Preparation method and application of tumor cell vaccine

Abstract

The invention relates to the technical field of vaccines, and particularly discloses a preparation method and application of a tumor cell vaccine, the preparation method comprises the steps of collecting breast cancer cells in a growth index phase, preparing a cell suspension, performing radiation treatment, uniformly mixing the cells and the like, and single-time high-dose (40Gy) radiation is adopted as an inactivation mode; the completeness of a cell structure and the diversity of antigens can be kept to the greatest extent while the proliferation capacity of tumor cells is thoroughly destroyed. The prepared vaccine can strongly activate organism specific T cell immunity and significantly increase the number of IFN gamma < + > CD8 < + > T cells in peripheral blood and tumor local microenvironments, so that an immunosuppressive'cold tumor 'microenvironment is reversed to an immunoactivated'hot tumor' microenvironment, and resistance of breast cancer brain metastasis to immunotherapy is overcome.

Description

Technical Field

[0001] This invention belongs to the field of vaccine technology, specifically relating to a method for preparing and applying a tumor cell vaccine. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women, and brain metastasis is a major cause of extremely poor prognosis. Currently, the standard clinical treatment for breast cancer brain metastases mainly includes surgical resection and radiotherapy. However, these treatments have significant limitations: high recurrence rates after surgery, radiotherapy damages normal brain tissue and has limited overall efficacy, and the median survival of patients remains short. Therefore, there is an urgent clinical need to develop novel treatment strategies that can effectively control and prevent breast cancer brain metastases.

[0003] In recent years, tumor immunotherapy, especially immune checkpoint inhibitors (such as PD-1 / PD-L1 antibodies), has made groundbreaking progress in the treatment of various malignant tumors.

[0004] However, breast cancer brain metastases are often considered a "cold tumor," characterized by a lack of sufficient tumor-infiltrating T lymphocytes in the tumor microenvironment. This insufficient infiltration of immune cells means that immune checkpoint inhibitors lack key effector cells as targets, leading to widespread resistance to the therapy and minimal efficacy.

[0005] Among them, autologous tumors are considered the best antigens to activate the body's immune response. Whole tumor cells have complete tumor cell antigens, containing a large number of specific and non-specific T cell epitopes, which can simultaneously activate CD4+ helper T lymphocytes and CD8+ cytotoxic T lymphocytes, and are more effective in activating T cell immune responses than single antigens or peptides.

[0006] Currently, conventional physical inactivation methods for preparing whole-cell tumor vaccines include ultraviolet irradiation, repeated freeze-thaw cycles, and high-temperature inactivation. However, these traditional methods have significant drawbacks: while often destroying cell proliferation capacity, they also excessively damage the overall cell structure and immunogenicity, resulting in low efficiency of antigen-presenting cells recognizing and processing the prepared vaccine in vivo, making it difficult to elicit a strong and durable tumor-specific T-cell immune response. Therefore, vaccines prepared by these methods generally have unsatisfactory clinical effects. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing and applying a tumor cell vaccine, in order to solve the problem mentioned in the background art that the vaccines prepared by the current whole tumor cell vaccine preparation methods have weak immunogenicity and are difficult to induce a strong and lasting T cell immune response.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A method for preparing a tumor cell vaccine and its application, comprising the following steps:

[0010] S1. Collect breast cancer cells in the growth exponential phase and prepare a solution with a concentration of 1×10⁻⁶. 6 A cell suspension of 1 cell / 100 μl PBS was prepared, and the cell suspension was placed on ice.

[0011] S2. The cell suspension described in S1 is subjected to a single 40 Gy ray irradiation treatment;

[0012] S3. Mix Poly-ICLC with the irradiated cells from S2 at a ratio of 5 μl of immunoadjuvant Poly-ICLC per 100 μl of PBS cell suspension.

[0013] S4. Store the mixed product at 2-8℃.

[0014] Among them, tumor cell vaccines are used in the preparation of drugs for treating brain metastases of breast cancer.

[0015] Preferably, the drug formulation for treating brain metastases of breast cancer is an injection.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] This invention employs a single high-dose (40 Gy) radiation as the inactivation method, which can completely destroy the proliferative capacity of tumor cells while maximally preserving the integrity of cell structure and antigen diversity. Compared with traditional ultraviolet irradiation, repeated freeze-thaw cycles, or high-temperature inactivation, this method can better preserve tumor-associated antigens. By coupling the high-dose radiation-inactivated tumor cells with the highly effective immune adjuvant Poly-ICLC, the vaccine prepared by this invention can strongly activate the body's specific T-cell immunity. It significantly increases the number of IFNγ+CD8+ T cells (i.e., activated cytotoxic T cells) in the peripheral blood and local tumor microenvironment, thereby reversing the immunosuppressive "cold tumor" microenvironment to an immune-activated "hot tumor" microenvironment and overcoming the resistance to immunotherapy in breast cancer brain metastases.

