CN109908334B

CN109908334B - Tumor vaccine and preparation method and application thereof

Info

Publication number: CN109908334B
Application number: CN201910217710.2A
Authority: CN
Inventors: 魏霞蔚; 魏于全; 余佳耘
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2019-03-21
Filing date: 2019-03-21
Publication date: 2022-02-15
Anticipated expiration: 2039-03-21
Also published as: CN109908334A

Abstract

The invention relates to a tumor vaccine, a preparation method and application thereof, belonging to the field of medicines. The invention provides a tumor vaccine, which is prepared by inactivating lactic acid-stimulated tumor cells. The invention also provides a preparation method and application of the tumor vaccine. The mouse transplanted tumor model experiment proves that the vaccine obtained by stimulating the tumor cells with the lactic acid shows obvious anti-tumor effect in both preventive and therapeutic experiments, can inhibit the growth of tumors, prolong the survival time of experimental mice, improve the survival rate, and has the effect obviously superior to that of inactivated tumor cells which are not stimulated with the lactic acid. The invention provides a new drug selection for clinically preventing and treating tumors and a new tumor treatment strategy.

Description

Tumor vaccine and preparation method and application thereof

Technical Field

The invention relates to a tumor vaccine, a preparation method and application thereof, belonging to the field of medicines.

Background

Tumor vaccines have been studied for decades and are currently considered as an effective anti-tumor therapy. Recent studies have attempted a variety of different vaccine sources, including DNA vaccines, protein and antigen-specific polypeptide vaccines, bacterial or viral vector vaccines, tumor whole cell vaccines, and the like. The immune system recognizes tumors through unique Tumor Associated Antigens (TAAs) that promote the immune system in recognizing tumors and enhance anti-tumor therapy. Compared to single targeted vaccines, tumor cells can provide a full range of TAAs, which contain antigenic determinants of CD8+ cytotoxic T lymphocytes and CD4+ helper T cells, and can activate both innate and adaptive immune responses. In addition, the tumor whole cell vaccine can promote the generation of long-term CD8+ T lymphocyte memory through CD4+ T cells and enhance the anti-tumor response, and compared with a vaccine with a single determinant, the tumor whole cell vaccine can also greatly reduce the chance of tumor escape immune response. And the tumor whole cell vaccine can provide all tumor antigens, so that antigen loss is avoided. There are currently several whole tumor vaccines, including: tumor-derived exosomes, dead tumor whole cells, tumor whole cell necrosis lysates, irradiated tumor whole cell vaccines, viral oncolytic lysates, dendritic cell/tumor fusion vaccines, dendritic cells presented by whole tumor RNA, and can enhance the immunogenicity of the dead tumor whole cells through oxidative modification.

Normal cells under different oxygen concentrations metabolize glucose through different pathways to produce Adenosine Triphosphate (ATP) and lactate or carbon dioxide. Even under oxygen-rich conditions, tumor cells tend to metabolize glucose by glycolysis. Glycolysis produces ATP less efficiently than oxidative phosphorylation reactions. This process results in higher lactate concentrations in tumor tissues than in normal tissues. Lactic acid is an important by-product in tumor metabolism, and its concentration is up to 40mmol/L in tumor tissues. The previous research shows that the role of lactic acid in tumors includes: promoting metastasis, suppressing anti-immune response, and increasing angiogenesis.

So far, no report about that lactic acid can inhibit tumor growth exists, and no tumor vaccine prepared by stimulating tumor cells with lactic acid exists.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. To this end, the present invention aims to provide a tumor vaccine. The invention also aims to provide a preparation method and application of the tumor vaccine.

The invention provides a tumor vaccine, which is prepared by inactivating lactic acid-stimulated tumor cells.

Furthermore, the lactic acid-stimulated tumor cells are obtained by culturing lactic acid and tumor cells in a mixed manner.

Further, the tumor cells were cultured in a carbon dioxide cell incubator.

Further, the culture time is 19.2-28.8 h.

Preferably, the incubation time is 24 h.

Further, the concentration of the lactic acid is 5mmol/L or more.

Preferably, the concentration of the lactic acid is 20mmol/L or more.

Preferably, the concentration of the lactic acid is 5-30 mmol/L.

Preferably, the concentration of the lactic acid is 20 mmol/L.

Further, the tumor cell is a lymphoma cell or a breast cancer cell.

Preferably, the lymphoma cell is a T-cell lymphoma cell.

Further, the inactivation is irradiation inactivation.

Preferably, the radiation inactivation is X-ray radiation inactivation.

Preferably, the operating voltage of the X-ray is 220kV, and the current is 40 mA.

Preferably, when the tumor cells are T-cell lymphoma cells, the irradiation dose is 75 Gy. Preference is given to

When the tumor cells are breast cancer cells, the irradiation dose is 50 Gy.

