A personalized tumor vaccine using bacteria as a carrier and its preparation method

Abstract

The application belongs to the technical field of tumor vaccines, and more particularly relates to a tumor personalized vaccine with bacteria as a carrier and a preparation method thereof.The tumor personalized vaccine comprises engineered positive charge bacteria, training immune activators loaded on the engineered positive charge bacteria, and tumor antigens electrostatically adsorbed and adhered to the surface of the engineered positive charge bacteria.By loading the training immune activators on the engineered positive charge bacteria and electrostatically adsorbing the tumor personalized antigens, experiments prove that the tumor personalized vaccine can avoid immune tolerance, improve the retention time of the antigens at an injection site, enhance the recruitment of DCs and monocytes / macrophages at a vaccination site, enhance the uptake and antigen presentation of immune cells, significantly enhance the activation reaction of tumor-specific T cells, weaken the tumor immunosuppressive microenvironment, has an obvious tumor inhibition effect, and realizes precise postoperative treatment for different tumor patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor vaccines, and more specifically, relates to a personalized tumor vaccine using bacteria as a carrier and a preparation method thereof. Background Art

[0002] In recent years, tumor immunotherapy has held great promise for anti-cancer treatment. Various tumor immunotherapy strategies have been extensively studied and used alone or in combination with other conventional approaches, such as surgery, chemotherapy, and radiotherapy. Furthermore, postoperative immunotherapy, such as immune checkpoint blockade (ICB), has been reported to reduce the risk of cancer recurrence and metastasis. However, its effectiveness remains to be improved.

[0003] Tumor vaccination offers unique advantages and holds enormous potential for future cancer treatment. Vaccines include whole-cell tumor vaccines, dendritic cell vaccines, DNA vaccines, viral vector vaccines, mRNA vaccines, and classic peptide vaccines. However, the development of tumor vaccines is hampered by shortcomings such as poor immunogenicity of tumor antigens, difficulty in inducing sustained immune responses, and poor safety. Several disadvantages currently limit the development of tumor vaccines from various sources. For example, whole-cell tumor vaccines alone have low immunogenicity and can only stimulate a low immune response, requiring combination with other appropriate adjuvants to enhance their immunogenicity. Dendritic cell vaccines require in vitro stimulation with tumor antigens and cytokines to mature DCs, which are then infused back into the body. This complex preparation process requires the activation of large numbers of DCs, which is time-consuming and labor-intensive. DNA tumor vaccines combine fragments encoding tumor-specific antigens with plasmid DNA vectors, enabling them to express the desired antigen and thereby induce a specific immune response. However, DNA tumor vaccines also require further optimization, as plasmid DNA rarely enters the cell nucleus and expresses tumor antigens, and the tissue injection site is not well suited to induce APC maturation and stimulate T cell activation. The active components of tumor vaccines include tumor antigens, formulations, immune adjuvants, and delivery vehicles. Numerous antigens, adjuvants, delivery strategies, and formulations have been investigated, but the effective response rate in clinical practice remains relatively low. Therefore, identifying the most effective antigen-adjuvant type and delivery method remains a key area of ​​research.

[0004] The history of tumor vaccine research and development spans over 120 years. In 1893, William B. Coley, the father of cancer immunotherapy, discovered that a toxin secreted by Streptococcus pyogenes could induce tumor regression in patients, thus laying the foundation for the development of tumor vaccines. In 2006, the world's first HPV vaccine was developed by Merck and approved by the US FDA. In 2010, Provenge, a DC cell vaccine for prostate cancer, became the first novel therapeutic tumor vaccine approved by the US FDA. In recent years, numerous studies have focused on developing various strategies to prepare tumor vaccines and enhance their immune responses in vitro and in vivo. David Mooney and colleagues developed a mesoporous silica microrod (MSR) that sequentially adsorbs polyethyleneimine (PEI) and a neoantigen through electrostatic interactions. The adsorbed PEI and MSR synergistically activate APCs and induce the secretion of inflammatory cytokines. In in vivo tumor inhibition experiments, the vaccine platform demonstrated strong anti-tumor activity when loaded with tumor-specific antigens. Professor Ma Guanghui reported a novel microsphere tumor vaccine formulation that exhibits unique self-healing properties after encapsulating tumor antigens. After vaccination, the antigens in the microspheres at the injection point can be effectively released and recruit more antigen-presenting cells, effectively improving the utilization rate of antigens, promoting the maturation of antigen-presenting cells, and improving the level of antigen presentation, thereby achieving effective T cell immune activation, effective tumor suppression, anti-tumor metastasis, and prevention of postoperative recurrence.

[0005] Bacteria and their derivatives can mobilize the immune system to respond to exogenous "danger signals" through innate immune responses. Bacteria possess a rich array of pathogen-associated molecular patterns, which can effectively activate immune cells, enhance specific immune response recognition, and eliminate tumor cells, even within the immunosuppressive tumor microenvironment. Bacteria possess unique targeting capabilities for hypoxic sites, enabling them to colonize tumors and achieve robust immune activation. For example, bacterial peptidoglycan, lipopolysaccharide, DNA, flagella, and RNA can bind to pattern recognition receptors on antigen-presenting cells, thereby triggering a corresponding immune response. Studies have reported the development of nano-cancer vaccines using Escherichia coli cytoplasmic membranes and tumor cell membranes from autologous tumor tissue as adjuvants and antigens. This vaccine induces dendritic cell maturation, thereby activating T cells. It has demonstrated anti-tumor efficacy in CT26 colon cancer models and 4T1 breast tumor mouse models. However, the vaccine requires a complex process for extracting bacterial cell membranes and fusion of the bacterial cell membrane with tumor antigens through methods such as ultrasound or extrusion. Fusion efficiency and stability require further improvement. Bacterial outer membrane vesicles (OMVs) are attractive immunostimulatory adjuvants due to their abundance of immunostimulatory antigens and lack of infectivity. Currently, OMVs are considered ideal components for tumor vaccines. However, several unfavorable factors limit their development, including the difficulty in controlling stable OMV production; the cumbersome process of inducing tumor antigen expression through genetic engineering; the difficulty in controlling quality of OMVs, which are naturally secreted by bacteria; and the challenges of large-scale preparation. Combining these advantages and disadvantages of tumor vaccines, further breakthroughs in their limitations will further advance their development.

[0006] How to improve vaccine safety, select appropriate adjuvants to more efficiently enhance the immune response in vivo, and improve the tumor immunosuppressive microenvironment are currently the key issues in improving tumor vaccine design. Summary of the Invention

[0007] In response to the shortcomings of the existing technology, the present invention provides a personalized tumor vaccine using bacteria as a carrier and a preparation method thereof, which solves the technical problems of the existing tumor vaccine such as low immunogenicity, low level of immune response of the body, induction of immune tolerance, and difficulty in scale-up preparation process.

