inositol; ④ salts: such as sodium chloride; ⑤ polyethylene glycols; ⑥ macromolecular compounds: such as 2-hydroxypropyl-β-cyclodextrin, albumin, serum protein (HAS), etc.; ⑦ hydrochlorides of histidine, glycine, glutamic acid and lysine, etc.

[0075] As used herein, the term "effective amount" refers to the therapeutic amount required to alleviate the symptoms of cancer or tumors, and relates to a sufficient amount of a pharmaceutical composition to provide the desired effect. Therefore, the term "therapeutically effective amount" refers to a therapeutic amount sufficient to cause a specific effect when administered to a typical subject. In various contexts, an effective amount as used herein also includes an amount sufficient to delay the development of cancer, change the course of a tumor condition (for example, but not limited to, slowing the progression of a cancer condition), or reverse a tumor condition. It should be understood that there are many ways known in the art to determine the effective amount for a given application. For example, pharmacological methods for dosage determination can be used in the treatment context. In the context of therapeutic or preventive applications, the amount of the composition administered to the subject will depend on the type and severity of the disease and the characteristics of the individual, such as overall health, age, sex, weight, and tolerance to the drug. It also depends on the extent, severity, and type of the disease. Those skilled in the art will be able to determine the appropriate dosage based on these and other factors.

[0076] A physician or veterinarian with ordinary skills in the art can easily determine and prescribe the effective amount of the treatment of the required composite vaccine. For example, a physician or veterinarian can start with a dosage of the composite vaccine that is lower than the level required for achieving the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved." Therapeutically effective amount" represents the concentration of the compound that is sufficient to cause the desired therapeutic effect. It is generally understood that the effective amount of the composite vaccine will vary according to the weight, sex, age and medical history of the subject. Other factors affecting the effective amount may include, but are not limited to, the severity of the patient's cancer condition, the stability of the tumor volume and (if necessary) another type of therapeutic agent administered together with the composite vaccine of the present invention. A larger total dose can be delivered by multiple administrations of the medicament. The method for determining efficacy and dosage is well known to those skilled in the art.

[0077] Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intrahepatic, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. Administration can be systemic or local.

[0078] Typically, the suitable daily dose of the microbial vaccine used in the composite vaccine composition and method of the present invention will be the amount of the composite vaccine at the lowest dose effective for producing a therapeutic effect on a specific tumor. Such an effective dose will generally depend on the factors as described above.

[0079] If desired, the effective daily / weekly / monthly dose of the combination vaccine may be administered as one, two, three, four, five, six or more sub-doses administered separately, optionally in unit dosage form, at appropriate intervals throughout the day. In certain embodiments of the invention, the combination vaccine may be administered twice or three times daily / weekly / monthly. In other embodiments, the combination vaccine will be administered once weekly / monthly.

[0080] Patients receiving such treatment are any animal in need thereof, including primates (particularly humans); and other mammals (such as horses, cattle, pigs, sheep, cats, and dogs); poultry; and pets in general.

[0081] When the composite vaccine of the present invention is administered to a subject, it will generate a strong immune response in the body, including a humoral immune response to produce sufficient neutralizing antibodies against multiple types of viruses / bacteria, and it is not excluded that it also includes inducing a cellular immune response to eliminate tumor cells.

[0082] The composite vaccine of the present invention is not targeted at specific cancers such as malignant tumors, but is non-specific and broad-spectrum. It can be applied to subjects suffering from two or more cancers / tumors at the same time to reduce or even eliminate cancer cells, or inhibit the progression and metastasis of cancer, reduce tumor volume, or at least alleviate cancer symptoms, allowing cancer patients to survive with tumors.

[0083] As used herein, the term "cancer" generally refers to a class of diseases or conditions in which abnormal cells divide uncontrollably and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymphatic systems. There are several main types of cancer. Carcinomas are cancers that originate in the skin or tissues that line or cover internal organs. Sarcomas are cancers that originate in bone, cartilage, fat, muscle, blood vessels, or other connective or supporting tissues. Leukemias are cancers that begin in blood-forming tissues (such as the bone marrow) and cause large numbers of abnormal blood cells to be produced and enter the blood. Lymphomas and multiple myeloma originate in cells of the immune system. Central nervous system cancers are cancers that originate in the tissues of the brain and spinal cord.

[0084] In some embodiments of any aspect, the cancer is a primary cancer. In some embodiments of any aspect, the cancer is a malignant cancer. As used herein, the term "malignant" refers to a cancer in which a group of tumor cells exhibit one or more uncontrolled growth (i.e., division beyond the normal range), invasion (i.e., invasion and destruction of adjacent tissues), and metastasis (i.e., spread to other locations of the body through lymph or blood). As used herein, the term "metastasis" refers to the spread of cancer from one part of the body to another. Tumors formed by cells that have spread are called "metastatic tumors" or "metastasis." Metastatic tumors contain cells similar to those in the original (primary) tumor.

[0085] As used herein, the term "benign" or "non-malignant" refers to a tumor that may grow larger but does not spread to other parts of the body. Benign tumors are self-limited and usually do not invade or metastasize.

[0086] "Cancer cell" or "tumor cell" refers to a single cell of a cancerous growth or tissue. A tumor generally refers to a mass or lesion formed by an abnormal growth of cells, which can be benign, precancerous, or malignant. Most cancer cells form tumors, but some (such as leukemias) do not necessarily form tumors. For those cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.