Further, the tumor vaccine is an injection vaccine.

The invention provides a preparation method of the tumor vaccine, which comprises the following steps: directly contacting tumor cells with lactic acid, collecting tumor cells, and inactivating.

The invention provides application of the tumor vaccine in preparing a medicament for preventing and treating tumors.

The invention provides an application of lactic acid as an active ingredient in preparing a medicament for preventing and treating tumors.

Further, the medicine is a tumor vaccine.

Preferably, the tumor vaccine is a tumor whole cell vaccine.

Preferably, the tumor is lymphoma or breast cancer.

Preferably, the lymphoma is a T-cell lymphoma.

Further, the tumor vaccine is an injection vaccine.

In the present invention, the tumor vaccine is preferably prepared using homologous tumor cells.

The invention provides a novel tumor vaccine. The mouse transplanted tumor model experiment proves that the vaccine obtained by stimulating the tumor cells with the lactic acid shows obvious anti-tumor effect in both preventive and therapeutic experiments, can inhibit the growth of tumors, prolong the survival time of experimental mice, improve the survival rate, and has the effect obviously superior to that of inactivated tumor cells which are not stimulated with the lactic acid. The invention provides a new drug selection for clinically preventing and treating tumors and a new tumor treatment strategy.

Drawings

FIG. 1 is a graph showing the effect of different concentrations of lactic acid on the growth of tumor cells in example 1;

FIG. 2 is a graph showing the phagocytosis of the whole tumor cell vaccine of the present invention by dendritic cells in example 2;

FIG. 3 is a graph showing the results of the preventive experiment in example 3;

FIG. 4 is a graph showing the results of the therapeutic experiment in example 3;

FIG. 5 is a graph showing the results of the cellular and humoral immunity experiments in example 4;

FIG. 6 is a graph showing the results of the antibody blocking assay in example 5;

FIG. 7 is a graph showing the results of the in vitro cytotoxicity test in example 6;

FIG. 8 is a graph showing the results of measuring IFN-. gamma.levels in spleen lymphocytes of example 7.

Detailed Description

The present invention has been completed based on the following findings of the inventors: low pH and lactate accumulation are characteristic manifestations of the tumor microenvironment. However, the present inventors have observed in their studies that lactic acid has a function of inhibiting tumor growth, and the inhibitory effect is enhanced with increasing concentration. Based on this, the present invention attempts to enhance the antitumor effect of inactivated tumor cells by lactic acid stimulation.

Further, irradiation is a commonly used physical method for inactivating tumor cells, which can enhance the expression of MHC class I molecules, promote antigen presentation, and recognition of irradiated cells by cytotoxic T cells, but the irradiated cells have very limited inhibition of tumor growth. The invention utilizes lactic acid to enhance the immunogenicity of the irradiated tumor whole cell vaccine, thereby enhancing the anti-tumor effect of the vaccine.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Materials and methods

1. Animal models and cell lines

The mice used in the following experiments were 6-8 weeks old BALB/C, C57BL/6J female mice and nude mice, 16-18 g, purchased from Vitamin River Laboratories (VRL) of Beijing Wintolite laboratory animal technology Inc., and bred in SPF-level animal houses of the laboratory.

The cell lines used in the following experiments were mouse T cell lymphoma cell line E.G7, mouse breast cancer cell line 4T1, anti-CD8 monoclonal antibody hybridoma (clone 2.43, rat IgG), anti-CD4 monoclonal antibody hybridoma (clone GK1.5, rat IgG) and anti-NK monoclonal antibody hybridoma (clone PK136), all available from ATCC (American type Culture Collection) of America. G7 was cultured in RPMI medium 1640 (Thermo Fisher Scientific Co., U.S.A.) containing 10% fetal bovine serum (Invitrogen Co., U.S.A.) and 0.4mg/ml G418 (MP Biomedicals Co., U.S.A.). 4T1 cells were cultured in RPMI Medium 1640 containing 10% fetal bovine serum. Hybridoma cells (clone 2.43) were cultured in DMEM medium containing 10% fetal bovine serum. PK136 cells were cultured in Hybri-care medium (ATCC, USA) containing 10% fetal bovine serum. GK1.5 cells were cultured in Iscove's modified Dulbecco's medium (Invitrogen, USA) containing 10% fetal bovine serum.

Separation and culture of dendritic cells: bone marrow-derived Dendritic Cells (DCs) were isolated according to the previous study (see: Li, M., et al, Effective inhibition of melanoma tuberculosis and growth via a new complex vaccine based on NY-ESO-1-alum-polysaccharide-HH2.mol Cancer,2014.13: p.179.). Bone marrow of C57BL/6J mouse tibia is extracted, cultured in 1640 medium, and 10ng/ml GM-CSF, 500ng/ml beta-hydroxy ethanol, 500ng/ml sodium pyruvate and 10ng/ml IL-4 are added to maintain the cell concentration at 2X 10⁶One per ml. Mature DCs were collected for use on day 6 or day 7 by changing 50% of the medium at day 3 and day 5 after cell isolation.

