Tumor vaccine as well as preparation method and application thereof

Abstract

The invention discloses a tumor vaccine as well as a preparation method and application thereof, and belongs to the technical field of tumor immunity. Aiming at the defects of long research and development period, high cost, non-obvious single-drug treatment effect and the like of the tumor vaccine at present, the invention provides a brand-new method for generating the tumor vaccine in a mouse body, a mode of pretreating the mouse by using an immune checkpoint inhibitor is utilized, and then tumor cells are inoculated subcutaneously; and after the tumor fades, the mouse can obtain strong immunocompetence to specific tumor cells, and an effect similar to that of a tumor vaccine is generated. Through verification, the mouse tumor vaccine prepared by the method provided by the invention can achieve very good tumor prevention and treatment effects. Meanwhile, the method is easy and convenient to operate, high in stability and low in cost, a brand new scheme for preparing the tumor vaccine is provided, and the possibility is provided for expanding to clinical tumor vaccines in the future.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of tumor immunotherapy, in particular to a tumor vaccine and a preparation method and application thereof. BACKGROUND

[0002] According to the purpose, the tumor vaccine is divided into therapeutic vaccine and preventive vaccine. At present, there is no report that the preventive vaccine directly acts on the tumor itself, but prevents the occurrence of related cancers by preventing some microbial infections, such as HPV vaccine, which can effectively prevent cervical cancer caused by HPV infection. The first approved therapeutic tumor vaccine is Provenge, which is the first therapeutic cancer vaccine for advanced prostate cancer. It proves that the concept of personalized immunotherapy is feasible, but the effect is relatively mild, which can prolong the survival period for several months.

[0003] According to the technical route, the tumor vaccine is mainly divided into mRNA vaccine, dendritic cell (DC) vaccine, polypeptide vaccine, virus vector vaccine, etc. At present, most of the therapeutic vaccines are still in the clinical research stage, and the tumor vaccine is often combined with immune checkpoint inhibitors (such as PD-1 / PD-L1 inhibitors), chemotherapy, radiotherapy, etc. to overcome the inhibition of tumor microenvironment and synergistically enhance the effect.

[0004] The tumor vaccine belongs to the emerging tumor immunotherapy scheme, and still has many deficiencies, which leads to the treatment effect far lower than the expectation, mainly with the following challenges.

[0005] Tumor heterogeneity: within the same tumor or between the primary lesion and the metastatic lesion, the cancer cells may have great differences in gene and protein expression. This means that the vaccine designed for one or a group of antigens may only eliminate a part of the cancer cells expressing the antigen, while the other cells not expressing the antigen will continue to survive and proliferate (referred to as "immune editing" or "antigen escape"), leading to treatment failure or recurrence.

[0006] Tumor immune microenvironment inhibits the effect of vaccine: tumor is a very "cunning" organization, which actively creates a microenvironment that inhibits the function of immune cells. This environment is full of: inhibitory cells: such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs). Inhibitory molecules: such as PD-L1, CTLA-4, IL-10, TGF-β, etc. Even if the vaccine successfully activates T cells, these activated "soldiers" may be "disarmed" or "paralyzed" after entering the tumor battlefield, and cannot effectively attack cancer cells. This is the key reason for the limited effect of single vaccine therapy.

[0007] Target selection difficulty: tumor-associated antigens (TAA) are mostly proteins that are also lowly expressed on normal cells (such as CEA, MAGE), only the tumor cells express more. They can trigger autoimmune toxicity to normal tissues, and the immune response can be weaker due to central tolerance.

[0008] Technical process and cost challenges: tumor vaccines, especially personalized vaccine processes, are complex and costly. Personalized neoantigen vaccines need to be performed individually for each patient, and the procedure is complex. Time-consuming: the entire process can take several months. For patients with rapidly progressing advanced cancer, the disease may change significantly during this period, and the vaccine production may not be completed. Extremely high cost: the current treatment cost of personalized vaccines can be as high as hundreds of thousands of dollars, far beyond the affordability of most patients.