[0087] A subject having a cancer or tumor is one in which there are objectively measurable cancer cells present in the subject's body. This definition includes malignant, actively proliferating cancers, as well as potentially dormant tumors or micrometastases. Cancers that migrate from their original location and implant in other vital organs will eventually lead to the death of the subject through functional deterioration of the affected organs. Hematopoietic cancers (e.g., leukemias) can outcompete the subject's normal hematopoietic compartment, leading to hematopoietic failure (in the form of anemia, thrombocytopenia, and neutropenia), ultimately leading to death.

[0088] Examples of cancer include, but are not limited to, malignant epithelial tumors, lymphomas, blastomas, sarcomas, leukemias, basal cell carcinomas, bile duct cancer; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancers; digestive system cancers; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer (including gastrointestinal cancer); glioblastoma (GBM); liver cancer; hepatoma; intraepithelial neoplasia; kidney cancer; laryngeal cancer; leukemias; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma cancers of the ovary; oral cavity (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urological cancer; vulvar cancer; and other carcinomas and sarcomas; and B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-dividing cell NHL; bulky EHL (bulky disease NHL); mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs syndrome.

[0089] "Cancer cells" are cancer cells, precancerous cells, or transformed cells in vivo, in vitro, or in tissue culture that have spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can be caused by infection with a transforming virus and the incorporation of new genomic nucleic acids, or the uptake of exogenous nucleic acids, it can also occur spontaneously or after exposure to a carcinogen, thereby mutating endogenous genes. Transformation / cancer involves, for example, morphological changes, cell immortalization, abnormal growth control, focus formation, anchorage independence, malignancy, loss of contact inhibition and growth density restriction, growth factor or serum independence, tumor-specific markers, invasiveness or metastasis, and tumor growth in a suitable animal host (e.g., nude mice).

[0090] The object can be a subject that has previously been diagnosed with or is identified as having a disease (for example, cancer) that needs treatment or one or more complications related to such disease, and (alternatively but not necessarily) has experienced the treatment of a certain disease or one or more complications related to the disease. Alternatively, the object can also be a subject that has not previously been diagnosed as having a disease that needs treatment or one or more complications related to this disease. For example, the object can be a subject that shows one or more disease risk factors or one or more complications related to the disease, or does not show a risk factor. A "subject in need of treatment" of a particular disease can be a subject that suffers from the disease, is diagnosed as suffering from the disease, or is at risk of developing the disease.

[0091] The terms "reduce", "reduce", "lower" or "inhibit" are all used herein to indicate a statistically significant reduction. In some embodiments, "reduce", "reduce" or "reduce" or "inhibit" generally refers to a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent), and can include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more. As used herein, "reduce" or "inhibit" does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. For individuals without a given disease, reduction can preferably drop to an acceptable level within the normal range.

[0092] We used the new formula composite vaccine of the present invention to conduct more than 60 groups of tumor inhibition experiments on mouse models including liver cancer, sarcoma, bladder cancer, melanoma, etc. The tumor inhibition rate was 25.9%-73.7%, and the tumor inhibition rate in 60% of the experiments exceeded 50%, showing a broad spectrum of anti-tumor effects.

[0093] As a typical example, one of the inventors was diagnosed with ductal cell carcinoma (stage II) in 2006 and underwent liver lobectomy. This type of liver tumor is insensitive to radiotherapy and chemotherapy, and the median survival of patients does not exceed 2 years. The inventor did not receive radiotherapy and chemotherapy. Based on the concept of composite vaccine treatment of tumors constructed by the research group, he only received composite vaccine injection treatment as a test subject for 2 years. The composite vaccine used is similar to or identical to the formula in the present invention. The inventor has lived healthily for 18 years with "no recurrence". Although he is old, his various physiological indicators are normal. In 2011, the inventor's sister suffered from the same ductal cell carcinoma, but it was multiple and accompanied by portal lymph node metastasis, making surgical resection impossible. The patient refused radiotherapy and chemotherapy and only received composite vaccine treatment. Within the next 5 years, the intrahepatic tumor "disappeared" in imaging, and the patient had "progression-free survival" for 4 years. Moreover, the safety of the injected composite vaccine has also been confirmed. The inventor received injections of the composite vaccine more than 1,000 times and did not experience any toxic reactions except for local redness and swelling of the skin at the injection site and a short-term mild fever (which is necessary for the production of immune efficacy).

[0094] Regarding the effectiveness and safety of the composite vaccine of the present invention, the inventors disclosed in the document A New Tumor Therapeutic Vaccine: A Real-World Survey on Treatment of 68 Patients with Advanced Cancer. Kecheng Xu, et al., Clinics in Surgery. March 3, 2022, Volume 7, Article 3432, the following facts regarding the composite vaccine (including Formulas 1, 2, and 3 of the present invention) tested on volunteers with tumors:

[0095] Tumor therapeutic vaccine (TTV) is a complex composed of multiple bacterial or toxin vaccines plus adjuvants. One of the authors of this article is a "volunteer" who received this complex vaccine TTV. In 2008, the authors of this article followed up patients with advanced cancer who had used the vaccine 10 years ago. These patients were all patients with advanced tumors that were proven to be incurable. Through home visits and telephone inquiries, a total of 38 cases were followed up, 28 of whom were still alive at the time of follow-up; the survival period was 2-16 years, and 23 survived for more than 10 years; 10 patients died, and only 2 died of cancer recurrence. After 2010, the inventors prospectively observed the efficacy of the complex vaccine on volunteers. 68 patients with advanced cancer met the following conditions: (1) those who lost the opportunity for surgical treatment and / or failed chemoradiotherapy; (2) those with pathological diagnosis; (3) those who were expected to survive no more than 1 year. Results: The survival period ranged from 8 to 204 months, with a median of 48 months, and 33 patients were still alive at the time of follow-up.