2. Vaccine preparation

After passage of tumor cells, 20mmol/L Lactic acid (Lactic acid) is added and placed in CO₂The cells were incubated in a constant temperature incubator for 24 hours (see: 1, Wu, H., et al, Central role of lactic acid in cancer cell resistance activation-induced cell death. J Pathol,2012.227(2): p.189-99; 2, Fischer, K., et al, inhibition effect of tumor cell-derived lactic acid on human T cells. blood,2007.109(9): p.3812-9.). The cells were subsequently harvested, centrifuged, the supernatant discarded and washed 3 times with PBS, finally resuspended in the corresponding serum-free, antibiotic-free medium and counted using a cell counting plate (mean number of cells in each square × 10)⁴I.e., the number of cells per ml of liquid) and adjusted to a cell concentration of 1X 10⁷Cells/1 ml (1X 10)⁶Cells/100. mu.l). Inactivation was then performed by irradiation with X-rays at 220kV/40mA (Rad source Rs2000 irradiator). The irradiation dose of G7 cells was 75Gy, and the irradiation dose of 4T1 cells was 50 Gy. In the following experiments, the total dose per mouse was 200. mu.l.

3. Reagents and antibodies

OVA 257-264 and OVA 323-339 were purchased from InvivoGen, USA. Geneticin (G418) was purchased from MPBiomecicals, USA. The lymphocyte separation liquid is purchased from Shenzhenjindacae and is a biotechnology company. Leukocyte activation mixture, anti-CD4+ -APC monoclonal antibody, anti-CD8+ -FITC monoclonal antibody, anti-CD 11b + -FITC monoclonal antibody, anti-PHK 67-PE monoclonal antibody, anti-IFN-gamma + -FITC monoclonal antibody were purchased from BD company of America. anti-NK, anti-CD 4and anti-CD8 monoclonal antibodies were extracted from PK136, GK1.5 and clone 2.43 hybridoma cells, respectively.

4. Statistical analysis

The statistical analysis software used in this experiment was SPSS 23.0. The statistical method of the experimental data adopts one-factor analysis of variance. The Log-rank test was used for survival curve analysis. P values <0.05 are statistically different.

EXAMPLE 1 Effect of lactic acid on tumor cell growth

1. Experimental methods

The effect of lactic acid on tumor cell proliferation was analyzed by the MTT method. Collecting log-phase grown E.G7 cells, centrifuging to remove supernatant, resuspending and adjusting cell concentration to 2 × 10⁴And/ml. Dividing the resuspended E.G7 cells into 5 groups, and adding 0mmol/L,5mmol/L,10mmol/L,20mmol/L and 30mmol/L lactic acid respectively. The prepared cell suspension was measured in a volume of 200. mu.l per well in a sterile 96-well plate, each set of 3 replicate wells, and the marginal wells were filled with sterile physiological saline. The above 96-well plate was incubated in a 5% CO2 incubator at 37 ℃ for 24 hours. Subsequently, 20. mu.l of MTT solution (5mg/ml) was added to each well, and the mixture was incubated in an incubator for 4 hours. Taking out the 96-well plate, centrifuging for 5min at 250g by a centrifuge, removing the supernatant, adding 150 μ l of dimethyl sulfoxide into each well, and placing the 96-well plate on a shaking table to shake for 10min at low speed to fully dissolve crystals. The 96-well plate was placed on a microplate reader, and absorbance was measured at a wavelength of 570 nm. Analyzing the detection result: the inhibition rate of cell proliferation is equal to the absorbance of the experimental group/the absorbance of the control group × 100%.

2. Results

In order to study the influence of lactic acid on tumor cells, the experiment adds lactic acid with different concentrations to stimulate the tumor cells when the tumor cells are cultured in vitro, and observes the growth and proliferation of the tumor cells. After 24h, the proliferation of the tumor cells was observed under a microscope. As a result, it was found that lactic acid at various concentrations inhibited the proliferation of tumor cells, and the intensity of the inhibition was proportional to the lactic acid concentration (FIGS. 1A-E), and in the 20mmol/L concentration group, the tumor cells hardly proliferated, while in the 30mmol/L concentration group, the tumor cells were almost completely apoptotic. MTT was also used in this experiment to detect the inhibition of tumor cell proliferation (FIG. 1F), and it was found that the lactic acid concentration was increased and the inhibition of cell proliferation was enhanced, and that the 20mmol/L concentration group had significant statistical differences compared with the two concentration groups of 5mmol/L and 10mmol/L, but no statistical differences compared with the 30mmol/L concentration group (P <0.05or P < 0.01; ANOVA).