[0018] Furthermore, in a mouse model of breast cancer brain metastases, the vaccine monotherapy of this invention demonstrated potent anti-tumor activity, significantly reducing intracranial tumor burden and markedly prolonging the overall survival time of the tumor-bearing mice. This proves the direct efficacy of this vaccine strategy in controlling refractory breast cancer brain metastases.

[0019] Furthermore, the vaccine provided by this invention exhibits a significant synergistic effect when used in combination with existing immune checkpoint inhibitors (such as anti-PD-L1 antibodies). The T-cell immunity pre-activated by the vaccine provides the basis for the action of the PD-L1 antibody, and the combination of the two can further significantly prolong the survival time of mice, providing a highly promising combination therapy strategy for solving the problem of drug resistance in clinical immunotherapy.

[0020] Furthermore, the preparation method of the present invention is simple in steps and the conditions are controllable. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the anti-brain metastasis effect of the vaccine of the present invention. Regions AB represent the effect of the vaccine prepared by 40 Gy radiation on tumor burden; Region C represents the effect of the vaccine prepared by 40 Gy radiation combined with anti-PD-L1 treatment on the survival time of tumor-bearing mice.

[0022] Figure 2 This diagram illustrates the immune microenvironment of breast cancer brain metastases remodeled by the vaccine activating CD8+ T cells. Region A: Survival time of tumor-bearing mice in the vaccine and control groups. Region B: Survival time of FVB-bearing mice after neutralizing CD8 antibodies. Region C: CD8+ T cell count at the injection site in FVB mice 5 days after injection with / without irradiated tumor cells. Region D: Difference in the number of IFNγ+CD8+ T cells in peripheral blood between the control and vaccine groups 7 days after treatment. Region E: Difference in the number of tumor-infiltrating CD8+ T cells between the control and vaccine groups 12 days after treatment. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1:

[0025] Preparation of novel tumor cell vaccines

[0026] Cell culture: Mouse breast cancer cell line 4T1 (or other cell lines suitable for constructing brain metastasis models) was placed in RPMI-1640 complete medium containing 10% fetal bovine serum and cultured routinely in a cell culture incubator at 37°C and 5% CO2.

[0027] Cell collection: When the cells grow to the logarithmic growth phase (with a confluence of about 80%-90%), they are digested with trypsin and collected.

[0028] Cell resuspending: Wash cells twice with sterile phosphate-buffered saline (PBS), and finally resuspend in PBS, adjusting the cell concentration to 1 × 10⁻⁶. 7 cells / mL, i.e., prepared into 1×10 6 Prepare a cell suspension of 1 cell / 100 μL PBS and keep the suspension on ice for later use.

[0029] Radiation inactivation: The above cell suspension was placed under a radiation instrument and subjected to a single dose of 40 Gy of gamma rays to completely inactivate the cell's proliferative capacity.

[0030] Adjuvant coupling: Add the immune adjuvant Poly-ICLC to the irradiated cell suspension at a ratio of 5 μL Poly-ICLC per 100 μL of cell suspension, and gently pipette to mix it evenly.

[0031] Vaccine preservation: The prepared novel tumor cell vaccine is stored in an environment of 2-8℃ and used for subsequent animal experiments within 30 minutes.

[0032] Experimental Analysis:

[0033] Establishment and grouping of a mouse model of breast cancer brain metastasis

[0034] Model establishment: 6-8 week old female BALB / c mice were selected, and 4T1-Luc breast cancer cells that stably express luciferase were injected into the mice to establish a breast cancer brain metastasis model.

[0035] Model validation: On day 4 after modeling, mice were injected intraperitoneally with a fluorescein substrate (60 mg / kg). Approximately 10 minutes later, the mice were deeply anesthetized, and bioluminescent signals in the brain region were detected using a small animal in vivo imaging system.

[0036] Experimental grouping: Based on the detected intensity of tumor fluorescence signals, mice that failed to develop tumors were excluded, and the mice that successfully developed tumors were randomly divided into the following two groups:

[0037] Control group: injected with PBS.

[0038] Vaccine treatment group: injected with the tumor cell vaccine prepared in this invention.

[0039] Vaccine treatment efficacy evaluation

[0040] Dosage regimen: After model validation and grouping (i.e., day 4 post-modeling), mice in the vaccine treatment group received their first treatment. Intradermal injections were administered bilaterally into the medial thighs of the mice, with a dose of 1 × 10⁻⁶ mcg per side. 6 1 cell / 5μL Poly-ICLC / 100μL PBS. Control mice were injected with the same volume (100μL) of PBS at the same site.

