Tumor composite vaccine for treating tumors by combining PD-1 with pyrimidine antimetabolic drugs and preparation method of tumor composite vaccine
By mixing lyophilization of αPD-1, cytarabine and inactivated bacterial solution, a tumor complex vaccine with PD-1 combined with pyrimidine anti-metabolic drugs was prepared, which solved the shortcomings of tumor vaccines in activate immune response and overcome the immunosuppressive microenvironment, significantly inhibited tumor growth and improved anti-tumor effect.
Patent Information
- Application Number
- CN202411868758.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-18
- Publication Date
- 2025-03-25
AI Technical Summary
Tumor vaccines face issues such as immune tolerance, antigen heterogeneity, immunosuppressive microenvironment, clinical trial complexity, market acceptance and economic burden, impact on the immune system, and regulatory challenges when treating tumors, resulting in limited efficacy and application.
A tumor complex vaccine for treating tumors with PD-1 combined with pyrimidine anti-metabolic drug cytarabine that inhibits cell synthesis and αPD-1 that relieves the immunosuppressive effect and activates T cell function was prepared.
This tumor complex vaccine significantly inhibits tumor growth, improves the anti-tumor effect, and solves the shortcomings of traditional tumor vaccines in activate immune response and overcomes the immunosuppressive microenvironment.
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Figure CN119679929A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a tumor composite vaccine for treating tumors by combining PD-1 with pyrimidine anti-metabolite drugs and a preparation method thereof. Background Art
[0002] Tumor vaccines are therapeutic vaccines that activate the body's immune system to attack tumor cells. Unlike traditional vaccines that prevent infectious diseases, tumor vaccines are mainly used to treat existing cancers. The basic principle of tumor vaccines is to use tumor cell-specific antigens to stimulate the patient's immune response, thereby identifying and destroying cancer cells. Although the research on tumor vaccines started relatively late, with the deepening of understanding of the immune system, especially the rapid development of tumor immunology, the types and applications of tumor vaccines have continued to increase. At present, tumor vaccines are mainly divided into two categories: preventive vaccines and therapeutic vaccines. Preventive vaccines such as HPV vaccines and hepatitis B vaccines are mainly used to reduce the occurrence of certain types of cancer; while therapeutic vaccines target existing tumors and aim to improve patients' survival and quality of life.
[0003] In recent years, the clinical application of tumor vaccines has gradually increased. Taking Sipuleucel-T as an example, this vaccine has been approved by the FDA for the treatment of prostate cancer. Other vaccines under study have also shown certain efficacy, such as vaccines for melanoma, lung cancer and breast cancer. In general, the clinical application of tumor vaccines is mainly concentrated in the following aspects: (1) Monotherapy: In some cases, tumor vaccines can be used as the only treatment, especially in patients with early tumors; (2) Combination therapy: Tumor vaccines are often used in combination with other treatments (such as chemotherapy, radiotherapy, immune checkpoint inhibitors, etc.) to improve efficacy; (3) Personalized treatment: By analyzing the patient's tumor characteristics, personalized tumor vaccines are developed to improve the treatment effect. The advantages of tumor vaccines are: (1) Strong specificity: Tumor vaccines can target specific tumor antigens and reduce damage to normal cells; (2) Persistence: Once activated, the immune system can remember tumor antigens and provide long-term protection; (3) Relatively small side effects: Compared with traditional chemotherapy, tumor vaccines usually have fewer side effects and patients have a relatively higher quality of life.