[0096] A "real-world" study enrolled 68 patients with progressive solid cancers who underwent conventional treatment and were treated with TTV injections alone. Results: The duration of TTV treatment ranged from 3 to 96 months, with a median of 24 months. Complete tumor response (CR) was achieved in 44.1% of patients, partial response (PR) in 36.7%, and stable disease (SD) in 19.1%. Overall survival (OS) from the start of TTV treatment ranged from 8 to 204 months, with a median of 48 months. At final follow-up, 48.5% of patients were alive. This study demonstrates that this simple and safe treatment effectively promotes tumor regression or stabilization, prolonging patient survival in some cases. TTV represents a promising alternative treatment option for progressive cancers.

[0097] To make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings. Those skilled in the art should understand that the following embodiments are only used to illustrate the present invention and are not intended to limit the present invention.

[0098] Example

[0099] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned therein, unless otherwise specified, refer to the percentage by mass.

[0100] In the examples herein, if no specific description is given for the operating temperature, the temperature generally refers to room temperature (15-35° C.).

[0101] The various composite vaccine preparations used in the examples were prepared by Shaoxing Yueran Biopharmaceutical Technology Co., Ltd. Any organization or individual may obtain these preparations for verification of the present invention. However, they may not be used for other purposes, including development and utilization, scientific research and teaching, without the permission of Shaoxing Yueran Biopharmaceutical Technology Co., Ltd., and may not be used for the treatment of cancer patients or cancer-affected animals.

[0102] The animal model construction and drug administration studies used in the examples were commissioned to Shanghai Institute of Pharmaceutical Industry Co., Ltd.

[0103] Statistical Analysis: All numerical variables in this study are expressed as mean ± standard error. Two-group comparisons were performed with the two-tailed Student's t-test, and three-group comparisons were performed with ANOVA. Statistical differences were considered when P < 0.05.

[0104] Example 1: Preparation of composite vaccine

[0105] 1. Preparation of microbial inactivation preparations

[0106] Various inactivated suspension preparations of microorganisms are prepared using techniques well known to those skilled in the art. The microorganisms include Bordetella pertussis, Salmonella typhi, Salmonella paratyphi A, Salmonella paratyphi B, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Proteus, lactic acid bacteria, Bifidobacterium longum, Diphtheria and Tetanus, Clostridium acetobutylicum, Salmonella typhimurium, and Streptococcus pyogenes.

[0107] Taking the well-known Escherichia coli, i.e., Escherichia coli, as an example, the preparation method of the inactivated bacterial suspension preparation thereof may include the following steps:

[0108] A single colony was selected from an LB plate of Escherichia coli (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2. LB solid medium was supplemented with 15 g / L agar powder) and inoculated into 5 ml of LB medium. The culture was then inoculated into a 1000 ml shake flask containing 100 ml of TB medium (24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K2HPO4.3H2O, 2.31 g / L KH2PO4, 5 g / L glycerol, pH 7.0-7.5) at a ratio of 1% v / v. The culture was carried out at 37°C and 220 rpm for 20-24 h. The cells were harvested by centrifugation, washed three times with an equal volume of physiological saline, harvested by centrifugation, suspended with physiological saline, and the bacterial concentration was adjusted to obtain a concentration of 1 billion to 10 billion cells / ml (10 10-11 / mL) of bacterial suspension.

[0109] Place the suspension in a sterilizer and heat at 121°C for about 15 minutes to obtain an inactivated preparation of Escherichia coli.

[0110] Inactivated preparations of all microorganisms were prepared in a similar manner.

[0111] 2. The solution of the mixture of various excipients is subjected to heat and pressure sterilization or filtration treatment to obtain a sterile excipient mixture solution.

[0112] According to the proportion of each component in the formula, the predetermined proportion of the microbial inactivation preparation and the sterile excipient mixture solution are mixed in a sterile environment such as a clean workbench to obtain a sterile compound vaccine.

[0113] The aforementioned Formula 1A, Formula 1B, Formula 1C, Formula 2A, Formula 2B, Formula 2C, Formula 2D, Formula 3A, Formula 3B, and Formula 3C have been prepared by Shaoxing Yueran Biopharmaceutical Technology Co., Ltd. and placed in a 4°C refrigerator for later use.

[0114] Example 2: Safety assessment of various composite vaccines

[0115] Shanghai Institute of Pharmaceutical Industry Co., Ltd. was commissioned to conduct toxicity studies on all compound vaccines, demonstrating that all compound vaccines of the present invention, including the formulation prepared in Example 1, are safe and non-toxic. A brief description of Formulation 3A is provided below.

