Vaccine composition for treating tumors by combining inactivated bacterial liquid with alphaPD-1 and preparation method of vaccine composition

Abstract

The invention discloses a vaccine composition for treating tumors by combining inactivated bacterial liquid with alphaPD-1 and a preparation method of the vaccine composition, and belongs to the technical field of biological medicines. The preparation method comprises the following steps: mixing and dissolving soybean lecithin, DSPE-PEG2000, cholesterol, alphaPD-1, an inactivated bacterial liquid and a solvent to obtain a mixed solution; sequentially carrying out ultrasonic treatment and extrusion on the mixed solution to obtain a pretreatment solution; after the pretreatment liquid is subjected to dialysis treatment, deionized water is added to fix the volume to 10 mL, then a freeze-drying protective additive is added for mixing, and the vaccine composition is obtained through freeze-drying. The vaccine composition with safety and an anti-tumor effect is obtained by mixing and freeze-drying inactivated bacterial liquid and alphaPD-1 which relieves an immunosuppressive effect and activates a T cell function.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a vaccine composition for treating tumors by combining an inactivated bacterial solution with αPD-1 and a preparation method thereof. Background Art

[0002] In recent years, with the improvement of people's living standards, changes in living habits and environmental pollution, the incidence of tumors has increased year by year. Malignant tumors have become one of the most important killers threatening human health and life. Under current technical conditions, although surgery can effectively remove tumors visible to the naked eye, it cannot guarantee the eradication of all tumor cells, nor can it change the internal environment of tumor occurrence. In addition to serious toxic side effects, many tumors are insensitive to or resistant to radiotherapy and chemotherapy. With existing methods, it is still difficult to ensure that malignant tumor cells are completely eliminated, so tumor recurrence is a core issue that threatens the patient's life. Therefore, it is urgent to find new adjuvant treatment methods. With the in-depth study of the molecular mechanisms of tumor occurrence and development and the rapid development of biotechnology, biological therapy has become the fourth mode of comprehensive tumor treatment and has received more and more attention. Tumor biotherapy is a new therapy for tumor prevention and treatment using modern biotechnology and its products. It achieves anti-tumor effects by mobilizing the host's natural defense mechanism or giving naturally produced highly targeted substances. It mainly includes somatic cell therapy and cytokine therapy, tumor vaccines, molecular targeted therapy, radioimmunotargeted therapy, tumor gene therapy and biochemotherapy. Tumor vaccines are an important part of tumor biotherapy and have developed rapidly in recent years. The establishment of transgenic cell lines has provided an effective way for the prevention and treatment of tumors.

[0003] Patent CN103446580A discloses a tumor vaccine and a preparation method thereof. The tumor vaccine includes cell vesicles derived from apoptotic tumor cells and an adjuvant. Specifically, the tumor cells are irradiated with ultraviolet light to cause apoptosis, and then the cell vesicles released by the apoptotic tumor cells are collected. The cell vesicles are mixed with the adjuvant to form a tumor vaccine, thereby achieving the immune targeting and tumor cell killing ability of the tumor vaccine.

[0004] Patent CN113679829A discloses a tumor vaccine for preventing postoperative recurrence of cancer and its preparation method and application. The tumor vaccine involved in the invention is a nanoparticle of a core material wrapped in a hybrid membrane, with the cell membrane in the solid tumor tissue from surgical resection in the hybrid membrane as the tumor antigen, and the Gram-negative bacterial inner membrane in the hybrid membrane as the immune adjuvant. The two can be delivered to the same dendritic cell together, and the uptake and presentation of tumor antigens can be utilized to synergistically enhance the body's innate immunity and specific immunity, and have a certain lymph node enrichment ability, with significant efficacy in preventing recurrence after tumor resection surgery, prolonging the patient's survival, and providing a long-term protection mechanism. The core material wrapped in the hybrid membrane of the tumor vaccine involved can help maintain the rigid structure and stability of the hybrid membrane, thereby maintaining the immune enhancement, lymph enrichment, and prevention of recurrence after tumor resection surgery of the hybrid membrane; at the same time, other bioactive components such as siRNA or chemotherapy drugs can be further encapsulated to achieve more functions.

[0005] Sindilimab binds to PD-1 and blocks the binding of PD-1 to PD-L1 and PD-L2, thereby relieving the immunosuppressive effect, activating T cell function, enhancing T cell immune surveillance and killing capabilities against tumors, and generating tumor immune responses. Inactivated bacterial vaccines can promote the body to produce a strong immune effect, thereby activating the body's immune system and further enhancing the body's immune system's ability to remove harmful substances.