[0004] Although tumor vaccines show good prospects in cancer treatment, their development and application still face some significant defects and challenges: (1) Immune tolerance: Tumor cells usually evade the attack of the immune system through various mechanisms, such as expressing immunosuppressive factors and downregulating major histocompatibility complex (MHC) molecules, which may result in the vaccine being unable to effectively activate the immune response; (2) Antigenic heterogeneity: The antigens within tumor cells are highly heterogeneous, that is, different patients with the same cancer or different tumor cells of the same patient may express different antigens. This diversity makes it difficult to develop a universally effective tumor vaccine for all patients. In addition, the antigen expression of tumor cells may also change as the disease progresses, thus affecting the effectiveness of the vaccine; (3) Immunosuppressive microenvironment: The tumor microenvironment is usually immunosuppressive, and tumor cells can suppress the response of the immune system by producing inhibitory cytokines and recruiting immunosuppressive cells (such as regulatory T cells and myeloid-derived suppressor cells). This means that even if the tumor vaccine can activate the immune system, the final effect may still be limited; (4) Complexity of clinical trials: The clinical trials of tumor vaccines are complex in design and usually take a long time to observe the efficacy. Due to the heterogeneity of cancer and individual differences among patients, it is more difficult to evaluate the effectiveness and safety of vaccines. In addition, different doses, routes of administration and combination treatments of vaccines will affect the results; (5) Market acceptance and economic burden: Although tumor vaccines show good potential, the number of tumor vaccines currently available on the market is limited and the prices are generally high. This results in some patients being unable to obtain the treatment they need, limiting the widespread application of tumor vaccines; (6) Impact on the immune system: The success of tumor vaccines depends on a healthy immune system, and some patients have impaired immune function due to age, underlying diseases or other treatments (such as chemotherapy, radiotherapy), which may reduce the effectiveness of the vaccine; (7) Regulatory challenges: The development and approval process of tumor vaccines is relatively complex and involves reviews by multiple regulatory agencies. Developers need to provide sufficient clinical data to demonstrate the safety and effectiveness of the vaccine, which is a huge challenge in terms of time and resources.
[0005] Combination therapy is used in the treatment of various diseases, but it may weaken the efficacy, become ineffective or even cause serious adverse reactions due to the interaction between different drugs. Therefore, designing a safe and effective tumor compound vaccine based on the concept of combination therapy is of great significance for expanding the application direction of tumor vaccines. Summary of the invention
[0006] In view of the deficiencies of the prior art, the present invention forms a tumor composite vaccine by mixing and lyophilizing an inactivated bacterial solution, a pyrimidine anti-metabolite drug cytarabine that inhibits cell synthesis, and αPD-1 that relieves the immunosuppressive effect and activates T cell function, thereby solving the technical problems raised in the background technology. Specifically, the technical solution of the present invention includes the following contents:
[0007] One of the purposes of the present invention is to provide a method for preparing a tumor composite vaccine for treating tumors by combining PD-1 with pyrimidine anti-metabolites, the preparation method comprising the following steps:
[0008] Soy lecithin, DSPE-PEG2000, cholesterol, αPD-1, inactivated bacteria solution and solvent are mixed and dissolved in a mass ratio of 100:1:50::2.6:1300:5000, and evaporated under reduced pressure to obtain a mixed solution containing a film;
[0009] The mixed solution containing the film is mixed with a pyrimidine antimetabolite drug, and subjected to ultrasound and extrusion in sequence to obtain a pre-treated solution;
[0010] After the pretreatment solution is dialyzed, deionized water is added to make the volume 10 mL, and then a freeze-drying protective agent is added and mixed, and the tumor composite vaccine is obtained by freeze-drying.
[0011] Furthermore, the inactivated bacterial solution has a concentration of 5×10 8 cfu / ml of Staphylococcus aureus solution and the concentration of inactivated solution was 5×10 8 The cfu / ml of Bifidobacterium longum bacterial liquid were mixed in a volume ratio of 2:1.
[0012] Furthermore, the strain number of the Staphylococcus aureus is CICC 21600, and the strain number of the Bifidobacterium longum is CICC 6190.