[0116] Maximum dosage experiment of Yueran tumor vaccine sample on ICR mice (toxicity test)

[0117] 1. Purpose of the study

[0118] To observe the toxic reactions and mortality caused by a single subcutaneous administration of Shaoxing Yueran tumor vaccine sample to ICR mice.

[0119] 2. Experimental Drugs

[0120] Sample name: Shaoxing Yueran tumor vaccine sample.

[0121] 3. Experimental Animals

[0122] Twenty ICR mice, half male and half female, weighing 20 ± 2 g, were provided by Shanghai Slake Laboratory Animal Co., Ltd., production license number: SCXK (Shanghai) 2017-0005; use license number: SYXK (Shanghai) 2019-0027.

[0123] 4. Dosage Method

[0124] 4.1 Dosage: 1 ml / mouse.

[0125] 4.2 Acceptable volume: 1ml.

[0126] 4.3 Number of doses: 1 time.

[0127] 5. Route of administration

[0128] Subcutaneous administration

[0129] 6. Preparation of drug solution: sample stock solution

[0130] 7. Test methods

[0131] Ten female ICR mice were divided into a normal group (n=5) and a drug-treated group (n=5). A single subcutaneous dose of 1 ml / mouse (maximum volume, maximum concentration) was administered. Immediately after administration, the animals were observed for various symptoms of poisoning, and deaths were recorded. Observation was continued for 14 days.

[0132] Ten male ICR mice were divided into a normal group (n=5) and a non-sterile group (n=5). A single subcutaneous dose of 1 ml / mouse (maximum volume, maximum concentration) was administered. Immediately after administration, the animals were observed for various symptoms of poisoning, and deaths were recorded. Observation was continued for 14 days.

[0133] 8. Observation indicators

[0134] After the animals were administered the drugs, their various manifestations and poisoning symptoms were observed immediately. During the experiment, the animals were observed twice a day (morning and afternoon).

[0135] Death: The number of mice that died during the observation period was recorded. Dead animals were immediately autopsied and their major organs (heart, liver, spleen, lungs, kidneys, etc.) were visually inspected for changes. If any abnormalities were visible, pathological examination was performed.

[0136] Toxic reactions: During the observation period, the behavior, skin, respiration, defecation, urination, appetite, and abnormal secretions from the nose, eyes, and mouth of male and female mice were recorded.

[0137] 9. Observation Period

[0138] After the observation period (14 days), all surviving mice were killed, and the internal organs of the test animals were dissected and grossly examined for abnormalities.

[0139] 10. Test results

[0140] This study aimed to investigate the toxicity and mortality resulting from a single subcutaneous administration of a sample to ICR mice. Twenty SPF-grade ICR mice, half male and half female, were used in the experiment and divided into a normal group and a treatment group, with 10 mice in each group, half male and half female. The sample was administered subcutaneously once to the ICR mice at a dose of 1 ml / mouse (maximum volume and maximum concentration) and the mice were observed for 14 days. The results showed that no mice died, and no adverse reactions, such as those related to diet and activity, occurred during the entire observation period. At the end of the observation period, all mice were sacrificed, and no abnormalities in their internal organs were found during autopsy and gross examination.

[0141] Table 1. Mouse mortality rates of sterilized and non-sterilized Shaoxing Yueran tumor vaccine samples

[0142] 11. Conclusion

[0143] The Yueran tumor vaccine sample was administered subcutaneously once at a dose of 1 ml per mouse. No mouse died and no obvious toxic reaction was observed during the 14-day observation period. At the end of the experiment, the mice were killed and their major organs were dissected and no abnormalities were found. The vaccine is safe within this dosage range.

[0144] Example 3: Study on the Tumor Inhibitory Effect of Composite Vaccine on Lung Cancer

[0145] Shanghai Pharmaceutical Industry Research Institute Co., Ltd. was commissioned to conduct a study on the inhibitory effect of the composite vaccine on lung cancer. The results demonstrated that the composite vaccine formulations prepared in Example 1 all had an inhibitory effect on lung cancer. Using formulation 3A as an example, the experimental report is as follows.

[0146] Study on the anti-tumor effect of Yueran anti-tumor vaccine on Lewis lung cancer in C57 mice

[0147] 1. Purpose

[0148] Observation of the anti-tumor efficacy of Shaoxing Yueran tumor vaccine against Lewis lung cancer in mice

[0149] 2 Test drugs

[0150] 3.1 Name

[0151] Anti-tumor vaccines

[0152] 3.3 Preparation method

[0153] The tumor vaccine concentrate was injected subcutaneously at 0.1 ml / animal and 0.2 ml / animal; and injected intramuscularly at 0.1 ml / animal.

[0154] 3 Experimental Materials

[0155] 3.1 Positive Control

[0156] Cisplatin powder injection 10mg / bottle, produced by Qilu Pharmaceutical Co., Ltd., batch number 0H0484B03.

[0157] 3.2 Tumor Origin

[0158] The Lewis lung cancer model was maintained by the Pharmacology Research and Evaluation Center of Shanghai Institute of Pharmaceutical Industry.

[0159] 4. Experimental Animals

[0160] Fifty C57BL / 6 mice, 20 ± 2 g, were purchased from the Tongxiang Branch of Zhejiang Weitonglihua Laboratory Animal Technology Co., Ltd. Production license number: SCXK(Zhejiang)2020-0002. Use license number: SYXK(Shanghai)2019-0027.