[0006] There are few studies on the preparation of tumor composite vaccines using inactivated bacteria combined with anti-tumor drugs, mainly because factors such as the immune system status, underlying diseases and genetic background of different patients will affect the intensity and durability of the immune response, and will produce strong adverse reactions such as high fever and general discomfort.

[0007] Therefore, designing a safe vaccine composition is of great significance for further enhancing the safe anti-tumor effect of the vaccine composition. Summary of the invention

[0008] In view of the deficiencies of the prior art, the present invention forms a vaccine composition by mixing and lyophilizing an inactivated bacterial solution 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:

[0009] One of the purposes of the present invention is to provide a method for preparing a vaccine composition for treating tumors by combining an inactivated bacterial solution with αPD-1, the preparation method comprising the following steps:

[0010] Soy lecithin, DSPE-PEG2000, cholesterol, αPD-1, inactivated bacteria solution and solvent are mixed and dissolved, and a mixed solution containing a film is obtained by evaporation under reduced pressure;

[0011] The mixed solution containing the film is subjected to ultrasound and extrusion in sequence to obtain a pre-treated solution;

[0012] 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 vaccine composition is obtained by freeze-drying.

[0013] 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.

[0014] Furthermore, the strain number of the Staphylococcus aureus is CICC 21600, and the strain number of the Bifidobacterium longum is CICC 6190.

[0015] 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:

[0016] 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.

[0017] 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:

[0018] 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.

[0019] Furthermore, the solvent is chloroform.

[0020] Furthermore, the mass ratio of the soybean lecithin, DSPE-PEG2000, cholesterol, αPD-1, inactivated bacteria solution and solvent is 100:1:50::2.6:1300:5000.

[0021] Furthermore, the ultrasonic conditions include ultrasonic power of 300W, ultrasonic temperature of 30°C and ultrasonic time of 120s.

[0022] Furthermore, the filter membrane used for extrusion is a polycarbonate membrane with a pore size of 100 nm.

[0023] Furthermore, the filter membrane used in the dialysis is a polycarbonate membrane with a pore size of 30 nm.

[0024] Furthermore, the freeze-drying protective agent is a sucrose solution with a mass concentration of 5%.

[0025] Furthermore, the amount of the lyophilization protectant added is 0.5 g per 10 ml.

[0026] The second purpose of the present invention is to provide a vaccine composition prepared by a method for preparing a vaccine composition for treating tumors using a combination of inactivated bacterial solution and αPD-1.

[0027] The third purpose of the present invention is to provide a vaccine composition prepared by a method for preparing a vaccine composition for treating tumors by combining an inactivated bacterial solution with αPD-1, and its application in the field of tumors.

[0028] Furthermore, the application comprises the following steps:

[0029] The vaccine composition and physiological saline are mixed and shaken at a mass ratio of 1:1 to obtain a suspension, and the suspension is dispersed at an ultrasonic power of 100 W for 30 seconds to 120 seconds to obtain an αPD-1+Neo-BCV vaccine solution;

[0030] The αPD-1+Neo-BCV vaccine solution was injected subcutaneously to complete the treatment.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention creatively mixes an inactivated bacterial liquid consisting of an inactivated Staphylococcus aureus liquid and an inactivated Bifidobacterium longum liquid with αPD-1 to prepare a vaccine composition having anti-tumor effects and being safe for the human body, and through the synergistic cooperation of the inactivated bacterial liquid and αPD-1, the therapeutic effect of αPD-1 on tumor treatment is further improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a statistical chart of the safety test results of the αPD-1+Neo-BCV vaccine solution prepared by the present invention;

[0034] Figure 2 This is a statistical diagram of the therapeutic effect experiment of the αPD-1+Neo-BCV vaccine solution prepared by the present invention. Figure 2 A in the figure is a schematic diagram of mouse model treatment; Figure 2 B in the figure is the growth curve of tumor volume in each group of mice. The growth of the group treated with αPD-1+Neo-BCV vaccine solution was obviously slow. Figure 2 C in the figure is the macroscopic image of the tumor of mice in each group; Figure 2 D in the figure is the tumor weight of mice in each group. The tumor weight of the group treated with αPD-1+Neo-BCV vaccine solution was significantly reduced. Figure 2 E in the figure is the macroscopic image of spleen of mice in each group; Figure 2 F in the figure is the change graph of spleen index of mice in each group; Figure 2 G in the figure is the weight change curve of mice in each group. DETAILED DESCRIPTION

[0035] 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.