[0013] Furthermore, the concentration of the inactivated bacterial solution is 5×10 8 The method for preparing a Staphylococcus aureus bacterial solution of cfu / ml comprises the following steps:
[0014] Staphylococcus aureus was inoculated into a sterile broth medium and cultured at 37°C for 24 h to obtain a Staphylococcus aureus bacterial solution. Formaldehyde was added to the Staphylococcus aureus bacterial solution according to a mass ratio of Staphylococcus aureus bacterial solution to formaldehyde of 1:0.003, and the solution was shaken and inactivated in a 37°C water bath environment for 24 h. After the inactivation, the concentration of the bacterial solution was adjusted to 5×10 8 cfu / ml to obtain the inactivated bacterial solution concentration of 5×10 8 cfu / ml of Staphylococcus aureus solution.
[0015] Furthermore, the concentration of the inactivated bacterial solution is 5×10 8 The method for preparing a Bifidobacterium longum bacterial solution of cfu / ml comprises the following steps:
[0016] Bifidobacterium longum was inoculated into a sterile broth medium and cultured at 37°C for 24 h to obtain a Bifidobacterium longum bacterial solution. Formaldehyde was added to the Bifidobacterium longum bacterial solution according to a mass ratio of Bifidobacterium longum bacterial solution to formaldehyde of 1:0.003 and the solution was inactivated by shaking in a 37°C water bath for 24 h. After the inactivation, the concentration of the bacterial solution was adjusted to 5×10 8 cfu / ml to obtain the inactivated bacterial solution concentration of 5×10 8 cfu / ml of Bifidobacterium longum bacterial solution.
[0017] Furthermore, the solvent is chloroform.
[0018] Furthermore, the pyrimidine antimetabolite drug is a cytarabine solution with a mass concentration of 20 mg / ml.
[0019] Furthermore, the volume ratio of the mixed solution containing the film to the pyrimidine antimetabolite drug is 1:1.
[0020] Furthermore, the ultrasonic conditions include ultrasonic power of 300W, ultrasonic temperature of 30°C and ultrasonic time of 120s.
[0021] Furthermore, the filter membrane used for extrusion is a polycarbonate membrane with a pore size of 100 nm.
[0022] Furthermore, the filter membrane used in the dialysis is a polycarbonate membrane with a pore size of 30 nm.
[0023] Furthermore, the lyophilization protective agent is a sucrose solution with a mass concentration of 5%, and the amount of the lyophilization protective agent added is 0.5 g per 10 ml.
[0024] The second purpose of the present invention is to provide a tumor composite vaccine prepared by a method for preparing a tumor composite vaccine for treating tumors by combining PD-1 with pyrimidine anti-metabolite drugs.
[0025] Compared with the prior art, the present invention has the following beneficial effects:
[0026] The present invention creatively mixes an inactivated bacterial solution composed of an inactivated Staphylococcus aureus solution and an inactivated Bifidobacterium longum solution, αPD-1 and a pyrimidine anti-metabolite drug cytarabine that inhibits cell synthesis, and the prepared tumor composite vaccine has a good anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 This is a statistical diagram of the effectiveness experiment of the tumor composite vaccine solution prepared by the present invention, wherein: Figure 1 A in the figure is a schematic diagram of mouse model treatment; Figure 1 B in the figure is the macroscopic image of mouse tumors in the control group and the treatment group; Figure 1 C in the figure is the tumor weight of mice in the control group and the treatment group. The tumor weight of the treatment group is significantly smaller than that of the control group. Figure 1 D in the figure is the tumor volume growth curve of the two groups, and the treatment group grew slowly. DETAILED DESCRIPTION
[0028] The technical solution of the present invention will be clearly and completely described below through the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.
[0029] Unless otherwise specified, the raw materials and reagents used in the present invention are commercially available or can be prepared by known methods.