[0008] To achieve the above objectives, the present invention provides a personalized tumor vaccine using bacteria as a carrier, comprising engineered positively charged bacteria and a trained immune activator loaded thereon, and also comprising a tumor antigen adhered to the surface of the engineered positively charged bacteria by electrostatic adsorption; the trained immune activator is a substance capable of activating trained immunity.

[0009] Preferably, the engineered positively charged bacteria are positively charged bacteria obtained by inactivating and charge-reversing the bacteria.

[0010] Preferably, the tumor antigen is a tumor patterned antigen or a tumor personalized antigen; wherein: the tumor patterned antigen is chicken ovalbumin, alpha-fetoprotein, carcinoembryonic antigen or squamous cell carcinoma antigen; the tumor personalized antigen is a tumor antigen derived from tumor tissue of colorectal cancer, melanoma, breast cancer, lung cancer, ovarian cancer, choriocarcinoma, cervical cancer, liver cancer, bladder cancer or skin cancer.

[0011] Preferably, the trained immune activator is a polysaccharide or polypeptide capable of activating trained immunity, more preferably one or more of β-glucan, muramyl dipeptide, and muramyl tripeptide.

[0012] Preferably, the final concentration of the engineered positively charged bacteria in the tumor personalized vaccine is (1.5*10 8 ~2*10 9 ) CFU / mL, the final concentration of the tumor antigen in the tumor personalized vaccine is 100-500 μg / mL, and the final concentration of the trained immune activator in the tumor personalized vaccine is 100-500 μg / mL.

[0013] According to another aspect of the present invention, a method for preparing the personalized tumor vaccine is provided, comprising the following steps:

[0014] S1: performing charge reversal on the bacteria to obtain the engineered positively charged bacteria;

[0015] S2: digesting and lysing the tumor tissue, collecting cell lysates, and obtaining the tumor antigen;

[0016] S3: The trained immune activator is mixed with the engineered positively charged bacteria and the tumor antigen and incubated so that the engineered positively charged bacteria are loaded with the trained immune activator and the surface of the engineered positively charged bacteria adsorbs the tumor antigen by electrostatic adsorption, thereby obtaining a personalized tumor vaccine.

[0017] Preferably, in step S1, the bacteria are mixed with an amino compound, a carboxyl activator and an amino acid protective agent, and then shaken and incubated to achieve charge reversal of the bacteria; wherein the amino compound is PEI, chitosan or polylysine, the carboxyl activator is EDC, and the amino acid protective agent is NHS.

[0018] Preferably, in step S2, the digestion treatment is: adding RPMI 1640 culture medium containing type I collagenase to the tumor tissue and incubating.

[0019] Preferably, the incubation temperature in step S2 is 25-37° C., and the incubation time is 10-120 min; the incubation temperature in step S3 is 25-37° C., and the incubation time is 10-120 min.

[0020] According to another aspect of the present invention, a drug for treating tumors is provided, which includes the personalized tumor vaccine using bacteria as a carrier.

[0021] In general, the above technical solutions conceived by the present invention have the following beneficial effects compared with the prior art:

[0022] (1) The present invention provides a personalized tumor vaccine using bacteria as carriers, which includes engineered positively charged bacteria and a trained immune activator loaded on the surface of the bacteria, as well as a tumor antigen wrapped on the surface of the bacteria by electrostatic adsorption. The engineered positively charged bacteria are obtained by flipping the bacterial charge, and the engineered positively charged bacteria are mixed and incubated with the tumor antigen and the trained immune activator to obtain a personalized tumor vaccine. Experiments have shown that personalized tumor vaccines can avoid immune tolerance, improve the retention of antigens at the injection site, promote DC recruitment, antigen uptake and presentation at the inoculation site, significantly enhance tumor-specific T cell activation responses, and weaken the tumor immunosuppressive microenvironment.

[0023] (2) The present invention uses bacteria as vaccine carriers to form an antigen library, thereby improving the retention of antigens at the injection site; and bacteria can act as adjuvants to provide PAMPs signals for tumor antigens, thereby enhancing the uptake and presentation of antigens by immune cells.

[0024] (3) Bacterial vaccines are mainly based on the fact that bacteria can be used as adjuvants. However, a major problem with bacterial vaccines is that they can induce immune tolerance during use. That is, after multiple subcutaneous injections of the vaccine, macrophages will no longer respond or respond very poorly, resulting in a decrease in the production of inflammatory cytokines by macrophages, which in turn causes a decrease in the amount of DCs recruited at the injection site, weakening the immune activation effect. On the other hand, trained immune activators represented by dextran can promote T cell proliferation and activate the immune phagocytic response. However, when a single trained immune activator is used as an adjuvant for tumor vaccines, it is easily metabolized and degraded, and still cannot improve the tumor's immunosuppressive microenvironment, leading to the induction of infiltration of suppressive immune cells, turning DCs into dysfunctional DCs, and continuously enhancing the tumor's immunosuppressive microenvironment. However, the present invention loads trained immune activators represented by dextran on the surface of bacteria, and further electrostatically adsorbs tumor antigens. Experiments have found that trained immune activators such as dextran may be more likely to reside in the injection site due to their ability to promote the release of more inflammatory factors, significantly enhancing the recruitment of DCs and monocytes / macrophages at the inoculation site, increasing interaction with macrophages, and significantly improving the therapeutic effect.

[0025] (4) There are a large number of neoantigens in the tumor tissues of clinical tumor patients that are formed by gene mutations and lack in normal tissues, which makes it difficult for traditional tumor vaccines that deliver tumor-related antigens to achieve efficient tumor-specific T cell activation responses for clinical tumor patients. The technical solution of the present invention can prepare engineered bacteria and trained immune activators in advance. When used, personalized tumor antigens are obtained by using the tumor tissue removed from the tumor patient's surgery, and then simply mixed with the pre-prepared engineered bacteria and trained immune activators to obtain a personalized vaccine for the tumor patient and directly administer it to the patient, thereby exerting a very good function of inhibiting tumor recurrence and metastasis. It is quick to use and can achieve precise postoperative treatment for different tumor patients, exerting a very good function of inhibiting tumor recurrence and metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a flow chart of the preparation method of the personalized tumor vaccine using bacteria as a carrier according to the present invention.