[0036] Unless otherwise specified, the raw materials and reagents used in the present invention are commercially available or can be prepared by known methods.

[0037] Preparation Example 1:

[0038] The preparation of the inactivated Staphylococcus aureus liquid specifically includes the following processes:

[0039] 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.

[0040] Preparation Example 2:

[0041] The preparation of the inactivated Bifidobacterium longum bacterial solution specifically includes the following steps:

[0042] 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 5×10 8 cfu / ml of Bifidobacterium longum bacterial solution.

[0043] Preparation Example 3:

[0044] The preparation of the inactivated bacterial solution specifically includes the following processes:

[0045] 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.

[0046] Embodiment 1:

[0047] The preparation of a vaccine composition for treating tumors by combining an inactivated bacterial solution with αPD-1 specifically comprises the following steps:

[0048] 100 mg of soybean lecithin, 1 mg of DSPE-PEG2000, 50 mg of cholesterol, 2.6 mg of αPD-1, 1300 mg of the inactivated bacteria solution obtained in Preparation Example 3 and 5000 mg of chloroform were mixed and dissolved, and then evaporated under reduced pressure in a sample bottle until a thin film was formed on the surface to obtain a mixed solution;

[0049] The mixed solution was ultrasonically treated for 120 seconds in an ultrasonic environment at an ultrasonic power of 300 W and an ultrasonic temperature of 30°C. After the ultrasonic treatment, it was placed in a liposome extruder and extruded through a polycarbonate membrane with a pore size of 100 nm to obtain a pre-treated solution. The pre-treated solution was dialyzed with a nanodialysis device containing a pore size of 30 nm and a filter membrane made of polycarbonate, and then deionized water was added to make the volume 10 mL, and then 0.5 mg of a sucrose solution with a mass concentration of 5% was added and mixed, and the vaccine composition was obtained by freeze-drying.

[0050] Preparation Example 1:

[0051] Process for preparing the vaccine composition into an injectable αPD-1+Neo-BCV vaccine solution:

[0052] The vaccine composition 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 at an ultrasonic power of 100 W for 30 seconds to 120 seconds to obtain an αPD-1+Neo-BCV vaccine solution;

[0053] Treatment was completed by subcutaneous injection of αPD-1+Neo-BCV vaccine solution.

[0054] Verification Example 1:

[0055] Safety experiment of αPD-1+Neo-BCV vaccine solution:

[0056] (1) Experimental process:

[0057] The tumor-bearing mice were randomly divided into a control group (without any intervention treatment), a low-dose group (each mouse was injected with 0.2 mL of αPD-1 + Neo-BCV vaccine solution), a normal treatment dose group (each mouse was injected with 0.4 mL of αPD-1 + Neo-BCV vaccine solution), a medium-high dose group (each mouse was injected with 0.6 mL of αPD-1 + Neo-BCV vaccine solution), and a high-dose group (each mouse was injected with 0.8 mL of αPD-1 + Neo-BCV vaccine solution), with 4 mice in each group. The αPD-1 + Neo-BCV vaccine solution was injected subcutaneously in the left armpit of mice in the low-dose group, the normal treatment dose group, the medium-high dose group, and the high-dose group, respectively. The administration was divided into four times, with an interval of 48 hours between each administration. The animals were sacrificed 48 hours after the last administration, and the mice were dissected to obtain whole blood, subcutaneous transplanted tumors, hearts, kidneys, and livers. The samples were fixed and embedded in paraffin according to conventional methods. The results are shown in Table 1. Figure 1 .

[0058] (2) Statistical analysis of results:

[0059] During the observation period, some mice administered the vaccine experienced hair loss, skin swelling and ulceration at the injection site (see Figure 1 -A);

[0060] The fur of mice in the control group, low-dose group, normal treatment dose group, and medium- and high-dose groups was smooth and well combed, while the fur of mice in the high-dose group was messy, listless, and less active (see Figure 1 -B, the left side of the figure shows mice in the high-dose group; the right side shows mice in the normal treatment dose group);

[0061] Compared with the control group, the nuclei of the heart, liver, and kidney cells of the mice in the normal treatment dose group were blue, the cytoplasm was pink, and the cell morphology and tissue structure remained intact, indicating that the αPD-1+Neo-BCV vaccine solution prepared by the present invention showed no obvious toxicity to the organs (see Figure 1 -C).

[0062] An independent sample t-test was performed to study the effects of αPD-1+Neo-BCV vaccine solution on the white blood cell count, lymphocyte count, monocyte count, neutrophil count, red blood cell count and hemoglobin in the whole blood of mice in different groups. The results are shown in Table 1.