The above experimental results show that lactic acid can inhibit the growth of tumor cells in vitro.

Example 2 phagocytosis of dendritic cells by the tumor Whole cell vaccine of the present invention

1. Experimental methods

In order to study whether the phagocytosis of the whole tumor cell vaccine by the DC is enhanced, the experiment stains the irradiated tumor cells which are stimulated by lactic acid with different concentrations by PHK67, then the cells are mixed with the DC for overnight culture, and the percentage of the cells with double positive CD11c and PHK67 is detected after the DC is screened by a flow cytometer. The specific experimental method is as follows:

g7 cells were stimulated with a lactate concentration gradient, comprising: 0mmol/L,5mmol/L,10mmol/L,20mmol/L, and 30mmol/L, and irradiating. With Diluent Diluent C (100. mu.lper 2X 10)⁵cells), adding 0.4. mu.l of PKH67 staining solution into each 100. mu.l of Diluent C, repeatedly sucking and beating for 10 times, and incubating at room temperature for 2-5 min. The reaction was neutralized with 1640 complete medium and washed 2 times with 10ml of complete medium each time. The cells were resuspended in the medium in which the DCs were cultured and counted. The prepared tumor cells were mixed with DC cells at a ratio of 1: 1, mixed culture overnight. The percentage of cells that were double positive for CD11c and PHK67 was determined using flow cytometry and the results are shown in fig. 2A, 2B.

To further study the phagocytic function of DCs, 4 experimental groups were set up: group 1: culturing DCs alone; group 2: co-culturing DCs with untreated e.g7 cells; group 3: co-culturing DCs and the irradiated E.G7 cells; group 4: DCs were co-cultured with 20mmol/L lactate-stimulated and irradiated E.G7 cells. The percentage of cells that were double positive for CD11C and PHK67 was determined using flow cytometry and the results are shown in fig. 2C, fig. 2D.

2. Results

As can be seen from FIGS. 2A and 2B, at lactic acid concentrations of 0mmol/L,5mmol/L,10mmol/L,20mmol/L and 30mmol/L, the percentage of DCs phagocytosed by tumor cells to the total amount of DCs was 12.22% + -0.078%, 16.415% + -0.32%, 16.89% + -0.88%, 18.28% + -1.10%, 31.69% + -1.46%, and the difference was statistically significant (P <0.05, P <0.01) compared to lactic acid concentration of 20 mmol/L. The experiment can find that the phagocytosis of the DC cells on the treated tumor cells is enhanced along with the increase of the concentration of the lactic acid. To avoid the effect of apoptotic tumor cells on the experimental results, the following experiment chose a lactate concentration of 20mmol/L as the experimental concentration.

As can be seen from fig. 2C and fig. 2D, the percentage of tumor cell phagocytized DC in

groups

1, 2 and 3 to the total DC was 0.45% ± 0.071%, 6.845% ± 0.16%, 12.22% ± 0.078%, respectively, and the difference was statistically significant compared to group 4 (. P <0.05,. P < 0.01). The experiment can find that the phagocytosis of DC to tumor cells can be increased by irradiation, and the phagocytosis of DC to tumor cells irradiated after being stimulated by lactic acid is stronger.

Example 3 prevention experiment and treatment experiment of the tumor Whole cell vaccine of the present invention

1. Experimental methods

The experiment selects a C57BL/6J female mouse (used for an E.G7 mouse T cell lymphoma model) or a BALB/C mouse (used for a 4T1 mouse breast cancer model) with the age of 6-8 weeks and the weight of 16-18 g. Each model was divided into 3 groups of 10 mice each. The specific grouping is as follows: 1) saline group (saline): 200. mu.l of physiological saline; 2) control group: 1X 10⁶Mu.l or 1X 10 cells/100. mu.l of 75Gy irradiated E.G7 cells⁶Cells/100 μ l of 4T1 cells irradiated to 50Gy in 200 μ l; 3) experimental groups: 1X 10⁶Cells/100 μ L of E.G7 cells stimulated with 20mmol/L lactic acid (lactic acid) for 24h and irradiated with 75Gy at a dose of 200 μ L or 1X 10⁶Cells/100. mu.l of 4T1 cells stimulated with 20mmol/L lactate for 24h and irradiated to 50 Gy.

Prevention animal model: selecting corresponding fruitThe mice were tested and randomly divided into the above three groups, and on the 0 th, 14 th and 21 st days of the test, the mice were administered subcutaneously at the axilla and groin at multiple points, the physiological saline group was administered, and the control group and the test group were administered 2X 10 times as described above⁶The prepared tumor cells. Mice were inoculated subcutaneously 3X 10 on day 28⁶G7 cells or 1X 10⁶4T1 cells.