[0009] Due to the above challenges, tumor vaccines have not been effective in tumor prevention and treatment, and most tumor vaccines are still in the laboratory stage, making it difficult to pass the drug regulatory department's approval and not be widely used.

[0010] So how to solve the challenges faced by current tumor vaccines? If a new method can be found to make animals produce strong specific tumor immunity, its effect is equivalent to a special tumor vaccine. And if this tumor vaccine production scheme does not require a complex antigen preparation process, nor does it require a complex delivery system and the assistance of vaccine adjuvants, and this new method of tumor vaccine production has a significant preventive and therapeutic effect on a variety of tumors, then this new tumor vaccine production scheme has great potential to move from the laboratory to clinical application. SUMMARY

[0011] The purpose of the present application is to provide a tumor vaccine and its preparation method and application, to solve the problems existing in the prior art. It has been verified that the mouse tumor vaccine prepared by the method provided by the present application can achieve very good tumor prevention and treatment effects. At the same time, the method is simple to operate, has high stability, and is low in cost. Therefore, the present application provides a new scheme for preparing a tumor vaccine, which provides the possibility for expanding to a clinical tumor vaccine in the future.

[0012] To achieve the above-mentioned purpose, the present application provides the following scheme:

[0013] The present application provides a preparation method of a tumor vaccine, characterized in that it comprises the following steps:

[0014] The mouse is treated with an immune checkpoint inhibitor, and then tumor cells are inoculated to form a tumor;

[0015] After the tumor completely regresses, the PBMC cells of the mouse are extracted, and the tumor vaccine is obtained.

[0016] Preferably, the mouse is treated with the immune checkpoint inhibitor at a dosage of 200 μg per mouse, once every 3 days, for a total of 4 times.

[0017] Preferably, the immune checkpoint inhibitor comprises a CTLA-4 antibody and a PD-L1 antibody.

[0018] Preferably, the inoculation of tumor cells comprises subcutaneous inoculation and intraperitoneal inoculation.

[0019] Preferably, the tumor cells comprise mouse hepatoma cells, mouse melanoma cells, and mouse ovarian cancer cells.

[0020] The application also provides use of the PBMC cells prepared according to the preparation method in preparation of a medicine for preventing and / or treating tumors.

[0021] The application also provides a tumor vaccine prepared according to the preparation method.

[0022] The application discloses the following technical effects:

[0023] In view of the long research and development cycle, high cost, and insignificant single-drug treatment effect of the existing tumor vaccines, the application provides a novel method for generating a tumor vaccine in a mouse body, which uses an immune checkpoint inhibitor to pretreat the mouse, then subcutaneously inoculates tumor cells, and after the tumor subsides, the mouse can obtain strong immunity to specific tumor cells, and the method has the effect similar to that of a tumor vaccine. Meanwhile, the method is simple to operate, high in stability, and low in cost, and therefore, the application provides a novel method for preparing a tumor vaccine, and provides a possibility for future expansion to a clinical tumor vaccine.

[0024] The application sets a control group of mice and a treatment group of mice, and after the mice in both groups are subcutaneously inoculated with the same number of Hepa1-6 cells in the right armpit, the growth of tumors is observed, and the results show that: after 14 days of tumor implantation, the tumors in the control group of mice grow obviously, and the average volume exceeds 500 mm 3 However, the mice in the treatment group do not form tumors from the beginning to the end. After 10 weeks, the mice in the treatment group are inoculated with the same number of Hepa1-6 cells again, and still remain in a state of 100% inability to form tumors; after 50 weeks, the mice in the treatment group are inoculated with the same number of Hepa1-6 cells again, and still remain in a state of 100% inability to form tumors.

[0025] Furthermore, 5×10 5PBMC cells, and then the mice were injected with the PBMC cells through the tail vein, the mice were able to prevent the growth of Hepa1-6 tumors. Further, the PBMC cells of the peripheral blood of the mice in the treatment group in the tumor regression period were injected into the Hepa1-6 tumor-bearing mice (tumor volume less than 500 mm 3 ) through the tail vein, and the tumors of the tumor-bearing mice were also completely eliminated.