[0041] Tumor burden monitoring: Starting from the first administration, the intensity of bioluminescent signals in the brains of two groups of mice was detected and recorded weekly using a small animal in vivo imaging system to quantify changes in tumor burden.

[0042] Survival analysis: The survival status of mice was observed daily, and the time from the start of modeling to the death of mice due to excessive tumor burden or reaching the humane endpoint was recorded. Survival curves were plotted, and the total survival time of the two groups of mice was compared.

[0043] Experimental results: such as Figure 1 As shown in AB, compared with the PBS control group, the intracranial tumor growth of mice in the vaccine-treated group was significantly inhibited, and the tumor burden was significantly reduced. Meanwhile, as... Figure 1 C and Figure 2 As shown in Figure A, the overall survival time of mice in the vaccine-treated group was significantly prolonged compared to the control group.

[0044] Combination therapy with vaccines and anti-PD-L1 antibodies

[0045] Grouping and Treatment: A combined treatment group was added. Mice in this group received the same intradermal injection of vaccine as the vaccine treatment group, followed by intraperitoneal injection of anti-PD-L1 antibody (100 μg per mouse, twice a week for 2 weeks).

[0046] Efficacy assessment: Efficacy was also assessed by monitoring tumor burden and survival time.

[0047] Experimental results: such as Figure 1 As shown in Figure C, compared with the use of the vaccine alone or the use of the anti-PD-L1 antibody alone, the combination of vaccine and anti-PD-L1 antibody therapy showed a significant synergistic effect, which could most effectively control tumor growth and prolong the survival time of tumor-bearing mice to the greatest extent.

[0048] Vaccine immunization mechanism verification

[0049] To elucidate the mechanism of action of the vaccine of the present invention, the following experiments were conducted:

[0050] In vivo T-cell activation assay: On day 5 post-vaccination, skin tissue was collected from the injection site of mice, and the locally infiltrated immune cells were analyzed by flow cytometry. Results are as follows: Figure 2 As shown in Figure C, the vaccine injection site recruited and activated a large number of CD8+ T cells.

[0051] Peripheral blood immune response assay: On day 7 after the start of treatment, peripheral blood was collected from mice, and the proportion of IFNγ-positive CD8+ T cells was detected by flow cytometry. Results are as follows: Figure 2As shown in Figure D, the proportion of IFNγ+CD8+ T cells in the peripheral blood of mice in the vaccine-treated group was significantly higher than that in the control group.

[0052] Tumor microenvironment immune cell infiltration analysis: On day 12 after the start of treatment, mice were sacrificed, brain metastases were harvested, and single-cell suspensions were prepared for flow cytometry analysis. Results are as follows: Figure 2 As shown in E, the tumor tissue of the vaccine-treated mice showed more CD8+ T cells infiltrating.

[0053] CD8+ T cell function verification: During treatment, a group of mice receiving the vaccine were added, and simultaneously injected with neutralizing anti-CD8 antibodies to eliminate CD8+ T cells in their bodies. Results were as follows... Figure 2 As shown in Figure B, the survival benefit from the vaccine completely disappeared after CD8+ T cells were eliminated.

[0054] In summary, this invention utilizes a novel tumor cell vaccine prepared by high-dose radiation-coupled with the immune adjuvant Poly-ICLC to activate CD8+ T cells in mice, thereby inducing an anti-tumor immune response. This significantly prolongs the survival time and reduces the tumor burden in tumor-bearing mice, and also enhances the anti-brain metastasis effect of PD-L1 antibody, providing a new strategy for the treatment of breast cancer brain metastases.

[0055] It should be understood that numerous specific implementation decisions can be made during the development of any practical implementation, such as in any engineering or design project. Such development efforts may be complex and time-consuming, but for those skilled in the art who benefit from this disclosure, the development effort will be a routine work of design, manufacturing, and production without requiring much experimentation.

[0056] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a tumor cell vaccine, characterized in that, Includes the following steps: S1. Collect breast cancer cells in the growth exponential phase and prepare a solution with a concentration of 1×10⁻⁶. 6 A cell suspension of 1 cell / 100 μl PBS was prepared, and the cell suspension was placed on ice. S2. The cell suspension described in S1 is subjected to a single 40 Gy ray irradiation treatment; S3. Mix Poly-ICLC with the irradiated cells from S2 at a ratio of 5 μl of immunoadjuvant Poly-ICLC per 100 μl of PBS cell suspension. S4. Store the mixed product at 2-8℃.

2. The use of the tumor cell vaccine prepared according to claim 1 in the preparation of a drug for treating brain metastases of breast cancer.

3. The application according to claim 2, characterized in that: The drug formulation for treating brain metastases from breast cancer is an injectable preparation.

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