Treatment group animal models: selecting corresponding experimental mice, randomly dividing into three groups, and inoculating 3 × 10 mice on the 0 th day of experiment⁶G7 cells or 1X 10⁶4T1 cells. 5-7 days after inoculation, when the tumor size is about 2mm, the injection is subcutaneously administered at the armpit and the groin of the mouse, the normal saline group is administered with normal saline, and the control group and the experimental group are respectively administered with 2 multiplied by 10⁶The prepared tumor cells. 1 time a week.

After the tumor of the inoculated mouse grows out, measuring the size of the tumor every 2 days, wherein the calculation formula of the tumor volume is as follows: major diameter x minor diameter²×0.52。

2. Results

2.1 tumor-inhibiting Effect in preventive experiments

Two animal models, the E.G7 mouse T cell lymphoma model and the 4T1 mouse Breast Cancer model, were first established in this experiment (see: Zeng, S., et al, Formulation, Characterisation, and anticancer Properties of Trans-and Cis-Citral in the 4T1Breast Cancer xenogeneft mouse model, 2015.32(8): p.2548-58.). As can be seen from FIG. 3A, the tumor growth volumes of the E.G7 control group (E.G7) and the E.G7 experimental group (E.G7/lactic acid) mice were significantly reduced compared to the saline group, and the tumor volumes of the two groups were 1039 + -174 mm³And 215 + -45 mm³The saline group and the control group had significant statistical differences (P) compared with the experimental group<0.05or**P<0.01; ANOVA). As can be seen from FIG. 3B, the survival time of the experimental group mice was significantly prolonged. In the 4T1 animal model group (fig. 3D and 3E), similar results were observed, and the tumor growth was significantly inhibited and the survival time was significantly prolonged in the mice of the experimental group compared to the saline group and the control group.

As can be seen from fig. 3C, on day 7 after tumor inoculation in the e.g. g7 animal model, the tumor free rate of the mice in the saline group was 0%, the tumor free rate of the mice in the e.g. g7 control group and the e.g. g7 experimental group was 20%, the tumor free rate of the mice in the saline group was 0% when day 43 after tumor inoculation, and the tumor free rate of the mice in the e.g. g7 control group and the e.g. g7 experimental group was 30% and 80%, respectively. In both the control and experimental groups, 80% of mice had tumors outgrowth after tumor inoculation. However, as time goes by, the tumor of the mice in the experimental group gradually shrinks and disappears, and only 20% of the mice are loaded with the tumor, while the tumor rate of the control group is 70%. In the 4T1 animal model group (FIG. 3F), the tumor-free rates of the saline group and the control group were 0%, while the tumor-free rate of the experimental group was 40%.

The experimental results show that the tumor cells after being stimulated and irradiated by lactic acid have the anti-tumor effect, have the inhibition effect on the growth of subcutaneous solid tumors, and can prolong the survival time of experimental mice after inoculation and increase the survival without tumors.

2.2 tumor-inhibiting Effect in therapeutic experiments

In order to further research whether the tumor whole cell vaccine has therapeutic effect, animal models of two therapeutic experimental groups are established, and the tumor growth and survival period of the mice in the experiment are evaluated. In the E.G7 animal model (FIG. 4A, FIG. 4B), mice died and tumor detection was stopped at day 29, and the mean tumor volume of 2750 + -240 mm was reached in the saline group mice at day 26 after inoculation³The tumor volumes of mice in the E.G7 control group (E.G7) and the E.G7 experimental group (E.G7/lactic acid) are 1567 +/-229 mm³And 1141. + -. 327mm³The tumor volume of the mice in the normal saline group is obviously larger than that of the experimental group, and the difference has statistical significance (. about.P)<0.05or**P<0.01; ANOVA). All mice were tumor bearing, but the survival of the experimental group tumor bearing mice was significantly prolonged. Similar results can be seen in the 4T1 animal model (fig. 4C, fig. 4D).

Example 4 the approach of the tumor whole cell vaccine of the present invention to exert antitumor effect

1. Experimental methods

To clarify the role of cellular and humoral immunity in the antitumor process, splenic lymphocytes were isolated in this experiment, and plasma was extracted from mice of the vaccine immunization group or the control group and then infused before tumor inoculation.

To examine the antitumor effect of humoral immunity, 16-18 g of C57BL/6J female mice 6-8 weeks old were selected, divided into three groups according to the protocol of example 3, and the mice were inoculated subcutaneously with 3X 10 cells on day 1⁶G7 cells were injected into tail vein of mice of each group on day 0 and day 2 with 150 μ l of serum (serum of mice of the corresponding group of example 3), and thereafter injected 2 times per week for 3 weeks.