[0027] Figure 2 Figure 1 shows the antigen attachment of the BG / OVA@EcN vaccine prepared in Example 1. Figure A shows a confocal laser image of the antigen-loaded BG / OVA@EcN vaccine, with a scale of 20 μm. Figure B shows the zeta potential of the antigen-loaded BG / OVA@EcN vaccine. Figure C shows a TEM image of the antigen-loaded BG / OVA@EcN vaccine, with a scale of 1 μm.

[0028] Figure 3 The attached status of antigens loaded on BG / Ag@EcN vaccine prepared in Example 1. (A) is the laser confocal microscope image of antigens loaded on BG / Ag@EcN vaccine, with a scale of 20 μm; (B) is the zeta potential of antigens loaded on BG / Ag@EcN vaccine.

[0029] Figure 4 Figure 3. Antigen retention of the BG / OVA-Cy5.5@EcN vaccine at the subcutaneous injection site. (A) shows subcutaneous antigen retention as observed by small animal imaging; (B) shows the fluorescence ratio of retained antigen; (C) shows the ratio of antigen uptake by macrophages at the subcutaneous injection site; and (D) shows the ratio of antigen uptake by dendritic cells at the subcutaneous injection site.

[0030] Figure 5 The number of monocytes / macrophages and dendritic cells recruited at the subcutaneous injection site after subcutaneous inoculation of the vaccine BG / OVA@EcN in vivo. Figure 5 Content (A) corresponds to the number of monocytes recruited at the subcutaneous injection site; Figure 5 Content (B) corresponds to the number of macrophages recruited at the subcutaneous injection site; Figure 5Content (C) corresponds to the number of dendritic cells recruited at the subcutaneous injection site.

[0031] Figure 6 Figure 1 shows the increase in the number of monocytes and macrophages in the blood and the increase in the proportion of trained monocytes and macrophages after inoculation with the BG / OVA@EcN vaccine. (A) shows the change in the number of monocytes in the blood; (B) shows the change in the number of macrophages in the blood; (C) shows the change in the proportion of trained monocytes in the blood; and (D) shows the change in the proportion of trained macrophages in the blood.

[0032] Figure 7 The changes in the number of activated dendritic cells and activated CD8 + Changes in the number of T cells. Content (A) shows the changes in the number of activated dendritic cells in the lymph nodes; Content (B) shows the changes in the number of activated CD8 + Changes in T cell numbers.

[0033] Figure 8 Figure 3: The efficacy of the BG / OVA@EcN vaccine in preventing tumor growth and prolonging mouse survival in a subcutaneous B16-OVA melanoma tumor model. (A) shows the tumor treatment effect; (B) shows the survival extension effect in tumor-bearing mice.

[0034] Figure 9 The therapeutic effect and survival extension effect of vaccine BG / OVA@EcN in treating subcutaneous B16-OVA melanoma tumor model. Content (A) is the tumor treatment effect; content (B) is the effect of prolonging the survival of tumor mice. Content (CF) is the CD3 + T cell count, CD8 + T cell number, activated IFNγ + CD8 + T cell number, activated CD69 + CD8 + T cell count.

[0035] Figure 10 Figure 3: The efficacy of the vaccine BG / Ag-4T1@EcN in inhibiting recurrence and prolonging survival in an orthotopic 4T1 breast tumor resection model. (A) shows the efficacy in inhibiting recurrence after surgery; (B) shows the efficacy in prolonging survival in tumor-bearing mice.

[0036] Figure 11 Content (A) and content (B) are the numbers of monocytes / macrophages in the blood of the surviving mice in Example 5, and content (C) and content (D) are the proportions of trained monocytes / macrophages in the blood.

[0037] Figure 12 Content (A) and content (B) are the proportions of memory T cells in the blood of the surviving mice in Example 5, and content (C) and content (D) are the activation proportions of T cells in the blood after antigen restimulation in vitro.

[0038] Figure 13 Figure 3: The efficacy of the MDP / Ag-H22@MG1655 vaccine in inhibiting recurrence and prolonging survival in a subcutaneous H22 liver cancer model after resection. (A) shows the efficacy in inhibiting recurrence after surgery; (B) shows the efficacy in prolonging survival in mice bearing tumors.

[0039] Figure 14 Content (A) and content (B) are the numbers of monocytes / macrophages in the blood of the surviving mice in Example 6, and content (C) and content (D) are the proportions of trained monocytes / macrophages in the blood.

[0040] Figure 15 Content (A) and content (B) are the proportions of memory T cells in the blood of the surviving mice in Example 6.

[0041] Figure 16 Figure 1 shows the efficacy of the vaccine MDP / Ag-MC38@MG1655 in inhibiting recurrence and prolonging survival in a subcutaneous colon cancer MC38 tumor resection model. (A) shows the efficacy in inhibiting recurrence after surgery; (B) shows the efficacy in prolonging survival in tumor-bearing mice.

[0042] Figure 17 Content (A) and content (B) are the numbers of monocytes / macrophages in the blood of the surviving mice in Example 7, and content (C) and content (D) are the proportions of trained monocytes / macrophages in the blood.

[0043] Figure 18 Content (A) and content (B) are the proportions of memory T cells in the blood of the surviving mice in Example 7. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0045] The present invention provides a personalized tumor vaccine using bacteria as a carrier. The personalized tumor vaccine uses bacteria as a carrier, includes engineered positively charged bacteria and a trained immune activator loaded thereon, and also includes tumor antigens adhered to the surface of the engineered positively charged bacteria by electrostatic adsorption; the trained immune activator is a substance that can activate trained immunity.

[0046] The engineered positively charged bacteria described herein are obtained by inactivating and reversing the charge of the bacteria. The bacteria used in the bacterial-based personalized tumor vaccine of the present invention can theoretically be any bacterial carrier that is beneficial to the host. In some embodiments, the bacteria are Gram-positive and / or Gram-negative bacteria, wherein Gram-negative bacteria include, but are not limited to, at least one of Escherichia coli strain Nissle 1917 (ECN), Escherichia coli strain MG1655, and attenuated Salmonella VNP20009; and Gram-positive bacteria include, but are not limited to, at least one of Lactobacillus, Bifidobacterium, and probiotic spore-forming bacteria.

[0047] The tumor antigen described in the present invention can be any tumor patterned antigen or tumor personalized antigen isolated from tumor tissue that can play a role in tumor prevention or treatment. In some embodiments, the tumor antigen is a tumor patterned antigen or a tumor personalized antigen, and the tumor patterned antigen is chicken ovalbumin (OVA) or alpha-fetoprotein (AFP), carcinoembryonic antigen, squamous cell carcinoma antigen, etc.; the tumor personalized antigen is a tumor antigen derived from tumor cell tissue in colorectal cancer, melanoma, breast cancer, lung cancer, ovarian cancer, choriocarcinoma, cervical cancer, liver cancer, bladder cancer or skin cancer.