[0026] In summary, the mouse tumor vaccine prepared by the method provided by the application can achieve very good tumor prevention and treatment effects. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0028] Figure 1 Tumor growth of the three groups of mice in Example 1;

[0029] Figure 2 Tumor growth of the mice inoculated with Hepa1-6 cells and B16F10 cells again after 10 weeks of complete tumor regression in Example 1;

[0030] Figure 3 Tumor growth of the mice inoculated with Hepa1-6 cells again after 50 weeks of complete tumor regression in Example 1;

[0031] Figure 4 Tumor liver metastasis of the mice injected with Hepa1-6 cells in the spleen after 10 weeks of complete tumor regression in Example 1;

[0032] Figure 5 Prevention effect of the mice treated with CTLA-4 antibody in advance for different times on Hepa1-6 tumors in Example 2;

[0033] Figure 6 Immune ability of PD-L1 antibody pre-treatment on Hepa1-6 subcutaneous tumors in Example 3; A is the tumor growth of the mice in the IgG control group and the PD-L1 antibody treatment group; B is the tumor growth of the mice inoculated with Hepa1-6 cells again after 10 weeks of complete tumor regression;

[0034] Figure 7 Immune ability of the mice treated with CTLA-4 antibody in advance on B16F10 subcutaneous tumors in Example 4;

[0035] Figure 8 Immune capacity against ID8 intraperitoneal tumor with CTLA-4 antibody pretreatment in Example 5;

[0036] Figure 9 Immune capacity against Hepal-6 tumor with CTLA-4 antibody pretreatment in Example 6 was dependent on the dual protection of CD3 positive cell population and NK cell population.

[0037] Figure 10 Immune capacity against Hepal-6 tumor with CTLA-4 antibody pretreatment in Example 7 could prevent the growth of Hepal-6 subcutaneous tumor by PBMC transplantation into control mice;

[0038] Figure 11 Immune capacity against Hepal-6 tumor with CTLA-4 antibody pretreatment in Example 8 could treat Hepal-6 subcutaneous tumor by PBMC transplantation into control tumor-bearing mice. DETAILED DESCRIPTION

[0039] Various exemplary embodiments of the present application will now be described in detail with reference to the drawings. The detailed description is not intended to limit the present application, but to explain certain aspects, features, and embodiments of the present application.

[0040] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, the use of the term "about" in relation to a value or a range of values is intended to include each individual intermediate value and each smaller range that falls within the range of values. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not intended as an admission that the reference is prior art, but rather that the reference is part of the technical literature that is relevant to the disclosure.

[0042] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof, and it is to be understood that all such modifications and variations warrant the patentable subject matter under the patent laws. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0043] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” and the like are open-ended terms that are intended to be synonymous, and are generally used to permit for the inclusion of additional nonspecified elements or integers.

[0044] The prior art patent with application number 202211348248.8 and the title of “Use of MIF gene knockout tumor cells in the preparation of a tumor vaccine” is similar to the present application in that it also uses intact and active cells as the source of immunizing antigens, and does not add immune adjuvants and other auxiliary measures to trigger the immune response of the animal body to this type of tumor, including long-term immunity. The difference is that the patent uses gene knockout technology to modify the original tumor cells to achieve the effect of tumor immunity, which does not go through the process of first forming a tumor and then gradually regressing. The present application, on the other hand, uses immune checkpoint inhibitors to first activate the immune system in the body, and then inoculates unmodified tumor cells subcutaneously within a certain time window. Finally, this tumor cell goes through the process of first forming a tumor and then being cleared by the immune system, thereby obtaining long-term tumor immunity. Compared to the patent, the present application is more convenient to operate, as it directly uses unmodified tumor cells as antigens, and its immune process is more similar to the effect of natural immunity. In addition, the present application has found that after immunization, the NK cells and T cells in the PBMC cells of the mice also play a role in tumor immunity, and the transplantation of PBMC into control mice can enable the control mice to acquire tumor immunity, further verifying the stability and effectiveness of the tumor immunity function produced by the method of the present application. The specific research is shown in the following examples.