To examine the antitumor effect of cellular immunity, splenic lymphocytes were collected according to the previous study method (see: Hodgkins, S.R., et al, NO2 immunization analysis of cellular immunity of cellular lymphocytes CD11c + cell type chemotherapy CD4+ T cell polarization. Respir, 2010.11: p.102.). After the mice of example 3 were immunized, on the 28 th day of the experiment, the mice were sacrificed by cervical dislocation and the spleens of the mice were removed. The filter was placed in a clean petri dish, the mouse spleen was then placed in the filter, a small amount of lymphocyte separator was added, and the spleen was gently ground with the bottom of the syringe plunger. Transferring the lymphocyte separation liquid containing spleen lymphocytes into a 15ml centrifuge tube, and slowly adding 200-500 mu l of serum-free and antibiotic-free 1640 culture medium to cover the separation liquid. After centrifugation, the cells were harvested, resuspended again and counted. Selecting 6-8 week old 16-18 g C57BL/6J female mice, dividing into three groups according to the scheme of example 3, and inoculating the mice subcutaneously with 3 × 10 on day 1⁶G7 cells, injected in tail vein of mice of each group at day 0 by 1X 10⁷Splenic lymphocytes and injected 2 times a week after inoculation.

2. Results

The results suggest that lymphocytes extracted from mice immunized with lactic acid-stimulated + irradiated e.g. 7 cells significantly inhibited tumor growth compared to the control group (fig. 5A). However, there was no significant difference in tumor growth rates in mice receiving plasma infusions (fig. 5B). The antitumor effect of the tumor whole cell vaccine is not shown by humoral immunity.

Example 5 antibody blocking assay

The cell adoptive experiment and the humoral adoptive experiment of the embodiment 4 confirm that the cell immunity plays an important role in the anti-tumor effect of the tumor whole cell vaccine. In order to determine which immune cells occupy important positions in the anti-tumor effect, an E.G7 animal model is selected in the experiment, and CD4+, CD8+ and NK cell knockout mice are established.

1. Experimental methods

Recovering the anti-CD8 monoclonal antibody hybridoma (clone 2.43, rat IgG), the anti-CD4 monoclonal antibody hybridoma (clone GK1.5, rat IgG) and the anti-NK monoclonal antibody hybridoma (clone PK 136). Collecting three cells growing in logarithmic phase after passage, centrifuging at 2500rpm for 5min with a common high speed centrifuge, removing supernatant, collecting cells, repeatedly washing with serum-free and antibiotic-free 1640 culture medium for 3 times, counting with a cell counting plate, and adjusting cell concentration of the three cells to 2 × 10⁶Cells/ml (1X 10)⁶Cells/500. mu.l).

The nude mice were randomly divided into 3 groups of 10 mice each, and 500. mu.l/l of norphytane was intraperitoneally injected into each nude mouse on the 0 th day of the experiment to promote the formation of ascites in the nude mice. On the 7 th day of the experiment, three groups of nude mice were injected with three kinds of hybridoma cells intraperitoneally, and each nude mouse was injected with 800-⁶Cell/cell), gently kneading the abdomen of the nude mouse after injection to mix the cells evenly in the abdominal cavity of the mouse. Observing the growth condition of the ascites of the nude mice after injection, slowly expanding the abdomen of the nude mice 7-10 days later, extracting the ascites of the nude mice by using an injector, centrifuging for 3min at 1200rpm by using a common high-speed centrifuge, and collecting supernatant. Three groups of supernatants each contained anti-CD8⁺Monoclonal antibody, anti-CD4⁺Monoclonal antibodies and anti-NK cell monoclonal antibodies.

C57BL/6J mice were selected and randomly divided into 7 groups of a, b, C1, C2, C3, C4 and C5, 8 mice in each group were immunized subcutaneously on the 0 th, 14 th and 21 st days of the experiment, and the grouping was as follows: a: saline group, b: g7 control, c 1: e.g7 experimental group, c 2: g7 panel + anti-CD4+ cell monoclonal antibody panel, c 3: g7 panel + anti-NK cell monoclonal antibody panel, c 4: g7 panel + anti-CD8+ cell monoclonal antibody panel, c 5: g7 experimental group + IgG monoclonal antibody group.

The whole tumor cell vaccine of the present invention was prepared according to the method described in the materials and methods, and administered subcutaneously at multiple sites in the axilla and groin of mice on

days

0, 14 and 21 of the experiment in a total amount of 200. mu.l per mouse. Mice were given a total of 7 doses of antibody blocking at a dose of 500 μ g per mouse, 1 day before and 2 times per week after immunization. On day 28 of the experiment, mice were vaccinated 3X 10⁶And E, G7 cells, measuring the size of the tumor once every 2 days after the tumor of the inoculated mouse grows out, and calculating the tumor volume. The results of the experiment are shown in FIG. 6.