[0048] In some embodiments, the trained immune activator is a polysaccharide or polypeptide capable of activating trained immunity. In preferred embodiments, it is one or more of β-glucan, muramyl dipeptide, muramyl tripeptide, and the like.

[0049] In some embodiments, the final concentration of the engineered positively charged bacteria in the tumor personalized vaccine is (1.5*10 8 ~2*10 9 ) CFU / mL, the final concentration of the tumor antigen in the tumor personalized vaccine is 100-500 μg / mL, and the final concentration of the trained immune activator in the tumor personalized vaccine is 100-500 μg / mL.

[0050] The present invention also provides a method for preparing the personalized tumor vaccine using bacteria as a carrier, such as Figure 1 As shown, the following steps are included:

[0051] S1: culturing bacteria and performing charge reversal on the bacteria to obtain the engineered positively charged bacteria;

[0052] S2: digesting and lysing the tumor tissue, collecting cell lysates, and obtaining the tumor antigen;

[0053] S3: The trained immune activator is mixed with the engineered positively charged bacteria and the tumor antigen and incubated so that the engineered positively charged bacteria are loaded with the trained immune activator and the surface of the engineered positively charged bacteria adsorbs the tumor antigen by electrostatic adsorption, thereby obtaining a personalized tumor vaccine.

[0054] In some embodiments, the charge reversal of the bacteria in step S1 is specifically performed by incubating the bacteria with an amino compound, a carboxyl activator, and an amino acid protective agent in a shaking incubation, and resuspending by centrifugation to obtain the engineered positively charged bacteria, wherein the amino compound is PEI, chitosan, polylysine, etc., the carboxyl activator is EDC, and the amino acid protective agent is NHS. EDC is a carboxyl activator, but the ester it forms is easily hydrolyzed. After the addition of NHS, its hydroxyl group reacts with the active ester to form a relatively more stable active ester, which easily undergoes a substitution reaction with the amino group to form an amide bond. In addition, NHS can also improve coupling efficiency.

[0055] In some embodiments, the bacteria are subjected to charge reversal in step S1, wherein the amino compound is preferably PEI (polyethyleneimine), and its final concentration is preferably 2-4 mg / mL, the carboxyl activator is preferably EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide), and its final concentration is preferably 20-30 mg / mL, and the amino acid protective agent is preferably NHS (N-hydroxysuccinimide), and its final concentration is preferably 15-25 mg / mL.

[0056] In some embodiments, step S2 is to inoculate personalized tumor cells orthotopically into the mammary pad of Balb / c mice until the tumor volume reaches 250-300 mm 3 The tumor is surgically removed and used for subsequent preparation of personalized tumor antigens.

[0057] In some embodiments, in step S2, the digestion treatment is to mince the tumor tissue and then add RPMI 1640 culture medium containing type I collagenase and incubate, wherein the volume of RPMI 1640 culture medium is 2-4 mL and the concentration of type I collagenase is 0.7-0.9 mg / mL.

[0058] In some embodiments, the incubation temperature in step S2 and step S3 is 25-37° C., and the incubation time is 10-120 min.

[0059] In some specific embodiments, in step S2, the tumor tissue is digested with type I collagenase, mechanically minced to obtain small tumor tissue particles, and digested at 37°C for 30 minutes. After physical grinding, gauze filtration, and lysis with red blood cell lysis buffer, a single cell suspension is obtained. The cell suspension is repeatedly frozen and thawed in liquid nitrogen and a 37°C water bath, and then subjected to ultrasonic cell disruption to obtain a cell lysate, which is the tumor antigen.

[0060] The present invention also provides a drug for treating tumors, comprising the bacterial-based personalized tumor vaccine of the present invention. The vaccine can be administered by subcutaneous injection, and its dosage form includes, but is not limited to, a suspension.

[0061] Bacteria possess a rich array of pathogen-associated molecular patterns that can effectively activate immune cells even in the immunosuppressive microenvironment of tumors, enhancing specific immune responses to recognize and eliminate tumor cells. Furthermore, bacteria possess unique targeting capabilities for hypoxic sites, enabling specific colonization at tumor sites. Furthermore, bacteria possess strong immune-activating abilities and are easily engineered, making them ideal candidates for tumor immunotherapy. For example, bacterial peptidoglycan, lipopolysaccharide, DNA, flagella, and RNA can bind to pattern recognition receptors on antigen-presenting cells, thereby triggering a corresponding immune response. However, a major problem with bacterial vaccines is the induction of immune tolerance during use. This means that after multiple subcutaneous injections of the vaccine, macrophages no longer respond or respond only minimally, leading to a decrease in the production of inflammatory cytokines by macrophages, which in turn reduces the number of DCs recruited at the injection site and weakens the immune activation effect. The present invention involves inverting the charge of bacteria to produce engineered positively charged bacteria. These engineered positively charged bacteria are then mixed and incubated with tumor antigens and a trained immune activator to produce a personalized tumor vaccine consisting of a bacterial carrier, a trained immune activator loaded on the bacterial surface, and a tumor antigen electrostatically adsorbed and coated on the bacterial surface. The personalized tumor vaccine of the present invention can avoid immune tolerance, improve the retention of antigens at the injection site, promote DCs recruitment, antigen uptake and presentation at the vaccination site, significantly enhance tumor-specific T cell activation response, and weaken the tumor immunosuppressive microenvironment.

[0062] The personalized tumor vaccine of the present invention is also loaded with trained immune activators represented by β-glucan, which can activate innate immune cells such as monocytes / macrophages to train immunity, promote DCs recruitment, antigen uptake and presentation at the vaccination site, and significantly enhance tumor-specific T cell activation response; at the same time, β-glucan can activate peripheral blood monocytes to train immunity, increase the proportion of M1 TAMs in tumor tissue, weaken the tumor immunosuppressive microenvironment, and ultimately significantly inhibit tumor growth.

[0063] Mouse breast cancer 4T1 cells, mouse melanoma B16-OVA cells, mouse liver cancer H22 cells, and mouse colon cancer MC38 cells used in the following examples were all purchased from the China Center for Type Culture Collection (CCTCC); BALB / c mice and C57BL / 6 mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.; β-glucan was purchased from Invivogen; yeast extract and tryptone were purchased from Oxoid, UK; RPMI1640 medium, collagenase I, and PEI were purchased from Gibco, USA.