[0045] Example 1

[0046] The mice were treated with immune checkpoint inhibitors CTLA-4 antibodies, and Hepa1-6 mouse hepatoma cells were planted subcutaneously to make the mice acquire immunity to Hepa1-6 tumors, including inhibiting the formation of tumors after subcutaneous inoculation and the injection of liver metastatic tumors into the spleen.

[0047] 1. Experimental method

[0048] (1) Prepare 2 groups of 6-8 week old male C57bl / 6J mice, 5 mice in each group. The treatment group mice were injected intraperitoneally with CTLA-4 antibodies (BioxCcell company, item number: BP0164) 200 μg each time, and the control group mice were injected intraperitoneally with the same amount of IgG antibodies (BioxCell company, item number: BP0086). Inject every 3 days, a total of 4 times.

[0049] (2) On the second day after the end of antibody injection, each mouse was injected subcutaneously with 5×10 6Hepa1-6 cells (Hepa1-6 cells were in logarithmic growth phase, and the tumor cells used in the following injection were also in logarithmic growth phase).

[0050] (3) The tumor growth of the mice was observed. When the tumors of the mice in the treatment group disappeared, the mice were considered to have the ability to produce immunity to Hepa1-6 tumors.

[0051] (4) Ten 6-8-week-old male C57bl / 6J mice were prepared, and 5x10 6 Hepa1-6 cells were injected subcutaneously in the right axillary fossa of each mouse. When the tumor volume of the mice grew to about 100 mm 3 , each mouse was injected intraperitoneally with 200 μg of CTLA-4 antibody, once every 3 days, for a total of 4 times. Then the tumor growth of the mice was observed.

[0052] The above (1)-(3) are for injecting antibodies first and then injecting tumors, and (4) is for injecting tumors first and then injecting antibodies, to screen the injection order.

[0053] (5) The mice obtained through the steps (1)-(3) were subcutaneously inoculated with tumors again at the 10th week after obtaining the immunity. The mice were divided into two groups, 5 mice in each group. In one group, 5x10 6 Hepa1-6 cells were injected subcutaneously in the right axillary fossa of each mouse. In the other group, 2x10 6 B16F10 cells were injected subcutaneously in the right axillary fossa of each mouse. Then the tumor growth of the two groups of mice was observed. Another group of 5 normal mice was inoculated with the same amount of Hepa1-6 cells in the right axillary fossa as a control group for tumor growth.

[0054] (6) The mice obtained through the steps (1)-(3) were subcutaneously inoculated with tumors again at the 50th week after obtaining the immunity. Three mice were taken as one group, and 5x10 6 Hepa1-6 cells were injected subcutaneously in the right axillary fossa of each mouse. Then the tumor growth of the mice was observed.

[0055] (7) The tumor immune mice obtained through the steps (1)-(3) and normal C57bl / 6J mice (control) were anesthetized and subjected to laparotomy surgery at the 10th week after obtaining the immunity, and 30 μL of about 5x10 5 Hepa1-6 cells were injected into the mice through the spleen. The mice were sacrificed after 6 weeks, and the liver metastasis of tumors in the two groups of mice was observed.

[0056] 2. Experimental results

[0057] From Figure 1As can be seen, the mice in the pre-injection CTLA-4 antibody group, after subcutaneous implantation of Hepa1-6 cells, the tumors first grew to about 300 mm 3 within two weeks, then the tumors rapidly and completely regressed. The control group mice, however, the tumors kept growing until they exceeded the ethical limit of the animals. This result shows that the injection of CTLA-4 antibody can activate the immune system of the mice, thus causing the tumors to completely regress. In contrast, when the tumors grow to 100 mm 3 in size and the mice are then injected with the same dose of CTLA-4 antibody, the tumors of some of the mice (2 / 5) will regress, but the tumors of most of the mice (3 / 5) still cannot be controlled.

[0058] Figure 2 It is shown that after the tumors have regressed for 10 weeks, different tumors are again subcutaneously inoculated, and it is found that the mice inoculated with Hepa1-6 cells do not form tumors all the time, preventing the recurrence of tumors. The control group mice inoculated with Hepa1-6 cells can form tumors normally. The mice inoculated with B16F10 cells form tumors rapidly and keep growing, proving that the immune memory to the tumors formed by Hepa1-6 cells is specific to Hepa1-6 cells.