2. Results

As can be seen from fig. 6A, tumor volume was significantly greater than blocking CD4+ and blocking NK group 17 days after blocking CD8+ T cells, with a more significant difference at day 27. The tumor volumes shown in FIG. 6B were 3199.20. + -. 415.22mm in the above 7 groups of mice on day 27 after tumor inoculation³、689.41±184.28mm³、114.95±33.85mm³、103.13±16.79mm³、260.97±33.09mm³、1985.05±299.53mm³、147.50±24.82mm³. The results show that after the blocking of CD8+ T cells in mice, the mean tumor volume is significantly larger than that of the control group and smaller than that of the normal saline group, and the difference is statistically different (P)<0.01). The mean tumor volumes of the CD4+ T lymphocyte blocking, NK cell blocking and IgG blocking groups were not significantly increased compared to the experimental group. From the analysis in the graph, it can be found that the CD8+ T lymphocyte plays an important role in the specific anti-tumor immunity induced by the tumor whole cell vaccine of the invention.

Example 6 in vitro cytotoxicity assay

Through a cell adoptive experiment and an antibody blocking experiment, the CD8+ lymphocyte has an anti-tumor immunity effect in the tumor whole cell vaccine. Next, for this experiment⁵¹Cr release assay to detect killing activity of CTL.

1. Experimental methods

According to the previous research report⁵¹Cr release assay for detecting spleen lymph of E.G7 immunized miceCytotoxicity of cells (see: 1, Lu, Y., et al, Immunogene therapy of tumors with vaccine base on xenogenic epidermal growth factor receptor. J Immunol,2003.170(6): p.3162-70; 2, Liu, Z., et al, Il-21 enzymes NK cell activation and cytolytic activation and indexes Th17 cell differentiation in inflammatory bone tissue disk, 2009.15(8): p.1133-44). With 100. mu. Ci⁵¹Cr-labeled E.G7 cells, and adjusting the cell concentration to 1X 10⁵And/ml. Mixing 100 μ l⁵¹Cr-labeled target cells were added to 96-well plates, 3 replicate wells per sample, grouped as follows: 1) spontaneous release group: add 100. mu.l serum-free, antibiotic-free 1640 medium to each well; 2) maximum release group: add 100. mu.l of 0.5% TritonX-100 into each well; 3) CTL activity assay group: each well was measured according to effector cell: target cell ratio 100: 1. 50: 1. 25: 1 and 12.5: 1 to 100. mu.l of spleen lymphocytes were added. And (3) putting the 96-well plate mixed with the target cells and the effector cells into a 5% CO2 constant-temperature cell culture box at 37 ℃ for incubation for 4-6 hours, centrifuging for 5 minutes at 250g by using a centrifuge, slightly sucking out 100 mu l of supernatant of each well, and detecting the radioactivity of each well on a machine. Radioactivity calculation formula: cell-specific killing rate ═ [ (experimental group release-spontaneous release)/(maximum release-spontaneous release)]×100。

In addition, specific CTLs were again detected using Tetramer technology. Mice were grouped and immunized according to the procedure described in the experimental protocol and spleen T lymphocytes were extracted from the mice, stimulated with 50. mu.g/ml OVA protein for 72H, and tested for CD8+ and H-2K by flow cytometry^bOVA tetramer positive cells are OVA-specific CTL.

2. Results

As shown in fig. 7, when the ratio of target cells to effector cells is 1: at 100, the killing rate of spleen lymphocytes of the normal saline group, the E.G7 control group (E.G7) and the E.G7 experimental group (E.G7/lactic acid) to the E.G7 is 2.2 +/-0.48%, 3.81 +/-0.94% and 9.35 +/-1.07% respectively, when the ratio of target cells to effector cells is 1: at 50, the killing rate of the spleen lymphocytes in the three groups to the E.G7 is respectively 0.615 +/-0.06%, 1.93 +/-0.49% and 2.99 +/-0.91%, when the ratio of target cells to effector cells is 1: at 25, the killing rate of the spleen lymphocytes to E.G7 in the three groups is respectively 0.46 +/-0.11%, 0.53 +/-0.26% and 2.22 +/-0.53%, when the ratio of target cells to effector cells is 1: at 12.5, the killing rates of the spleen lymphocytes in the three groups to the E.G7 are respectively 0.16 +/-0.11%, 0.14 +/-0.03% and 1.18 +/-0.13%. The ratio of target cells to effector cells was 1: 100. 1: 50. 1: 25. 1: at 12.5, the killing activity of lymphocytes in the experimental group is obviously higher than that of the normal saline group and the control group, and the statistical difference is obvious (P <0.05, P < 0.01; ANOVA). And as the ratio of target cells to effector cells increases, the killing activity increases.

The experimental results show that the tumor whole cell vaccine can increase and induce specific cytotoxic T lymphocytes to kill tumor cells.