[0064] The following are examples:

[0065] Example 1

[0066] Construction of a personalized tumor vaccine using probiotics (ECN) as a carrier

[0067] Preparation of engineered positively charged bacteria:

[0068] Prepare LB medium by placing 5 g of yeast extract, 10 g of tryptone, and 10 g of sodium chloride in a conical flask. Dissolve the mixture in 900 mL of ultrapure water. Adjust the pH to 7.2 with 1 mol / L sodium hydroxide (NaOH). Then, dilute the volume to 1 L with ultrapure water. Autoclave the flask (121°C, 20 min) in a YM75 fully automatic vertical pressure steam sterilizer to prepare LB medium. Cool the flask and store at 4°C until ready for use.

[0069] Remove EcN from a -80°C freezer and place in a clean bench. Inoculate the culture at a 1:100 ratio into LB medium and incubate overnight at 37°C on a shaker at 180 rpm for activation. The activated culture was inoculated into LB medium at a 1:100 ratio and incubated at 37°C on a shaker at 180 rpm. When the OD600 of the activated EcN culture reached 1.0, aspirate 500 μL of the culture and centrifuge at 8000 rpm for 3 minutes in a high-speed refrigerated centrifuge. Discard the supernatant, resuspend the pellet in saline, wash three times by centrifugation, and resuspend in 1 mL of saline to obtain the bacteria.

[0070] Dissolve PEI in PBS buffer to prepare a 6.25 mg / mL PEI stock solution. Dilute the solution to a final concentration of 3.125 mg / mL when using. Resuspend the above bacteria in physiological saline, add EDC and NHS to a final concentration of 25 mg / mL and 20 mg / mL, respectively. Add PEI to the bacterial solution and mix well. Incubate at 37°C in a shaker at 180 rpm for 30 minutes. After incubation, centrifuge at 8000 rpm in a high-speed refrigerated centrifuge for 3 minutes, discard the supernatant, resuspend the precipitate in physiological saline and wash it three times by centrifugation to obtain engineered positively charged bacteria (PEI@EcN).

[0071] Preparation of tumor whole cell-derived antigen:

[0072] Preparation of breast cancer tumor tissue-derived antigen (abbreviated as Ag-4T1): Collect mouse breast cancer 4T1 cells in logarithmic growth phase, wash once with PBS and resuspend, dilute with PBS to a cell density of 5 x 10 6 mL -1 . 18-20 g female BALB / c mice, shave the breast area, use a disposable sterile insulin syringe to draw the cell suspension, inoculate 100 μL of the tumor cell suspension at the lower part of the mouse breast to establish a 4T1 orthotopic tumor model. When the tumor grows to 300 mm 3 , perform surgical resection of the tumor.

[0073] Preparation of hepatocarcinoma tumor tissue-derived antigen (abbreviated as Ag-H22): Collect mouse hepatocarcinoma H22 cells in logarithmic growth phase, wash once with PBS and resuspend, dilute with PBS to a cell density of 2 x 10 7 mL -1 . 18-20 g male BALB / c mice, use a disposable sterile insulin syringe to draw the cell suspension, inoculate 100 μL of the tumor cell suspension at the subcutaneous part of the right hind thigh of the mouse to establish a H22 subcutaneous hepatocarcinoma tumor model. When the tumor grows to 300 mm 3 , perform surgical resection of the tumor.

[0074] Preparation of colon cancer tumor tissue-derived antigen (abbreviated as Ag-MC38): Collect mouse colon cancer MC38 cells in logarithmic growth phase, wash once with PBS and resuspend, dilute with PBS to a cell density of 5 x 10 6 mL -1 . 18-20 g female C57BL / 6J mice, use a disposable sterile insulin syringe to draw the cell suspension, inoculate 100 μL of the tumor cell suspension at the subcutaneous part of the right hind thigh of the mouse to establish a MC38 subcutaneous colon cancer tumor model. When the tumor grows to 300 mm 3 , perform surgical resection of the tumor.

[0075] Freshly excised tumor tissue was washed with saline and placed in a 6-well plate. After mincing with surgical scissors, 3 mL of RPMI 1640 medium containing 0.8 mg / mL type I collagenase was added and incubated in a 37°C incubator for 30 min. After digestion, the tissue fragments and digestion solution were passed through a 200-mesh cell sieve, pressed with a syringe plunger, and the cell suspension was collected by rinsing with RPMI 1640 medium. After filtering twice through a 200-mesh nylon sieve, the suspension was centrifuged at 1500 rpm for 5 min at 4°C. The cell pellet was lysed with red blood cell lysis buffer, the supernatant was discarded, and the cell pellet was washed three times with PBS to obtain a cell pellet. Protease inhibitor solution (Roche, Cat. No. 04693159001) was prepared according to the manufacturer's instructions and the cell pellet was resuspended in the sterile protease inhibitor solution. The cell suspension was repeatedly frozen and thawed four times in liquid nitrogen and a 37°C water bath, and then centrifuged to obtain a cell lysate, which was then broken by an ultrasonic cell disruptor to obtain small granular cell lysate, which is the tumor whole cell antigen prepared from fresh tumor tissue.

[0076] Preparation of bacterial vector-based personalized tumor vaccines:

[0077] 40 μg of the above whole-cell tumor antigen (final concentration of 200 μg / mL) and 40 μg of β-glucan (final concentration of 200 μg / mL) and engineered positively charged bacteria (bacterial number of 3*10 8 After co-incubation of CFU)PEI@EcN for 30 minutes, the engineered positively charged bacteria are loaded with β-glucan, and the surface of the engineered positively charged bacteria adsorbs tumor whole-cell antigens through electrostatic adsorption, thus obtaining a personalized tumor vaccine.

[0078] At the same time, a model vaccine was prepared by mixing 40ug model antigen (OVA) with 40ug β-glucan and engineered positively charged bacteria PEI@EcN (the number of bacteria was 3*10 8 After incubation with 544 CFU (400 μg / mL) for 30 min, the engineered positively charged bacteria were loaded with β-glucan, and the surface of the engineered positively charged bacteria adhered to the model antigen through electrostatic adsorption, thus obtaining a tumor model vaccine (BG / OVA@EcN).