[0059] Figure 3 It is shown that after the tumors have regressed for 50 weeks, the mice inoculated with Hepa1-6 cells do not form tumors in the end, proving that the immune memory to the tumors formed by Hepa1-6 cells is long-term.

[0060] Figure 4 It is shown that, unlike the significant liver metastasis phenomenon that occurs in the control mice, after the tumors have regressed for 10 weeks, the inoculation of Hepa1-6 cells in other parts (spleen) does not result in significant liver metastasis. This proves that the immune memory of the mice is a systemic response and can prevent the liver metastasis of Hepa1-6 tumors.

[0061] The above results show that the tumor immunity obtained by the sequence of first injecting the antibody and then injecting the tumor is significantly better than that obtained by the sequence of first injecting the tumor and then injecting the antibody. The mice with tumor immunity to Hepa1-6 tumors obtained according to the method of the present application have tumor immune memory to Hepa1-6 cells, which is specific, long-term (still have tumor immunity after the tumors have regressed for 50 weeks), and systemic, and is significantly better than other tumor vaccines in the prior art.

[0062] Example 2

[0063] After the mice are treated with the immune checkpoint inhibitor CTLA-4 antibody at different times, the preventive effect of the mice on Hepa1-6 subcutaneous tumors is detected.

[0064] 1. Experimental method

[0065] (1) Prepare 3 groups of 6-8 week old male C57bl / 6J mice, 5 in each group. One group is the control group, and the other two groups are the CTLA-4 antibody pretreatment group inoculated with tumor 2 weeks after the end of the pretreatment (2 weeks group) and the CTLA-4 antibody pretreatment group inoculated with tumor 4 weeks after the end of the pretreatment (4 weeks group). The antibody treatment group is injected intraperitoneally with 200 μg of CTLA-4 antibody per mouse each time, and the control group is injected intraperitoneally with the same amount of IgG antibody. Inject every 3 days, a total of 4 times.

[0066] (2) 2 weeks after the end of antibody injection, the mice in the control group and the 2 weeks group are injected subcutaneously with 5×10 6 Hepa1-6 cells in the right axillary fossa; 4 weeks after the end of antibody injection, the mice in the 4 weeks group are injected subcutaneously with 5×10 6 Hepa1-6 cells in the right axillary fossa.

[0067] (3) Observe the tumor growth of the mice.

[0068] 2. Experimental results

[0069] The results are shown in Figure 5 After 2 weeks of CTLA-4 antibody pretreatment, the mice can still produce a complete immune response to the inoculated Hepa1-6 tumor, thereby completely clearing the tumor, while after 4 weeks of CTLA-4 antibody pretreatment, the mice cannot produce a complete immune response to the inoculated Hepa1-6 tumor, only a small part of the mice can produce tumor regression, and most of the mice tumors will continue to grow until they exceed the ethical limit. These results show that the method provided by the present application can prevent new tumors for a certain period of time, and once the period is exceeded, the tumor immunity decreases and the tumor cannot be completely cleared, which is exactly in line with the pharmacological characteristics of antibodies in vivo.

[0070] Example 3

[0071] The mice are treated with an immune checkpoint inhibitor PD-L1 antibody and supplemented with subcutaneous implantation of Hepa1-6 mouse hepatocarcinoma cells to enable the mice to have an immune ability to Hepa1-6 tumor.

[0072] 1. Experimental method

[0073] (1) Prepare 2 groups of 6-8 week old male C57bl / 6J mice, 5 in each group. The treatment group is injected intraperitoneally with 200 μg of PD-L1 antibody (Bioxcell Company, Item No: BE0101) per mouse each time, and the control group is injected intraperitoneally with the same amount of IgG antibody (Bioxcell Company, Item No: BP0090). Inject every 3 days, a total of 4 times.