[0063]

[0064]

[0065] As can be seen from Table 1, compared with the control group, there were no significant differences in various indicators in the normal treatment dose group (p>0.05), indicating that the αPD-1+Neo-BCV vaccine solution prepared by the present invention had no significant effect on the peripheral blood of mice and no obvious evidence of hematological toxicity.

[0066] Verification Example 2:

[0067] Therapeutic effect experiment of αPD-1+Neo-BCV vaccine solution:

[0068] (1) Experimental process:

[0069] A Lewis subcutaneous transplant tumor model was established, and tumor-bearing mice were randomly divided into a control group, a vaccine alone group (αPD-1 was not added to the αPD-1+Neo-BCV vaccine solution, denoted as Neo-BCV), an αPD-1 alone group, and an αPD-1+Neo-BCV vaccine solution group. Drug administration began on the 7th day after tumor inoculation.

[0070] Control group, vaccine alone group: Neo-BCV vaccine solution without αPD-1 was injected subcutaneously into the left armpit of mice at a dose of 0.4 mL / mouse, divided into four doses, with an interval of 48 hours between each dose;

[0071] αPD-1 monotherapy group: Sintilimab was intraperitoneally injected at a dose of 200 mg / kg, once a week, for a total of two injections;

[0072] αPD-1+Neo-BCV vaccine solution medication group: αPD-1+Neo-BCV vaccine solution was subcutaneously injected at a dose of 0.4 mL / head, with an injection interval of 48 hours, for a total of 4 injections.

[0073] Results Figure 2 .

[0074] (2) Result analysis:

[0075] Depend on Figure 2It can be seen that compared with the control group and the αPD-1 single-drug group, the tumor weight and volume of the αPD-1+Neo-BCV vaccine solution group were significantly reduced, and the difference was statistically significant. And compared with the other groups, the weight of mice in the αPD-1+Neo-BCV vaccine solution group did not decrease significantly, indicating that the αPD-1+Neo-BCV vaccine solution had no obvious signs of toxicity whether it was used alone (without adding αPD-1) or in combination with αPD-1 (i.e., the mixed αPD-1+Neo-BCV vaccine solution). It shows that the vaccine solution prepared by adding inactivated bacterial solution in the present invention shows effective anti-tumor activity, and plays a good role in enhancing the anti-tumor effect of immune checkpoint inhibitors; and the inactivated bacterial solution and Sindilimab αPD-1 are mixed to obtain the combined αPD-1+Neo-BCV vaccine solution, which significantly slows down tumor growth, and the tumor inhibition rate reaches 75.96%, indicating that the inactivated bacterial solution used in the present invention can enhance the effect of αPD-1 (Sindilimab) in treating tumors.

[0076] 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 vaccine composition for treating tumors by combining an inactivated bacterial solution with αPD-1, 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, and a mixed solution containing a film is obtained by evaporation under reduced pressure; The mixed solution containing the film is 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 vaccine composition is obtained by freeze-drying.

2. The method for preparing a vaccine composition for treating tumors by combining an inactivated bacterial solution with αPD-1 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 vaccine composition for treating tumors by combining an inactivated bacterial solution with αPD-1 according to claim 1, characterized in that: The solvent is chloroform.

4. The method for preparing a vaccine composition for treating tumors by combining an inactivated bacterial solution with αPD-1 according to claim 1, characterized in that: The mass ratio of the soybean lecithin, DSPE-PEG2000, cholesterol, αPD-1, inactivated bacteria solution and solvent is 100:1:50::2.6:1300:5000.

5. The method for preparing a vaccine composition for treating tumors by combining an inactivated bacterial solution with αPD-1 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.

6. The method for preparing a vaccine composition for treating tumors by combining an inactivated bacterial solution with αPD-1 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.

7. The method for preparing a vaccine composition for treating tumors by combining an inactivated bacterial solution with αPD-1 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.

8. The method for preparing a vaccine composition for treating tumors by combining inactivated bacteria liquid with αPD-1 according to claim 1, characterized in that: The freeze-drying protective agent is a sucrose solution with a mass concentration of 5%.

9. The method for preparing a vaccine composition for treating tumors by combining an inactivated bacterial solution with αPD-1 according to claim 1, characterized in that: The amount of the lyophilization protective agent added is 0.5 g per 10 ml.

10. A vaccine composition prepared by the method for preparing a vaccine composition for treating tumors by combining an inactivated bacterial solution with αPD-1 as claimed in any one of claims 1 to 9.

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