TW202015708A - Pharmaceutical combination of dendritic cell vaccines and immune checkpoint antibodies for treatment of brain tumors and use thereof for preparing the same - Google Patents

Abstract

The present invention provides a pharmaceutical combination for treatment of brain tumors in a subject in need thereof, comprising a dendritic cell vaccine containing dendritic cells pulsed with an irradiated antigen, and an antibody targeting immune checkpoint proteins of T cells. The present invention also provides an use of a dendritic cell vaccine and an antibody which targets immune checkpoint proteins on T cells in the preparation of a pharmaceutical combination for treatment of brain tumors.

Description

Dendritic cell vaccine and anti-T cell immune checkpoint protein antibody combination drug combination and its use in preparation of brain tumor tumor combination drug

The present invention relates to a combined drug combination for treating brain tumors and its use in the preparation of a combined drug for treating brain tumors, in particular to a combined use of dendritic cell vaccines combined with anti-T cell immune checkpoint protein antibodies for the treatment of brain tumors The combination of drugs, and the use of the same in the preparation of drugs for treating brain tumors.

Due to the different types of cells in the brain, malignant brain tumors can be divided into different forms according to their cell types, such as glioblastoma and medulloblastoma. Traditional treatments for brain tumors include surgery, radiation therapy, and chemotherapy. Because many tumor cells will spread to the surrounding tissues, they cannot be treated by traditional surgery. In this case, they can only be treated by the combination of radiation therapy and chemotherapy. However, if the brain is treated with radiation, it may have side effects, such as damaged neurons, and when treated with chemotherapy, large molecule drugs cannot be used because of the presence of the blood brain barrier. Unable to cycle into the brain. In addition, brain tumors such as glioblastoma may also be resistant to traditional therapies. Therefore, it is necessary to develop a new treatment strategy that can completely eradicate brain tumors.

One potential treatment in the current research is immunotherapy using dendritic cells. Dendritic cells are one of the most effective antigen-presenting cells in the immune system and can induce tumor-specific T cells. With this feature of dendritic cells, they can be used as a vaccine to trigger T cell responses to destroy tumor cells. Recent research has also pointed out that dendritic cell vaccine (hereinafter referred to as "DC vaccine") can prolong the survival time of cancer patients.

However, in the role of cancer, many tumor cells are known to be able to avoid the attack of the immune system by expressing specific surface proteins through their binding to immune checkpoint proteins on T cells. . For example, when the immune checkpoint protein PD-1 (programmed cell death protein 1) on T cells is combined with PD-L1 (programmed death-ligand 1) ligand on tumor cells, the expansion and proliferation of T cells, Survival and action, such as cytokine release and cytotoxicity, are inhibited, and this combination also induces apoptosis of tumor-specific action T cells. This effect of evading the attack of the immune system reduces the effectiveness of immunotherapy in treating cancer.

Therefore, in order to completely eliminate brain tumor cells when treating brain tumors with DC vaccines, it is necessary to develop a new therapeutic strategy that can promote the efficacy of DC vaccines on the one hand and inhibit tumor cells in the immune response on the other. Escape effect.

One of the objectives of the present invention is to provide a new medical combination, and then through the combination of DC vaccine and anti-T cell immune checkpoint protein antibody treatment, to achieve more effective brain tumor treatment results than before.

In order to achieve the aforementioned object, the present invention provides a use of dendritic cell vaccine and anti-T cell immune checkpoint protein antibody in the preparation of a drug for treating brain tumors, wherein the dendritic cell vaccine may include antigens stimulated by radiation-treated antigens Dendritic cells.

In an embodiment of the present invention, the dendritic cell vaccine includes dendritic cells capable of presenting glioma antigens, and the dendritic cell line is further treated with lysate of brain glioma cells treated with radiation A bone marrow cell is obtained after co-cultivation and differentiation.

In an embodiment of the invention, the brain glioma cells can be treated with X-rays at a dose of 10-30 Gy. In an embodiment of the present invention, the lysate of the brain glioma cells can be obtained after processing the brain glioma cells in a freeze-thaw cycle, and the dendritic cells can use lipopolysaccharide to convert the bone marrow cells Obtained by differentiation.

In an embodiment of the present invention, the anti-T cell immune checkpoint protein antibody may be an anti-PD1 antibody, an anti-CTLA-4 antibody, or an anti-CD25 antibody.

In an embodiment of the present invention, the dose of the anti-PD1 antibody or the anti-CD25 antibody may be 200 mg every two days for a total of 4 doses.

In another embodiment of the present invention, the anti-CTLA-4 antibody may be administered at a dose of 100 mg every three days for a total of 3 doses.

In still another embodiment of the present invention, the dendritic cell vaccine may include at least 1×10 ⁶ dendritic cells.