Example 7 Interferon-Gamma (INF-Gamma) levels in spleen and peritumoral lymph nodes

1. Experimental methods

16-18 g of C57BL/6J mice with the age of 6-8 weeks are selected, experimental mice are immunized according to the grouping and method, and spleen lymphocytes and inguinal lymph node lymphocytes of the mice are extracted on the 7 th day after the last immunization. Separating T lymphocytes, making into single cell suspension, adding 10 μ g/ml polypeptide OVA_323–339Or OVA_257–264Culturing for 24h, and adding a Golgi blocker (see: Martin, M.D., et al, The impact of pre-existing memory on differentiation of newly regulated negative CD 8T cells, 2011.187(6): p.2923-31) in The last 4-6 h. The proportion of lymphocytes of CD4+ IFN-. gamma. + and CD8+ IFN-. gamma. + was measured by FACScan flow cytometry (Becton Dickinson) and analyzed by Cell Quest (BD Co., USA) software.

The level of INF-gamma was further verified by enzyme-linked immunosorbent assay (ELISA) in this experiment. Extracting spleen lymphocytes and inguinal lymph node lymphocytes according to the above method, adding 10 μ g/ml polypeptide OVA_323–339Or OVA_257–264And culturing for 48h, and taking supernatant for ELISA detection.

2. Results

Extracting splenic lymphocyte and paraneoplastic lymph node cell, adding OVA_323–339Or OVA_257–264And (5) polypeptide culture. Flow cytometry results showed that lymphocytes of CD4+ IFN-. gamma. + and CD8+ IFN-. gamma. + were increased in both splenic and paraneoplastic lymphocytes in the experimental group of mice compared to the control group (FIG. 8A, FIG. 8B). The ELISA experiment also confirmed the above results (fig. 8C).

Conclusion

The present invention observes that lactic acid has the function of inhibiting tumor growth. The antitumor effect of lactic acid stimulated tumor cells was then investigated. In an E.G7 mouse T cell lymphoma model and a 4T1 mouse breast cancer model, the tumor vaccine treated by lactic acid stimulation and irradiation shows a more remarkable tumor growth inhibition function. In order to further research the action mechanism of the tumor vaccine, spleen lymphocytes or plasma are extracted from mice immunized by lactic acid + irradiated E.G7 cells and then injected into tumor-bearing mice, and the result indicates that cellular immunity rather than humoral immunity plays an anti-tumor role. Subsequently, the present invention investigated the antitumor effects of CD4+, CD8+ T lymphocytes and NK cells. Blocking CD8+ T lymphocytes can cause a significant decrease in antitumor activity; blocking the NK cells results suggested a mild effect of NK cells on anti-tumor activity; blocking CD4+ T lymphocytes against tumor activity. In addition, the invention passes the standard⁵¹The Cr release assay detects the cytotoxicity of spleen lymphocytes. OVA-specific CTLs were detected by flow cytometry in the lactic acid-stimulated e.g. 7 vaccine group as increased compared to the other groups. Finally, both flow cytometry and ELISA experiments suggested activated CD8+ T cells (CD8+, IFN-. gamma. +) and CD4+ T cells (CD 4) in the lactate group⁺,IFN-γ⁺) Is increased obviously compared with the control group. Based on the research, the lactic acid can enhance the immunogenicity of the tumor whole cell vaccine, and the tumor cell vaccine stimulated by the lactic acid can possibly play an anti-tumor role through CD8+ T lymphocytes.

It should be appreciated that the particular features, structures, materials, or characteristics described in this specification may be combined in any suitable manner in any one or more embodiments. Furthermore, the various embodiments and features of the various embodiments described in this specification can be combined and combined by one skilled in the art without contradiction.

Claims

1. A tumor vaccine characterized by: it is prepared by inactivating tumor cells stimulated by lactic acid; the lactic acid stimulated tumor cells are obtained by mixed culture of lactic acid and tumor cells for 24 hours; wherein the concentration of the lactic acid is 20mmol/L, the tumor cells are T cell lymphoma cells or breast cancer cells, and the inactivation is irradiation inactivation; when the tumor cells are T cell lymphoma cells, the irradiation dose is 75 Gy; when the tumor cells are breast cancer cells, the irradiation dose is 50 Gy.

2. The tumor vaccine of claim 1, which is characterized by: culturing the tumor cells in a carbon dioxide cell incubator.

3. The tumor vaccine of claim 1, which is characterized by: the irradiation inactivation is X-ray irradiation inactivation.

4. The tumor vaccine of claim 3, which is characterized by: the working voltage of the X-ray is 220kV, and the current is 40 mA.

5. The tumor vaccine of claim 1, which is characterized by: the tumor vaccine is an injection vaccine.

6. A method for preparing a tumor vaccine according to any one of claims 1 to 5, wherein: the method comprises the following steps: directly contacting tumor cells with lactic acid, collecting tumor cells, and inactivating.

7. Use of the tumor vaccine of any one of claims 1-5 in the preparation of a medicament for the prevention and treatment of T-cell lymphoma or breast cancer.