[0161] 5. Dose setting

[0162] Subcutaneous injection of the original solution: 0.1ml / animal, 0.2ml / animal; intramuscular injection: 0.1ml / animal

[0163] 6. Dosage regimen

[0164] Subcutaneous injection 1, 4, 7, and 10 days after vaccination.

[0165] 7. Experimental Control

[0166] The negative control group was given the same volume of normal saline as the test group, with 10 mice in each group. The positive control group was given cisplatin DDP 6 mg / kg, once every other day for three consecutive times.

[0167] 8 Main steps of the experiment

[0168] The Lewis cells that were growing vigorously were taken and inoculated into the subcutaneous tissue of C57BL / 6 mice with 0.2 ml / mouse (about 1-2×106 ), and the drug was administered according to the experimental design the next day. The experiment was terminated on the 15th day, and the animals in each group were sacrificed. The tumors were dissected and weighed, and the tumor inhibition rate was calculated according to the following formula:

[0169] Tumor inhibition rate % = [(average tumor weight of the control group - average tumor weight of the drug group) / average tumor weight of the control group] × 100%

[0170] 9. Experimental Results

[0171] The experimental results are shown in Table 1 and Figure 1. Compared to the negative control group, the tumor inhibition rate of the cisplatin (DDP) group was 80.5%, the low-dose (0.1 ml / mouse) group was 54.9%, the high-dose (0.2 ml / mouse) group was 75.2%, and the intramuscular injection (0.1 ml / mouse) group was 77.0%. Both the low- and high-dose groups, as well as the intramuscular injection group, showed high anti-tumor activity. During the experiment, the cisplatin group and the intramuscular injection group showed weight loss after administration. The intramuscular injection group showed more significant weight loss, and the leg injection site affected normal activities. No obvious toxic reactions were observed in the high- and low-dose groups, and the animals grew normally, with no deaths.

[0172] Table 1. Antitumor efficacy experiments of antitumor vaccines

[0173] *Vs negative control, ****p<0.0001.

[0174] Example 4: Study II on the Tumor Inhibitory Effect of Composite Vaccine on Lung Cancer

[0175] Shanghai Institute of Pharmaceutical Industry Co., Ltd. was commissioned to study the lung cancer inhibition effects of the composite vaccine, Formula A, prepared in Example 1. The vaccine was compared with a live bacterial preparation that did not inactivate Bordetella pertussis, Salmonella typhi, and Staphylococcus aureus to examine the difference in vaccine potency between live and killed bacteria. The experimental report is as follows.

[0176] 1. Purpose

[0177] Observation of the anti-tumor efficacy of Yueran tumor vaccine against Lewis lung cancer in mice

[0178] 2. Test drug

[0179] 3.1 Name

[0180] Yueran Tumor Vaccine: 1. Unsterilized, batch number N20230821-00M; 2. Sterilized, batch number N20230821-12N

[0181] 3.3 Preparation method

[0182] The non-sterile and sterilized tumor vaccine concentrates of Yueran were injected at 0.1ml / animal and 0.2ml / animal.

[0183] 3. Experimental Materials

[0184] 3.1 Positive Control

[0185] Cisplatin powder injection 10mg / bottle, produced by Qilu Pharmaceutical Co., Ltd., batch number 0H0484B03.

[0186] 3.2 Tumor Origin

[0187] The Lewis lung cancer model was maintained by the Pharmacodynamics Research and Evaluation Center of Shanghai Institute of Pharmaceutical Industry.

[0188] 4. Experimental Animals

[0189] Sixty C57BL / 6 mice, 20 ± 2 g, were purchased from the Tongxiang Branch of Zhejiang Weitonglihua Laboratory Animal Technology Co., Ltd. Production license number: SCXK(Zhejiang)2020-0002. Use license number: SYXK(Shanghai)2019-0027.

[0190] 5. Dose setting

[0191] The non-sterile and sterilized stock solutions were injected at a volume of 0.1 ml / animal or 0.2 ml / animal.

[0192] 6. Dosage regimen

[0193] Subcutaneous injection 1, 4, 7, and 10 days after vaccination.

[0194] 7. Experimental Control

[0195] The negative control group was given normal saline of the same volume and concentration as the high-dose test group, with 10 mice in each group. The positive control group was given cisplatin DDP 6 mg / kg, once every other day for three consecutive times.

[0196] 8. Main steps of the experiment

[0197] Take the actively growing tumor source and inoculate 0.2 ml / animal (about 1-2×10 6 ), and the drug was administered according to the experimental design the next day. The experiment ended on the 13th day, and the animals in each group were sacrificed. The tumors were dissected and weighed, and the tumor inhibition rate was calculated according to the following formula:

[0198] Tumor inhibition rate % = [(average tumor weight of the control group - average tumor weight of the drug group) / average tumor weight of the control group] × 100%

[0199] 9. Experimental Results

[0200] The experimental results are shown in Table 2 and Figure 2. Compared with the negative control group, tumor weights in the cisplatin group, the low- and high-dose groups of non-sterilized samples, and the low- and high-dose groups of sterilized samples were significantly reduced. The tumor inhibition rate of cisplatin was 74.6%, the non-sterile low-dose (0.1 ml / animal) had a tumor inhibition rate of 49.0%, the high-dose (0.2 ml / animal) had a tumor inhibition rate of 79.3%, the sterilized low-dose (0.1 ml / animal) had a tumor inhibition rate of 79.0%, and the sterilized high-dose (0.2 ml / animal) had a tumor inhibition rate of 82.8%. This experiment observed that the sterilized group had a superior anti-tumor effect compared to the non-sterilized group.