The present invention also provides a combined drug combination for treating brain tumors, including: a dendritic cell vaccine and an anti-T cell immune checkpoint protein antibody, wherein the dendritic cell vaccine may include a tree stimulated by a radioactive antigen Process cells.

In an embodiment of the present invention, the dendritic cell vaccine includes dendritic cells capable of presenting glioma antigens, and the dendritic cell line is further treated with lysate of brain glioma cells treated with radiation A bone marrow cell is obtained after co-cultivation and differentiation.

In an embodiment of the invention, the brain glioma cells can be treated with X-rays at a dose of 10-30 Gy.

In an embodiment of the invention, the lysate of the brain glioma cells can be obtained after processing the brain glioma cells in a freeze-thaw cycle.

In an embodiment of the present invention, the dendritic cells can be obtained by differentiating the bone marrow cells with lipopolysaccharide.

In another embodiment of the present invention, the anti-T cell immune checkpoint protein antibody may be an anti-PD1 antibody, an anti-CTLA-4 antibody, or an anti-CD25 antibody.

In an embodiment of the invention, the dose of the anti-PD1 antibody or the anti-CD25 antibody may be at least 200 mg.

In an embodiment of the invention, the dose of the anti-CTLA-4 antibody may be at least 100 mg.

In another embodiment of the present invention, the dendritic cell vaccine may include at least 1×10 ⁶ dendritic cells.

With the present invention, the anti-tumor cell effect of the DC vaccine, combined with the anti-T cell immune checkpoint protein antibody, can prevent cancer cells from evading the removal and poisoning effect of the relevant T cells, thus increasing their therapeutic effect. In particular, the DC vaccine made by dendritic cells prepared by radio-significantly treating the antigen of the present invention is more effective and significantly improves the survival rate of individuals with brain tumors.

The embodiments of the present invention will be further described below. The examples listed below are used to clarify the present invention, and are not intended to limit the scope of the present invention. Anyone who is familiar with this art, without departing from the spirit and scope of the present invention, Some changes and retouching can be done, so the scope of protection of the present invention shall be deemed as defined by the scope of the attached patent application.

In one embodiment of the present invention, an animal model with multiple glioblastoma characteristics in humans, namely mouse orthotopic brain glioma cell 261 (that is, GL261) model, was used to test the combination of dendritic cell vaccine (that is, DC vaccine). Treatment and immune checkpoint antibody treatment have excellent anti-cancer effects. In order to establish tumors and directly observe the tumor status in vivo through bioluminescence images, GL261 cells were implanted intracranially in C57BL/6JNarl mice of the animal model used, and the cell line was modified to stably express fluorescent Luminase (GL261-Luc2). Next, mice with GL261 tumors were periodically administered DC vaccine and anti-T cell immune checkpoint antibodies, and their survival rate was observed and analyzed. Examples of cell culture

Mouse glioma cell line GL261-Luc2 (Bioware® Ultra, PerkinElmer, Waltham, MA, USA) was cultured in humidified air containing 5% carbon dioxide at 37°C and supplemented with 10% fetal bovine serum protein (fetal bovine serum), glutamine, sodium pyruvate and non-essential amino acids of Dulbecco's Modified Eagle Medium (DMEM) ((GIBCO/Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA )) in the culture medium. Preparation of brain glioma cell freeze-thaw lysate

The aforementioned mouse brain glioma cell line GL261-Luc2 is irradiated with X-rays of 10-30 Gy, preferably 20 Gy, and then the culture is continued for another 24 hours. Then adjust the cell concentration to 1x10 ⁷ cells/ml, then perform six cycles of freeze-thaw reaction. Each reaction system is placed in liquid nitrogen for 3 minutes, and then placed in a 56°C water bath 3 minute. The aforementioned lysate obtained through the freeze-thaw reaction was centrifuged at 500 g for 5 minutes, and the supernatant was recovered and stored at -80°C until use. Stimulate the preparation of mature dendritic cells with tumor-specific antigens

First, the femurs of C57BL/6JNarl mice were immersed in 75% ethanol, and the bone marrow cells were washed out of the bones using Hank’s balanced salt solution (HBSS), and the red blood cells were removed. Then, the bone marrow cells were cultured in 10% fetal bovine serum protein, glutamic acid, sodium pyruvate, non-essential amino acids, 20ng/mL mouse recombinant granulocyte macrophage cell stimulating factor (GM-CSF) and 10 ng /mL mouse recombinant interleukin-4 (IL-4) in dendritic cell culture medium. After culturing for 4 days, 10 ml of fresh dendritic cell culture fluid was added to the aforementioned culture fluid. On the 6th day, the unattached cells, that is, immature dendritic cells were gently aspirated and incubated with the aforementioned supernatant of the freeze-thaw tumor cell lysate (100 μg) for 30 minutes. After that, differentiation was performed with lipopolysaccharides (LPS) for 24 hours. The dendritic cells thus obtained are mature dendritic cells, which are analyzed by flow cytometry. These cells can express surface antigens such as CD11c, CD40, CD80, and CD86. These stimulating mature dendritic cell lines with GL261 antigen were used as DC vaccines used in the following experiments. Animal experiment