[0079] Example 2

[0080] In order to detect whether the antigen is successfully attached to the bacteria, this example uses a fluorescently labeled model antigen FITC-OVA, which is co-incubated with PEI@EcN in a 37°C constant temperature shaker for 30 minutes, and then washed 3 times with PBS and centrifuged to obtain a precipitate. After the precipitate is resuspended with PBS, it is observed under a confocal microscope whether the bacteria are successfully loaded with the antigen. Control group 1 is EcN bacteria, control group 2 is BG@EcN (BG@EcN is prepared by engineering positively charged bacteria PEI@EcN loaded with β-glucan prepared in Example 1), and control group 3 is OVA@EcN (OVA@EcN is prepared by engineering positively charged bacteria PEI@EcN loaded with the model antigen OVA prepared in Example 1). The experimental group is the tumor model vaccine (BG / OVA@EcN) prepared in Example 1. Figure 2 Content A shows that fluorescently labeled bacteria (Dil-EcN, i.e., PEI@EcN labeled with the fluorescent dye Dil) and fluorescently labeled OVA antigen (FITC-OVA) successfully co-localize, indicating that the antigen is successfully attached to the bacteria. Figure 2 Content B shows the potential of vaccines with glucan and antigen attached. Control group 1 was EcN, control group 2 was BG@EcN, control group 3 was OVA@EcN, and the experimental group was BG / OVA@EcN. Figure 2 Content C Electron microscopy legend shows that the live EcN bacteria in the control group 1 have a rod-shaped structure with obvious pili. The surface of the bacteria in the experimental group is rougher after being coated with antigens, and is covered with a layer of dark substance.

[0081] The same method was used to verify whether breast cancer tumor whole cell antigen (abbreviated as Ag-4T1) was successfully attached to the bacteria. Figure 3 Figure A shows the successful colocalization of fluorescently labeled bacteria (DIO-EcN, i.e., PEI@EcN labeled with the fluorescent dye DIO) and fluorescently labeled Ag-4T1 antigen (PKH-26-Ag), indicating successful antigen attachment to the bacteria. Figure B demonstrates the potential of vaccines with attached dextran and antigen. Control group 1 was EcN, control group 2 was BG@EcN, control group 3 was Ag-4T1@EcN, and the experimental group was BG / Ag-4T1@EcN.

[0082] Example 3

[0083] Vaccine immune activation effect in vivo

[0084] In this example, OVA was used as a model antigen to measure the residence time of BG / OVA-Cy5@EcN at the injection site. Twenty-four hours after injection, cells from the injection site and lymph nodes were collected for flow cytometry analysis. Control group 1 consisted of OVA-Cy5, prepared by mixing and stirring the OVA antigen and Cy5-NHS for 24 hours; control group 2 consisted of a mixture of free BG and OVA-Cy5; and control group 3 consisted of OVA-Cy5@EcN, prepared by attaching PEI@EcN to the OVA-Cy5 antigen. The experimental group received the tumor model vaccine (BG / OVA@EcN) prepared in Example 1.

[0085] Injection site and lymph node processing procedures: The excised subcutaneous injection site was digested with type I collagenase and incubated for 30 minutes. After digestion, the cell suspension was collected by passing through a 200-mesh cell sieve, pressing with a syringe plunger, and rinsing with RPMI1640 medium. After filtering twice through a 200-mesh nylon mesh, the cell suspension was centrifuged at 1500 rpm for 5 minutes at 4°C. Freshly excised tumor LNs were washed with saline and placed in a 24-well plate. The cell suspension was collected by pressing with a syringe plunger and rinsing with PBS. The cell suspension was filtered twice through a 200-mesh nylon mesh, centrifuged at 2000 rpm for 5 minutes at 4°C, washed once with PBS, and resuspended.

[0086] The experimental results show that when bacteria are loaded with antigens, the residence time of antigens at the injection site can be significantly prolonged compared with free antigens ( Figure 4 Content (A) and Content (B)). Figure 4 Figures (C) and (D) demonstrate increased antigen uptake by macrophages and dendritic cells at the injection site, and a significant increase in the number of antigen-positive DCs within the lymph nodes. In summary, the experimental BG / OVA@EcN formulation prolonged antigen retention at the injection site and was efficiently taken up by innate immune cells, which then carried the antigen into the lymph nodes for presentation.

[0087] To evaluate whether the vaccine activates DCs and elicits a specific cellular immune response, 6-7-week-old C57BL / 6J mice were subcutaneously vaccinated. On the third day, the injection site, lymph nodes, and blood were removed from the mice to assess immune cell activation. Heparinized blood was collected and centrifuged at 2500 rpm for 5 minutes to obtain a cell pellet. The pellet was lysed three times with red blood cell lysis buffer and washed three times with PBS to obtain the desired cells. The resulting cells were stained with fluorescent antibodies and analyzed by flow cytometry. Figure 5 、 Figure 6 、 Figure 7 and Figure 8Control group 1 was the PBS group, control group 2 was the free β-glucan group, control group 3 was the free OVA antigen group, control group 4 was the simple mixture group of free β-glucan and free OVA antigen, and control group 5 was the EcN group; control group 6 was the BG@EcN group, which was obtained by PEI@EcN loading BG; control group 7 was the OVA@EcN group, which was obtained by PEI@EcN loading OVA; the experimental group was the BG / OVA@EcN group prepared in Example 1, which was obtained by PEI@EcN loading BG and OVA.

[0088] The results showed that BG / OVA@EcN significantly enhanced the recruitment of DCs and monocytes / macrophages at the inoculation site. Figure 5 Content (A), Content (B) and Content (C)) and activate DC cells and macrophages to mature. Mature DC cells complete antigen presentation and activate CD8 + T and CD4 + T cells, promote the proliferation and activation of T cells ( Figure 7 Content (A), Content (B)). The results of immune cell analysis in the blood showed that after vaccine administration, the number of monocytes / macrophages in the blood increased significantly compared with the blank mice or free antigen group ( Figure 6 Content (C), content (D)), the number of migratory monocytes / macrophages also increased significantly ( Figure 6 Content (A), Content (B)). The results of immune cell analysis in lymph nodes are shown in Figure 7 Content (A), Content (B). These results demonstrate that the BG / OVA@EcN vaccine is more effective at inducing specific T cell responses than free antigen. Furthermore, after LPS restimulation of blood cells in vitro, the proportion of cells expressing the inflammatory cytokine TNFa significantly increased compared to the blank control group, demonstrating the vaccine's ability to train monocytes and macrophages, leading to higher secretion of inflammatory factors after a second antigen restimulation. This is crucial for generating specific anti-tumor immune responses in the body and improving the tumor immune microenvironment.

[0089] In the preventive tumor model, mice were first immunized with the vaccine and then inoculated with tumors. The tumor inhibition effect showed that the vaccine could significantly inhibit tumor growth and prolong the survival of mice ( Figure 8 Content (A), content (B)), indicating that the specific immune response generated by vaccination has a protective effect against tumor cell inoculation.