[0201] Table 1. Antitumor efficacy of Yueran antitumor vaccine *Vs negative control, ****p<0.0001.

[0202] Example 5: Study 3 on the Tumor Inhibition Effect of Composite Vaccine on Lung Cancer

[0203] Shanghai Pharmaceutical Industry Research Institute Co., Ltd. was commissioned to conduct a study on the inhibition of lung cancer by the composite vaccine formula II A prepared in Example 1. The experimental report is as follows.

[0204] 1. Abstract: The sample of Yue Ran was administered subcutaneously at a dose of 0.1 ml / mouse to C57 / BL / 6 mice 1, 4, 7, and 10 days after tumor inoculation. The anti-tumor effect of the drug was tested in vivo on the tumor-bearing animals. The results showed that the tumor inhibition rate was 42.09%.

[0205] 2. Objective: To test the anti-tumor efficacy of Yueran samples on Lewis lung cancer in mice

[0206] 3 Test drugs:

[0207] 3.1 Name: Yueran Sample.

[0208] 3.2 Provider: Shaoxing Yueran Biopharmaceutical Technology Co., Ltd.

[0209] 3.3 Preparation method: Inject 0.1 ml of the stock solution per mouse.

[0210] 4 Experimental Materials:

[0211] 4.1 Solvent: Normal saline.

[0212] 4.2 Positive control: cisplatin powder injection 10 mg / bottle, produced by Qilu Pharmaceutical Co., Ltd., batch number 111024CF.

[0213] 4.3 Tumor source: Lewis lung cancer model was maintained by the Drug Evaluation and Research Center of Shanghai Institute of Pharmaceutical Industry.

[0214] 5. Experimental Animals:

[0215] 5.1 Source: C57 / BL / 6 mice were provided by Shanghai Slake Laboratory Animal Co., Ltd., Certificate No. SCXK 2012-0014. Our laboratory animal use license No. SYXK (Shanghai) 2004-0015.

[0216] 5.2 Weight: 18-22 grams.

[0217] 5.3 Gender: Female.

[0218] 5.4 Number of animals: There were 10 mice in each of the experimental group and the positive control group, and two negative control groups.

[0219] 6. Dosage setting: inject 0.1ml of stock solution per mouse.

[0220] 7. Dosage schedule: Subcutaneous injection 1, 4, 7, and 10 days after vaccination

[0221] 8. Experimental control: The negative control group was given normal saline of the same volume and concentration as the high-dose experimental group; the positive control group was given cisplatin DDP 7 mg / kg, once every other day for two consecutive times.

[0222] 9. Main steps of the test:

[0223] Take the actively growing tumor source and inoculate 0.2 ml / mouse (about 1-2×10 6 ), and the drug was administered according to the experimental design the next day. At the end of the experiment, the animals in each group were killed, the tumors were dissected and weighed, and the tumor inhibition rate was calculated according to the following formula:

[0224] Tumor inhibition rate % = [(average tumor weight of the control group - average tumor weight of the drug group) / average tumor weight of the control group] × 100%

[0225] 10. Experimental results:

[0226] In vivo anti-tumor testing was conducted on C57 / BL / 6 mice using a subcutaneous injection of 0.1 ml / mouse at 1, 4, 7, and 10 days after tumor inoculation. The results showed a 42.09% tumor inhibition rate (see Table 3 and Figure 3 for details).

[0227] Table 3. Anti-tumor efficacy test of Yueran samples against Lewis lung cancer in mice ***P value < 0.01 compared with the negative control group.

[0228] Example 6: Study on the tumor-suppressing effect of the composite vaccine on melanoma

[0229] Shanghai Institute of Pharmaceutical Industry Co., Ltd. was commissioned to study the melanoma inhibition effects of the composite vaccines formulated in Example 1, Formula 1A, Formula 1B, Formula 1C, Formula 2A, Formula 3A, Formula 3B, and Formula 3C. These seven composite vaccines were numbered "Yueran Samples 1, 2, 3, 4, 5, 6, and 7," respectively. The experimental report is as follows.

[0230] Anti-tumor efficacy test of Yueran samples on B16 melanoma in mice

[0231] 1. Abstract:

[0232] In vivo anti-tumor experiments were conducted on mice with B16 melanoma using samples of Yue Ran (No. 1, No. 2, No. 3, No. 4, No. 5, No. 6, and No. 7) at a dose of 0.1 ml / mouse, with subcutaneous injection at 1, 4, 7, and 10 days after inoculation. The results showed that the tumor inhibition rates of B16 melanoma were: No. 1 19.23%, No. 2 17.52%, No. 3 25.21%, No. 4 6.41%, No. 5 35.04%, No. 6 44.02%, and No. 7 48.72%, respectively.

[0233] 2. Purpose:

[0234] Anti-tumor efficacy test of Yueran samples on B16 melanoma in mice

[0235] 3. Test drug:

[0236] 3.1 Name: Yueran samples (No. 1, No. 2, No. 3, No. 4, No. 5, No. 6, No. 7).