[0074] (2) On the second day after the antibody injection, each mouse was subcutaneously injected with 5 × 10⁵ antibodies into the right axilla. 6 Hepa1-6 cells.

[0075] (3) Observe the growth of tumors in mice. All tumors in the untreated group of mice disappeared. At this time, the mice were considered to have acquired the ability to develop immunity to Hepa1-6 tumors.

[0076] (4) Ten weeks after the mice acquired immunity, they were again subjected to subcutaneous tumor implantation. A total of 5 mice were involved, and each mouse received a subcutaneous injection of 5 × 10⁵ tumor cells in its right axilla. 6 One group of Hepa1-6 cells. Another group of five normal mice were used as a control group for subcutaneous inoculation with Hepa1-6 cells.

[0077] 2. Experimental Results

[0078] Results of tumor size monitoring, such as Figure 6 As shown in Figure A, similar to the results of CTLA-4 antibody injection, after mice were injected with PD-L1 antibody and subsequently subcutaneously inoculated with mouse Hepa1-6 cells, the Hepa1-6 tumors in the mice also underwent a process of initial enlargement followed by complete regression. In contrast, the tumors in the control group mice continued to grow. This indicates that not only does the immune checkpoint inhibitor CTLA-4 antibody have the function of activating the mouse immune system, but another important immune checkpoint inhibitor, PD-L1 antibody, also has a similar function.

[0079] In addition, the results of Hepa1-6 tumor cell reimplantation also showed ( Figure 6 In mice treated with PD-L1 antibody (B), tumor regression was observed, but subsequent subcutaneous reimplantation of Hepa1-6 cells failed to effectively form tumors. In contrast, normal control mice were able to form tumors normally after subcutaneous reimplantation of Hepa1-6 cells. This result suggests that the tumor immune memory formed in PD-L1 antibody-treated mice can also be activated by reimplantation of Hepa1-6 cells.

[0080] The above results indicate that the activation of mouse immunity by immune checkpoint inhibitor treatment is not limited to CTLA-4 antibodies, but also applies to PD-L1 antibodies or other untested immune checkpoint inhibitors. This suggests that this phenomenon may be a common feature of the immune system, warranting further investigation into its underlying mechanisms.

[0081] Example 4

[0082] Mice were treated with the immune checkpoint inhibitor CTLA-4 antibody and then subcutaneously implanted with B16F10 mouse melanoma cells to induce immunity against B16F10 tumors.

[0083] 1. Experimental method

[0084] (1) Two groups of 6-8 week old male C57bl / 6J mice were prepared, 5 mice in each group. The mice in the treatment group were injected intraperitoneally with CTLA-4 antibody 200 μg each time, and the mice in the control group were injected intraperitoneally with the same amount of IgG antibody. The injection was performed every 3 days, and a total of 4 injections were performed.

[0085] (2) On the second day after the end of antibody injection, 2x10 6 B16F10 cells were injected subcutaneously in the right axillary fossa of each mouse.

[0086] (3) The tumor growth of the mice was observed.

[0087] The results are shown in Figure 7 , and the mice in the CTLA-4 antibody injection group had tumors that grew to about 150 mm 3 within two weeks after subcutaneous implantation of B16F10 cells, and then the tumors regressed. The tumors of the control group mice continued to grow. This shows that the treatment of CTLA-4 antibody is not only effective for Hepa1-6 tumor, but also has similar effects on other types of tumors. The results of this example verify that the treatment of CTLA-4 antibody in the present application can effectively activate the immune system of mice against various tumors, thereby causing tumor regression, indicating that the immune checkpoint inhibitor proposed in the present application has certain universality for immune activation of mouse tumors.

[0088] Example 5

[0089] The immune checkpoint inhibitor CTLA-4 antibody was used to treat mice, and ID8 mouse ovarian cancer cells were implanted intraperitoneally to make the mice have partial immunity to ID8 tumors.

[0090] 1. Experimental method

[0091] (1) Two groups of 6-8 week old female C57bl / 6J mice were prepared, 5 mice in each group. The mice in the treatment group were injected intraperitoneally with CTLA-4 antibody 200 μg each time, and the mice in the control group were injected intraperitoneally with the same amount of IgG antibody. The injection was performed every 3 days, and a total of 4 injections were performed.