[0090] Example 4

[0091] In order to evaluate the therapeutic effect of the vaccine on the tumor model, a subcutaneous melanoma model with high expression of OVA antigen was first constructed. The vaccine was then administered on the 6th, 9th, 12th, and 17th days, and the tumor size and survival period were observed and tested.

[0092] Construction of subcutaneous melanoma model: breast cancer B16-OVA cells in the logarithmic growth phase were collected, washed once with PBS and resuspended, and diluted to 5×10 6 mL -1 The back of 18-20 g female C57BL / 6J mice was shaved, and the cell suspension was drawn up using a disposable sterile insulin syringe. 100 μL of tumor cell suspension (approximately 5×10 5 Tumor cells per mouse) were used to establish a B16-OVA subcutaneous tumor model. Figures 5 to 7 The experimental results are as follows. Figure 9 As shown in content (A) and content (B), after the last vaccination, i.e., grafting of the experimental group vaccine, the tumor volume of the mice was significantly suppressed and the survival time of the mice was significantly prolonged.

[0093] On the 20th day, the blood, tumor tissue, lymph node tissue, and spleen tissue of the mice were collected and the components of the immune cells were detected. It was found that CD3 + T cells, CD8 + T cells, CD4 + The number of T cells increased significantly, and their activated IFNγ + T, CD69 + The number of T cells also increased significantly, indicating that after vaccination of the experimental group, the cytotoxic T cells infiltrating the tumor tissue had a significantly improved killing effect on the tumor compared with the blank group. Figure 9 Content (C), content (D), content (E) and content (F). And the proportion of M1 macrophages and MHCII in tumor tissue + The number of DC cells increased significantly, which may be due to the increase in the proportion of circulating monocytes in the peripheral blood.

[0094] The results showed that the number of monocytes / macrophages in the trained mice increased, and after infiltrating into the tumor tissue, they were more likely to transform into pro-inflammatory M1 macrophages and MoDCs with antigen-presenting ability, thereby recruiting more cytotoxic T cells to infiltrate the tumor and play a tumor-suppressing role.

[0095] Example 5

[0096] Tumor tissues of clinical cancer patients harbor numerous neoantigens, formed by genetic mutations and absent in normal tissues. This makes it difficult for tumor vaccines that deliver tumor-associated antigens to achieve effective anti-tumor immunotherapy for cancer patients. This necessitates the development of novel personalized tumor vaccines to address the urgent need for postoperative tumor treatment. Therefore, we used PEI@EcN carriers to electrostatically adsorb personalized whole-cell breast cancer tumor antigens to test the potential of personalized vaccines using this bacterial carrier.

[0097] The experimental method is the same as in Example 1. After the in situ 4T1 tumor model is established, the tumor tissue grows to 300 mm. 3 After about 10 days, 95% of the tumor tissue volume was surgically removed and the surgical site was sutured. The fresh tumor tissue was used to prepare tumor whole cell antigen (abbreviated as Ag-4T1). Then 40 μg of the above tumor whole cell antigen was mixed with 40 μg of β-glucan and engineered positively charged bacteria (the number of bacteria was 3*10 8 After incubation with PEI@EcN (CFU) for 30 minutes, the engineered positively charged bacteria were loaded with β-glucan, and the engineered positively charged bacteria surface electrostatically adsorbed tumor antigens, resulting in the personalized tumor vaccine BG / Ag-4T1@EcN. After vaccination with each vaccine component on days 3, 6, 9, and 14, the size of recurrent tumors in mice was measured. Figure 10 、 Figure 11 and Figure 12 Control group 1, control group 2, control group 3, control group 4, control group 5, control group 6, control group 7, and experimental group correspond to PBS, BG, Ag-4T1, BG+Ag-4T1, EcN, BG@EcN, Ag-4T1@EcN, and BG / Ag-4T1@EcN, respectively.

[0098] The results showed that the BG / Ag-4T1@EcN group had a significant tumor inhibition effect and significantly prolonged the survival of mice ( Figure 10 Content (A) and content (B)). In the blood of the three surviving mice, the number of monocytes / macrophages increased significantly ( Figure 11 Content (A) and content (B)), and after LPS restimulation in vitro, the proportion of cells secreting TNFa was significantly increased compared with that of blank mice ( Figure 11 Content (C) and content (D)). The proportion of memory T cells in the blood has increased significantly ( Figure 12 Content (A) and content (B)), the secretion of IFNγ by T cells after antigen restimulation in vitro and the proportion of activated specific T cells also increased significantly ( Figure 12Content (C) and Content (D)). This suggests that after vaccine training, the mononuclear macrophages in the mouse blood have a stronger ability to secrete inflammatory factors. The vaccine also induces a specific immune response in the mice, and after infiltrating into tumor tissue, they have more killer T cells, exerting a tumor-suppressing effect. Figure 11 and Figure 12 The control group consisted of healthy mice, and the experimental group consisted of mice that survived the BG / Ag-4T1@EcN in the above experimental group.

[0099] Example 6

[0100] Similarly, we engineered bacteria with chitosan to create the positively charged Chitosan@MG1655 bacteria. We electrostatically adsorbed personalized antigens derived from whole-cell liver cancer tissue onto the Chitosan@MG1655 carrier to test whether this bacterial-based vaccine could effectively inhibit liver cancer recurrence.

[0101] The experimental method is the same as in Example 1. After the subcutaneous liver cancer tumor model is established, the tumor tissue grows to 300 mm. 3 After about 10 min, 95% of the tumor tissue volume was surgically removed. After suturing the surgical site, the fresh tumor tissue was used to prepare tumor whole cell antigen (abbreviated as Ag-H22). Then 40 μg of the above tumor whole cell antigen (final concentration of 200 μg / mL) was mixed with 40 μg of muramyl dipeptide (abbreviated as MDP, final concentration of 200 μg / mL) and engineered positively charged bacteria (bacterial number of 3*10 8 After incubation with Chitosan@MG1655 (CFU) for 30 minutes, the engineered positively charged bacteria were loaded with muramyl dipeptide. Tumor antigens were electrostatically adsorbed onto the engineered positively charged bacterial surface, resulting in the personalized tumor vaccine MDP / Ag-H22@MG1655. The size of recurrent tumors in mice was measured after vaccination with each vaccine component on days 3, 6, 9, and 14. Figure 13 、 Figure 14 and Figure 15 Control group 1, control group 2, control group 3, control group 4, control group 5, control group 6, control group 7 and experimental group correspond to PBS, MDP, Ag-H22, MDP+Ag-H22, MG1655, MDP@MG1655, Ag-H22@MG1655, and MDP / Ag-H22@MG1655, respectively.