[0237] 3.2 Provider: Shaoxing Yueran Biopharmaceutical Technology Co., Ltd.

[0238] 3.3 Preparation method: Inject 0.1 ml of the stock solution per mouse.

[0239] 4. Experimental Materials:

[0240] 4.1 Solvent: Normal saline.

[0241] 4.2 Positive control: cisplatin powder injection 10 mg / bottle, produced by Qilu Pharmaceutical Co., Ltd., batch number 111024CF.

[0242] 4.3 Tumor source: The B16 melanoma model was maintained by the Pharmacological Evaluation Research Center of Shanghai Institute of Pharmaceutical Industry.

[0243] 5. Experimental Animals:

[0244] 5.1 Source: BL / 6 mice were provided by Shanghai Slake Laboratory Animal Co., Ltd., Certificate No. SCXK 2012-0014. Our laboratory animal use license No. SYXK (Shanghai) 2004-0015.

[0245] 5.2 Weight: 18-22 grams.

[0246] 5.3 Gender: Female.

[0247] 5.4 Number of animals: There were 10 mice in each of the experimental group and the positive control group, and two negative control groups.

[0248] 6. Dosage setting: inject 0.1ml of stock solution per mouse.

[0249] 7. Dosage schedule: Subcutaneous injection 1, 4, 7, and 10 days after vaccination

[0250] 8. Experimental control: The negative control group was given the same volume and concentration of the corresponding solvent as the high-dose experimental group; the positive control group was given cisplatin DDP 7 mg / kg, once every other day for two consecutive times.

[0251] 9. Main steps of the test:

[0252] Take the actively growing tumor source and inoculate 0.2 ml / mouse (about 1-2×10 6 ), and the drug was administered according to the experimental design the next day. At the end of the experiment, the animals in each group were killed, the tumors were dissected and weighed, and the tumor inhibition rate was calculated according to the following formula:

[0253] Tumor inhibition rate % = [(average tumor weight of the control group - average tumor weight of the drug group) / average tumor weight of the control group] × 100%

[0254] 10. Experimental results:

[0255] In vivo anti-tumor testing was conducted on B16 melanoma mice using samples of Yueran (No. 1, No. 2, No. 3, No. 4, No. 5, No. 6, and No. 7) at a dose of 0.1 ml / mouse via subcutaneous injection 1, 4, 7, and 10 days after inoculation. The results showed that the tumor inhibition rates for B16 melanoma were 19.23% for No. 1, 17.52% for No. 2, 25.21% for No. 3, 6.41% for No. 4, 35.04% for No. 5, 44.02% for No. 6, and 48.72% for No. 7, respectively. See Table 4 and Figure 4 for details.

[0256] Table 4. Anti-tumor efficacy test of Yueran samples against B16 melanoma in mice ***P value < 0.01 compared with the negative control group.

[0257] The experimental results show that the composite vaccine formulas 1, 2 and 3 of the present invention have significant anti-tumor efficacy against Lewis lung cancer in mice and B16 melanoma in mice, demonstrating their broad-spectrum anti-tumor properties and their safety and non-toxicity, suggesting that they can be used in clinical practice for cancer treatment.

[0258] The above description of the embodiments is intended to facilitate understanding and application of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the teachings of this invention that do not depart from the scope of the present invention should be considered within the scope of protection of the present invention.

[0259] It should also be noted that the listing and discussion of previously disclosed documents in this specification should not be regarded as an admission that the documents are prior art or common knowledge that is equivalent to or replaces the present invention.

Claims

1. The use of the following microorganisms in the preparation of anti-tumor composite vaccines, characterized in that: A combination of three or more microorganisms: Bordetella pertussis, Salmonella typhi, Salmonella paratyphi A, Salmonella paratyphi B, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Proteus, Lactobacillus, Bifidobacterium longum, Diphtheria and Tetanus, Clostridium acetobutylicum, Salmonella typhimurium, and Streptococcus pyogenes.

2. An anti-tumor composite vaccine, characterized in that: The active ingredient of the medicine comprises an inactivated preparation of the following three microorganisms among the microorganisms as claimed in claim 1, more preferably consists of an inactivated preparation of the following three microorganisms: Bordetella pertussis, Salmonella typhi, Staphylococcus aureus; or The active pharmaceutical ingredient comprises, in addition to Bordetella pertussis, Salmonella typhi, and Staphylococcus aureus among the microorganisms as claimed in claim 1, further comprising Salmonella paratyphi A and Salmonella paratyphi B, that is, the active pharmaceutical ingredient comprises an inactivated preparation of the following five microorganisms among the microorganisms as claimed in claim 1, and more preferably consists of an inactivated preparation of the following five microorganisms: Bordetella pertussis, Salmonella typhi, Salmonella paratyphi A, Salmonella paratyphi B, and Staphylococcus aureus; or The pharmaceutical active ingredient comprises an inactivated preparation of the following nine microorganisms among the microorganisms as described in claim 1, and more preferably consists of an inactivated preparation of the following nine microorganisms: Listeria, Escherichia coli, Proteus, Lactobacillus, Bifidobacterium longum, Diphtheria and Tetanus, Clostridium acetobutylicum, Salmonella typhimurium, and Streptococcus pyogenes.