[0092] (2) On the second day after the end of antibody injection, 2x10 6 ID8 cells were injected intraperitoneally in each mouse.

[0093] (3) The abdominal swelling of the mice was observed.

[0094] 2. Experimental results

[0095] The results are shown in Figure 8As shown, the mice in the CTLA-4 antibody injection group and the control group all developed obvious abdominal bulges accompanied by ascites. The difference is that the abdominal bulges in the CTLA-4 antibody injection group were delayed by about 15 days compared with the control group, indicating that the CTLA-4 antibody treatment not only caused the "hot tumor" Hepa1-6 to develop a tumor immune response, but also affected the "cold tumor" ID8.

[0096] Example 6

[0097] The tumor immune ability of the mice treated with the CTLA-4 antibody depends on the dual action of NK cells and CD3-positive T cells in the mice.

[0098] 1. Experimental method

[0099] (1) The mice with the ability to produce immunity to Hepa1-6 tumors were prepared according to the steps (1)-(3) of Example 1, and the mice with complete tumor regression for 10 weeks were divided into 6 groups, 5 mice in each group. The mice in the first group were treated with control IgG (Bioxcell Company, item number BE0085 and item number BE0091) antibodies, and each mouse was injected with 200 μg of each of the two control antibodies through the abdominal cavity, once every 3 days, a total of 4 times. The mice in the second group were treated with NK1.1 antibodies (Bioxcell Company, item number: BE0036), the treatment method was consistent with the control group, the mice in the third group were treated with CD3 antibodies (Bioxcell Company, item number BE0001-1), the mice in the fourth group were treated with mouse NK1.1 antibodies and mouse CD3 antibodies, the mice in the fifth group were treated with CD8 antibodies (Bioxcell Company, item number BE0061), and the mice in the sixth group were treated with mouse NK1.1 antibodies and mouse CD8 antibodies, and the treatment method was consistent with the control group.

[0100] (2) The next day after the antibody injection was completed, 5x10 6 Hepa1-6 tumor cells were implanted subcutaneously in all mice, and the tumor growth of the two groups of mice was observed.

[0101] 2. Experimental results

[0102] The tumor growth of the mice in each group is shown in Figure 9 3 The mice in the control group, the NK1.1 antibody injection group, and the CD3, CD8 antibody injection group could not form tumors, while the mice injected with CD8 and NK1.1 antibodies first formed tumors of about 100 mm

[0103] ​This result shows that the tumor immunity is protected by both NK cells and CD3 positive T cells. Removing NK cells alone or removing CD3 positive T cells alone cannot eliminate the tumor immunity of the mice, but removing both CD8 positive T cells and NK1.1 positive cells can inhibit the tumor immunity to some extent.

[0104] Example 7

[0105] PBMC cells were extracted from the peripheral blood of the mice with tumor immunity and were transferred to untreated mice, which also acquired immunity to subcutaneously implanted tumor cells.

[0106] 1. Experimental method

[0107] (1) Blood collection. A mouse with immunity to Hepa1-6 tumor was prepared according to the procedures of (1)-(3) in Example 1. The mouse was anesthetized with an isoflurane gas mask, and the chest was cut open after the limbs were fixed to expose the heart. Then, the heart was punctured with a 1 mL syringe pre-filled with 0.2 mL of 20 mM EDTA solution to collect about 1 mL of blood. The peripheral blood was diluted 1:1 with 1 mL of PBS solution and placed on ice.

[0108] (2) PBMC cells were separated from the peripheral blood of the mice by Percoll gradient centrifugation. 5 mL of 70% Percoll solution was added to the bottom of a 15 mL centrifuge tube, 5 mL of 40% Percoll solution was added to the upper layer, and 1 mL of peripheral blood diluted with PBS solution was added to the top layer. Then, gradient centrifugation was performed at the lowest speed. The centrifugation time was 20 min, and the centrifugal force was 500 g. After centrifugation, the white turbid cell layer enriched at the interface was carefully transferred to a new 15 mL centrifuge tube, and 10 mL of PBS solution was added for centrifugal washing. The centrifugation was set at 300 g for 5 min. The obtained cell pellet was completely lysed with red blood cell lysis solution (BD, Cat. No. 555899), and finally the obtained PBMC cells were counted and diluted to 1x10 7 cells / mL and placed on ice.