[0030] Preparation Example 1:
[0031] The preparation of the inactivated Staphylococcus aureus liquid specifically includes the following processes:
[0032] Staphylococcus aureus with strain number CICC 21600 was inoculated on sterile broth medium in a sterile room with a sterile loop, and then placed in an incubator at 37°C for 24 hours to obtain Staphylococcus aureus bacterial solution. According to the mass ratio of Staphylococcus aureus bacterial solution to formaldehyde of 1:0.003, formaldehyde was added to the Staphylococcus aureus bacterial solution and mixed evenly, and then placed in a 37°C water bath environment for 24 hours of shaking inactivation. After the inactivation, the concentration of the bacterial solution was adjusted to 5×10 8 cfu / ml to obtain a concentration of 5×10 8 cfu / ml of Staphylococcus aureus solution.
[0033] Preparation Example 2:
[0034] The preparation of the inactivated Bifidobacterium longum bacterial solution specifically includes the following steps:
[0035] Bifidobacterium longum with strain number CICC 6190 was inoculated on a sterile broth medium in a sterile room with a sterile loop, and then placed in an incubator at 37°C for 24 hours to obtain a Bifidobacterium longum bacterial solution. According to the mass ratio of Bifidobacterium longum bacterial solution to formaldehyde of 1:0.003, formaldehyde was added to the Bifidobacterium longum bacterial solution and mixed evenly, and then placed in a 37°C water bath environment for 24 hours of shaking inactivation. After the inactivation, the concentration of the bacterial solution was adjusted to 5×10 8 cfu / ml to obtain a concentration of 5×108 cfu / ml of Bifidobacterium longum bacterial solution.
[0036] Preparation Example 3:
[0037] The preparation of the inactivated bacterial solution specifically includes the following processes:
[0038] The concentration of the inactivated bacterial solution obtained in Preparation Example 1 was 5×10 8 cfu / ml of Staphylococcus aureus solution and the inactivated solution obtained in Preparation Example 2 had a concentration of 5×10 8 cfu / ml of Bifidobacterium longum bacterial solution were mixed evenly in a volume ratio of 2:1 to obtain an inactivated bacterial solution.
[0039] Embodiment 1:
[0040] The preparation of a tumor composite vaccine for treating tumors by combining PD-1 with pyrimidine anti-metabolites specifically comprises the following steps:
[0041] Take 100mg soybean lecithin, 1mg DSPE-PEG2000, 50mg cholesterol, 2.6mg αPD-1, 1300mg inactivated bacteria solution obtained in Preparation Example 3 and 5000mg chloroform, mix and dissolve, then evaporate under reduced pressure in a sample bottle until a thin film is formed on the surface to obtain a mixed solution; add an equal volume of 20mg / ml cytarabine solution to the mixed solution, mix, and then place it in an ultrasonic power of 300W and an ultrasonic temperature environment of 30°C for 120s. After the ultrasonic treatment, put it into a liposome extruder and extrude it through a polycarbonate membrane with a pore size of 100nm to obtain a pretreatment solution. After the pretreatment solution is dialyzed with a nanodialysis device containing a pore size of 30nm and a filter membrane made of polycarbonate, add deionized water to make the volume 10mL, then add 0.5mg of 5% sucrose solution and mix, and freeze-dry to obtain a vaccine composition.
[0042] Preparation Example 1:
[0043] The process of preparing the tumor compound vaccine into an injectable tumor compound vaccine solution:
[0044] The tumor composite vaccine obtained in Example 1 and physiological saline were mixed in a mass ratio of 1:1 and shaken to obtain a suspension, and the suspension was dispersed by ultrasound for 30s to 120s to obtain a tumor composite vaccine solution, which was recorded as Neo-BCV;
[0045] Treatment is completed by subcutaneous injection of the tumor compound vaccine solution.
[0046] Verification Example 1:
[0047] Effectiveness experiment of tumor composite vaccine solution (Neo-BCV):
[0048] (1) Experimental process:
[0049] The tumor-bearing mice were randomly divided into a control group (injected with PBS) and a treatment group (injected with a tumor compound vaccine solution). On the 1st, 4th, 7th, and 10th days, PBS and tumor compound vaccines were injected subcutaneously in the left flank of the mice, respectively, with an injection dose of 0.4 mL / mouse. During this period, the tumor volume was measured with a vernier caliper every 3 days, and the survival status of the tumor-bearing mice, local and systemic injection reactions, and the treatment effect of the subcutaneous tumor-bearing model mice were observed and recorded. The results are shown in Figure 1 .