3. The anti-tumor composite vaccine according to claim 2, characterized in that The inactivated preparation is an inactivated product of a microbial suspension, for example, an inactivated product of a microbial suspension in physiological saline or a suspension in PBS buffer for injection.

4. The anti-tumor composite vaccine according to claim 3, characterized in that The inactivation method of the microbial suspension is physical inactivation, which is selected from heating inactivation, ultraviolet inactivation, or a combination of heating inactivation and ultraviolet inactivation.

5. The anti-tumor composite vaccine according to claim 4, characterized in that Suspensions of different microorganisms are inactivated individually; or suspensions of different microorganisms are mixed and inactivated together; or suspensions of different microorganisms are mixed with vaccine dressings / adjuvants and inactivated together.

6. The anti-tumor composite vaccine according to claim 5, characterized in that Before inactivation, the concentration of the suspension of various microorganisms is 1-10 billion cells / ml, preferably 3-5 billion cells / ml.

7. The anti-tumor composite vaccine according to any one of claims 2 to 4, characterized in that: The dosage form is an injection selected from subcutaneous injection and intramuscular injection.

8. The anti-tumor composite vaccine according to claim 7, characterized in that The invention relates to a pharmaceutical composition, which includes, in addition to a microbial inactivation preparation as a pharmaceutically active ingredient, a pharmaceutically acceptable dressing / adjuvant for injectable vaccines, wherein the dressing / adjuvant includes but is not limited to the following ingredients: polyinosinic-acid, dextran, lecithin, oil for injection, vitamin A, aluminum stearate, sodium carboxymethyl cellulose, fat emulsion for injection, and Span-20.

9. The anti-tumor composite vaccine according to claim 8, characterized in that The anti-tumor composite vaccine is selected from the following formula: Formulation 1: 8-15 v / v% (preferably 10-12 v / v%) of an inactivated preparation of Bordetella pertussis, 5-15 v / v% (preferably 8-10 v / v%) of an inactivated preparation of Salmonella typhi, 5-15 v / v% (preferably 8-10 v / v%) of an inactivated preparation of Staphylococcus aureus, 0.05-0.5 wt% (preferably 0.1-0.2 wt%) of polyinosinic acid (polyI:C), 3-10 wt% (preferably 5-8 wt%) of dextran, and the remainder of normal saline to make up 100% by weight; Formulation 2: 6-15 v / v% (preferably 8-10 v / v%) of an inactivated preparation of Bordetella pertussis, 8-15 v / v% (preferably 10-12 v / v%) of an inactivated preparation of Salmonella typhi, 2-8 v / v% (preferably 3-5 v / v%) of an inactivated preparation of Salmonella paratyphi A, 2-8 v / v% (preferably 3-5 v / v%) of an inactivated preparation of Salmonella paratyphi B, 3-10 v / v% (preferably 5-8 v / v%) of an inactivated preparation of Staphylococcus aureus, 0.05-0.5 wt% (preferably 0.1-0.2 wt%) of polyinosinic acid (polyI:C), 5-15 wt% (preferably 8-10 wt%) of dextran, and the remainder of normal saline to make up 100% by weight; Formula 3: 3-10 v / v% (preferably 5-7 v / v%) of the inactivated preparation for Listeria monocytogenes, 3-10 v / v% (preferably 5-7 v / v%) of the inactivated preparation for Escherichia coli, 3-10 v / v% (preferably 5-7 v / v%) of the inactivated preparation for Proteus, 3-10 v / v% (preferably 5-7 v / v%) of the inactivated preparation for lactic acid bacteria, 3-10 v / v% (preferably 5-7 v / v%) of the inactivated preparation for Bifidobacterium longum, 4-10 v / v% (preferably 6-8 v / v%) of the inactivated preparation for Diphtheria and Tetanus, and 1-5 v / v% of the inactivated preparation for Clostridium acetobutylicum. , preferably 2-3v / v%, inactivated preparation of Salmonella typhimurium 1-5v / v%, preferably 2-3v / v%, inactivated preparation of Streptococcus pyogenes 0.5-5v / v%, preferably 1-2v / v%, Vitamin A 0.2-2wt%, preferably 0.5-1wt%, aluminum stearate 1-2wt%, preferably 1-2wt%, CMC-Na sodium carboxymethyl cellulose (pharmaceutical grade, viscosity 800-1200) 0.5-5wt%, preferably 1-2wt%, fat emulsion for injection 3-10v / v%, preferably 5-7v / v%, Span-20 0.5-5wt%, preferably 1-2wt%, and the remainder of normal saline is used to make up 100% by weight.

10. The use according to claim 1, wherein The tumor is one or more combinations of the following cancers / tumors: malignant epithelial tumors, lymphomas, blastomas, sarcomas, leukemias, basal cell carcinomas, bile duct cancer; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; digestive system cancers; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer, gastrointestinal cancer; glioblastoma; liver cancer; hepatoma; intraepithelial neoplasia; kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer; lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; melanoma; Myeloma; neuroblastoma; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urological cancer; vulvar cancer; and other carcinomas and sarcomas; as well as B-cell lymphoma; chronic lymphocytic leukemia; acute lymphocytic leukemia; hairy cell leukemia; chronic myeloid leukemia; and post-transplant lymphoproliferative disorder, as well as abnormal blood vessel proliferation, edema, and Meigs syndrome associated with nevus hamartomatosis.

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