[0102] The results showed that the MDP / Ag-H22@MG1655 group had a significant tumor inhibition effect and significantly prolonged the survival of mice ( Figure 13Content (A) and content (B)). In the blood of the four surviving mice, the number of monocytes / macrophages increased significantly ( Figure 14 Content (A) and content (B)), and after LPS restimulation in vitro, the proportion of cells secreting TNFa was significantly increased compared with that of blank mice ( Figure 14 Content (C) and content (D)). The proportion of memory T cells in the blood has increased significantly ( Figure 15 Content (A) and Content (B)). This suggests that after vaccine training, the mononuclear macrophages in the mouse blood have a stronger ability to secrete inflammatory factors. The vaccine also induces a specific immune response in the mice, and after infiltrating into tumor tissue, they have more killer T cells, exerting a tumor-suppressing effect. Figure 14 and Figure 15 The control group consisted of healthy mice, and the experimental group consisted of mice that survived the MDP / Ag-H22@MG1655 infection in the above experimental group.

[0103] Example 7

[0104] Similarly, we used the PEI@MG1655 carrier to adsorb personalized antigens derived from whole cells of colon cancer tumor tissue through electrostatic adsorption to verify whether this bacterial-based vaccine has the effect of inhibiting the recurrence of colon cancer.

[0105] The experimental method is the same as in Example 1. After the subcutaneous colon cancer tumor model is established, the tumor tissue grows to 300 mm. 3 After about 10 min, 95% of the tumor tissue volume was surgically removed. After suturing the surgical site, the fresh tumor tissue was used to prepare tumor whole cell antigen (abbreviated as Ag-MC38). Then 40 μg of the above tumor whole cell antigen was mixed with 40 μg of muramyl dipeptide (abbreviated as MDP) and engineered positively charged bacteria (the number of bacteria was 3*10 8 After incubation with PEI@MG1655 (CFU) for 30 minutes, the engineered positively charged bacteria were loaded with muramyl dipeptide, and the engineered positively charged bacterial surface electrostatically adsorbed tumor antigens, resulting in the personalized tumor vaccine MDP / Ag-MC38@MG1655. The size of recurrent tumors in mice was measured after vaccination with each vaccine component on days 3, 6, 9, and 14. Figure 16 、 Figure 17 and Figure 18 Control group 1, control group 2, control group 3, control group 4, control group 5, control group 6, control group 7 and experimental group correspond to PBS, MDP, Ag-MC38, MDP+Ag-MC38, MG1655, MDP@MG1655, Ag-MC38@MG1655 and MDP / Ag-MC38@MG1655, respectively.

[0106] The results show that the MDP / Ag-MC38@MG1655 group has obvious tumor inhibition effect and prolongs the survival of mice Figure 16 The number of mononuclear macrophages in the blood of the 3 surviving mice is obviously increased Figure 17 The proportion of cells secreting TNFa after LPS restimulation in vitro is also obviously increased Figure 17 The proportion of memory T cells in the blood is obviously increased Figure 18 It is indicated that the mononuclear macrophages in the blood of the mice have stronger ability to secrete inflammatory factors after vaccine training, and the vaccine causes specific immune response in the mice, and more killer T cells are produced after infiltration into the tumor tissue, thereby playing a tumor inhibition effect. Figure 17 and Figure 18 The control group in the above-mentioned experiment is the healthy mice, and the experimental group is the mice surviving in the above-mentioned experimental group MDP / Ag-MC38@MG1655.

[0107] Those skilled in the art will easily understand that the above-mentioned is only the preferred embodiment of the present application, and is not used to limit the present application, and any modification, equivalent replacement and improvement within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A personalized tumor vaccine using bacteria as a carrier, characterized in that: The invention comprises engineered positively charged bacteria and a trained immune activator loaded thereon, and also comprises a tumor antigen adhered to the surface of the engineered positively charged bacteria by electrostatic adsorption; the trained immune activator is a substance capable of activating trained immunity; The engineered positively charged bacteria are positively charged bacteria obtained by inactivating and reversing the charge of the bacteria; The training immune activator is one or more of β-glucan, muramyl dipeptide, and muramyl tripeptide.

2. The personalized tumor vaccine according to claim 1, characterized in that The tumor antigen is a tumor patterned antigen or a tumor personalized antigen; wherein: The tumor patterning antigen is chicken ovalbumin, alpha-fetoprotein, carcinoembryonic antigen or squamous cell carcinoma antigen; The tumor personalized antigen is a tumor antigen derived from tumor tissue of colorectal cancer, melanoma, breast cancer, lung cancer, ovarian cancer, choriocarcinoma, cervical cancer, liver cancer, bladder cancer or skin cancer.

3. The personalized tumor vaccine according to claim 1, wherein The final concentration of the engineered positively charged bacteria in the tumor personalized vaccine is (1.5*10 8 ~2*10 9 ) CFU / mL, the final concentration of the tumor antigen in the personalized tumor vaccine is 100-500 μg / mL, and the final concentration of the trained immune activator in the personalized tumor vaccine is 100-500 μg / mL.

4. A method for preparing a personalized tumor vaccine according to any one of claims 1 to 3, characterized in that: The steps include: S1: performing charge reversal on the bacteria to obtain the engineered positively charged bacteria; S2: digesting and lysing the tumor tissue, collecting cell lysates, and obtaining the tumor antigen; S3: The trained immune activator is mixed with the engineered positively charged bacteria and the tumor antigen and incubated so that the engineered positively charged bacteria are loaded with the trained immune activator and the surface of the engineered positively charged bacteria adsorbs the tumor antigen by electrostatic adsorption, thereby obtaining a personalized tumor vaccine.

5. The method for preparing a personalized tumor vaccine according to claim 4, wherein: Step S1: mixing bacteria with an amino compound, a carboxyl activator, and an amino acid protective agent, followed by shaking and incubation to achieve charge reversal of the bacteria; wherein the amino compound is PEI, chitosan, or polylysine, the carboxyl activator is EDC, and the amino acid protective agent is NHS.

6. The method for preparing a personalized tumor vaccine according to claim 4, wherein: In step S2, the digestion treatment is: adding RPMI 1640 culture medium containing type I collagenase to the tumor tissue and incubating.

7. The method for preparing a personalized tumor vaccine according to claim 6, wherein: The incubation temperature in step S2 is 25-37° C., and the incubation time is 10-120 min; The incubation temperature in step S3 is 25-37° C., and the incubation time is 10-120 min.

8. A drug for treating tumors, characterized in that: The invention comprises the personalized tumor vaccine using bacteria as a carrier as claimed in any one of claims 1 to 3.

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