[0050] (2) Result analysis:
[0051] from Figure 1 It can be found that the tumor weight of mice in the treatment group was significantly smaller than that in the control group; the tumor growth of mice in the treatment group was significantly slower than that in the control group (P < 0.001), indicating that the tumor composite vaccine solution prepared by the present invention significantly inhibits tumor growth.
[0052] The above-described embodiments provide a detailed description of the technical solutions and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected.
Claims
1. A method for preparing a tumor composite vaccine for treating tumors by combining PD-1 with pyrimidine anti-metabolites, characterized in that: The preparation method comprises the following steps: Soy lecithin, DSPE-PEG2000, cholesterol, αPD-1, inactivated bacteria solution and solvent are mixed and dissolved in a mass ratio of 100:1:50::2.6:1300:5000, and evaporated under reduced pressure to obtain a mixed solution containing a film; The mixed solution containing the film is mixed with a pyrimidine antimetabolite drug, and subjected to ultrasound and extrusion in sequence to obtain a pre-treated solution; After the pretreatment solution is dialyzed, deionized water is added to make the volume 10 mL, and then a freeze-drying protective agent is added and mixed, and the tumor composite vaccine is obtained by freeze-drying.
2. The method for preparing a tumor composite vaccine for treating tumors by combining PD-1 with pyrimidine anti-metabolites according to claim 1, characterized in that: The inactivated bacterial solution has a concentration of 5×10 8 cfu / ml of Staphylococcus aureus solution and the concentration of inactivated solution was 5×10 8 The cfu / ml of Bifidobacterium longum bacterial liquid were mixed in a volume ratio of 2:
1.
3. The method for preparing a tumor composite vaccine for treating tumors by combining PD-1 with pyrimidine anti-metabolites according to claim 1, characterized in that: The solvent is chloroform.
4. The method for preparing a tumor composite vaccine for treating tumors by combining PD-1 with pyrimidine anti-metabolites according to claim 1, characterized in that: The pyrimidine antimetabolite drug is a cytarabine solution with a mass concentration of 20 mg / ml.
5. The method for preparing a tumor composite vaccine for treating tumors by combining PD-1 with pyrimidine anti-metabolites according to claim 1, characterized in that: The volume ratio of the mixed solution containing the film to the pyrimidine antimetabolite drug is 1:
1.
6. The method for preparing a tumor composite vaccine for treating tumors by combining PD-1 with pyrimidine anti-metabolites according to claim 1, characterized in that: The ultrasonic conditions include an ultrasonic power of 300 W, an ultrasonic temperature of 30° C., and an ultrasonic time of 120 s.
7. The method for preparing a tumor composite vaccine for treating tumors by combining PD-1 with pyrimidine anti-metabolites according to claim 1, characterized in that: The filter membrane used for the extrusion is a polycarbonate membrane with a pore size of 100 nm.
8. The method for preparing a tumor composite vaccine for treating tumors by combining PD-1 with pyrimidine anti-metabolites according to claim 1, characterized in that: The filter membrane used in the dialysis is a polycarbonate membrane with a pore size of 30 nm.
9. The method for preparing a tumor composite vaccine for treating tumors by combining PD-1 with pyrimidine anti-metabolites according to claim 1, characterized in that: The freeze-dried protective agent is a sucrose solution with a mass concentration of 5%, and the amount of the freeze-dried protective agent added is 0.5 g per 10 ml.
10. A tumor composite vaccine prepared by the method for preparing a tumor composite vaccine for treating tumors by combining PD-1 with pyrimidine anti-metabolites as described in any one of claims 1 to 9.