[0109] (3) PBMC cells of the control group mice were extracted by the same procedure.

[0110] (4) 50 μL of PBMC cells of the control group and the immune group mice obtained in the above step were injected into 2 groups of untreated C57bl / 6J mice through the tail vein, 3 mice in each group. The next day, 5x10 6Hepa1-6 tumor cells, and then every 3 days to observe the tumor growth of the two groups of mice.

[0111] 2. Experimental results

[0112] The results are shown in Figure 10 Figure 2, which shows that the tumor cells of the mice receiving the PBMC cell infusion of the tumor immune mice were eliminated by the immune system after inoculation of the tumor cells, and thus could not further proliferate to form tumors. The tumors of the control group mice receiving the PBMC cell infusion of the control mice continued to grow until they exceeded the size limit allowed by the animal ethics committee. It is inferred that certain cell populations contained in the PBMC cells of the mice have a strong tumor growth prevention effect, can be activated by tumors in a new mouse, and thus produce a strong immune response to completely eliminate the newly inoculated tumor cells. This indicates that the mice produced by the immunization method used in the present application have very strong tumor prevention effects in their PBMC cells.

[0113] Example 8

[0114] PBMC cells were extracted from the peripheral blood of the mice with tumor immunity, and were transferred to untreated tumor-bearing mice, and the tumors of the tumor-bearing mice completely regressed.

[0115] 1. Experimental method

[0116] (1) The PBMC cells of the control group and the immune group mice were obtained according to the steps (1)-(3) of Example 7, and were placed on ice for use.

[0117] (2) 50 μL of the PBMC cells of the control group and the immune group mice obtained in the above step were injected into 2 groups of C57bl / 6J tumor-bearing mice through the tail vein of the mice, 3 in each group, and the volume of the tumor was about 300 mm 3 . The tumor growth of the two groups of mice was then observed.

[0118] 2. Experimental results

[0119] The results are shown in Figure 11 Figure 2, which shows that the tumor cells of the mice receiving the PBMC cell infusion of the tumor immune mice were eliminated by the immune system after inoculation of the tumor cells, and thus could not further proliferate to form tumors. The tumors of the control group mice receiving the PBMC cell infusion of the control mice continued to grow until they exceeded the size limit allowed by the animal ethics committee. It is inferred that certain cell populations contained in the PBMC cells of the mice have a strong tumor growth prevention effect, can be activated by tumors in a new mouse, and thus produce a strong immune response to completely eliminate the newly inoculated tumor cells. This indicates that the mice produced by the immunization method used in the present application have very strong tumor prevention effects in their PBMC cells.

[0120] The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application.

Claims

1. A method for preparing a tumor vaccine, characterized by, The method comprises the following steps: tumor cells are inoculated after the mice are treated with an immune checkpoint inhibitor to form tumors; PBMC cells are extracted from the mice after the tumors completely regress, thereby obtaining the tumor vaccine.

2. The production method according to claim 1, wherein The mice are treated with the immune checkpoint inhibitor at a dosage of 200 μg per mouse, and the treatment is performed every 3 days for 4 times.

3. The production method according to claim 1, wherein The immune checkpoint inhibitor comprises a CTLA-4 antibody and a PD-L1 antibody.

4. The production method according to claim 1, wherein The tumor cells are inoculated subcutaneously and intraperitoneally.

5. The production method according to claim 1, wherein The tumor cells comprise mouse hepatoma cells, mouse melanoma cells and mouse ovarian cancer cells.

6. The PBMC cells prepared by the preparation method of any one of claims 1-5 are used for preparing a drug for preventing and / or treating tumors.

7. A tumor vaccine, characterized by, The tumor vaccine is prepared according to the preparation method of any one of claims 1-6.

Images