The Department of Animal Experiments is conducted in accordance with the "Animal Care and Use Process" of China Medical University. 6-week-old female C57BL/6JNar1 mice (n = 99) were obtained from the National Laboratory Animal Breeding Center. To construct orthotopic tumor model, each C57BL / 6JNar1 mice were implanted with intracranial to 2x10 ⁴ cells of GL261-luc2. Hereinafter, the date of implantation will be referred to as day 0, and the date of any treatment to the mice thereafter will be referred to as a specific date after day 0. Every week after tumor cell implantation, the size of the tumor in the mouse brain was estimated using an in vivo imaging system (IVIS Spectrum, PerkinElmer, Waltham, MA, USA). In addition, the survival rate of mice with GL261 tumors was analyzed by Kaplan-Meier survival curve and log-rank test to calculate the average survival time and median survival time after tumor implantation. DC vaccine

On days 7, 10, and 14 after tumor implantation, mice with GL261 tumors were injected subcutaneously with the aforementioned dendritic cells (1×10 ⁶ cells) stimulated or unstimulated with the tumor-specific antigen. Antibody

Mice with GL261 tumors were further intraperitoneally injected with the following anti-T cell immune checkpoint protein antibodies that can block immune checkpoints (hereinafter referred to as "immunological checkpoint antibodies"): rat anti-mouse PD-1 monoclonal antibodies, For example, RMP1-14 strain (BioXcell), hamster anti-mouse CTLA-4 monoclonal antibody, such as 9H10 strain (BioXcell), and rat anti-mouse CD25 monoclonal antibody, such as PC-61.5.3 strain (BioXcell). For mice administered anti-PD-1 antibody or rat IgG2a isotype control antibody, a dose of 200 μg was given every 2 days from day 15 for 4 times. For mice given anti-CTLA-4 antibody or hamster IgG isotype control antibody, a dose of 100 μg was given every 3 days from day 7 for a total of 3 times. For mice given anti-CD25 antibody or rat IgG1 isotype control antibody, a dose of 200 μg was given every 2 days from day 15 for 4 times. The test time axis of the aforementioned administration conditions is shown in FIG. 1. Example 1 Example 1 Combination of DC vaccine and anti-PD-1 antibody treatment enhances the survival rate of mice with GL261 tumors

In order to evaluate the effect of the combined treatment of DC vaccine and anti-PD-1 antibody on survival rate, C57BL/6JNar1 mice implanted with mouse brain glioma cells GL261-luc2 were randomly divided into groups, and the following components were injected intraperitoneally: Stimulated dendritic cells (DC vaccine only), anti-PD-1 antibody, rat IgG2a isotype control antibody, combination of DC vaccine and anti-PD-1 antibody, or combination of DC vaccine and rat IgG2a isotype control antibody . After that, Kaplan-Meier survival curve was drawn for each group. The results are shown in Figure 2. According to the data, all mice receiving the combination of DC vaccine and anti-PD-1 antibody survived for at least 58 days, while mice receiving other treatments, such as DC vaccine alone or DC vaccine and rat IgG2a isotype control antibody Combination therapy only showed a fairly short-term survival status. The 120-day survival rate of mice treated with the combination of DC vaccine and anti-PD-1 antibody was 88.9%, while the 120-day survival rate of DC vaccine alone was 16.67%.

^a : 當估計值係由設限數據所獲得時，所觀察到的最長存活時間即為設限值。                                                         表2

實施例2 結合DC疫苗和anti-CTLA-4抗體治療對具GL261腫瘤小鼠存活率的增強作用The average survival time and median survival time of the treated mice can be estimated according to the results in FIG. 2. The results are shown in Table 1. In addition, the difference in survival between any of the above two groups was compared based on median survival time or survival rate using a log rank test. As shown in Table 2, compared to the treatment with DC vaccine alone (p<0.001), or the combination treatment with DC vaccine and rat IgG2a isotype control antibody (p=0.002), the DC vaccine and anti-PD-1 antibody The combination-treated mice showed a higher survival rate. This result proves that the combined treatment of DC vaccine and anti-PD-1 antibody can effectively improve the anti-tumor effect of DC vaccine. Table 1

^a : When the estimated value is obtained from the set limit data, the longest observed survival time is the set limit value. Table 2

Example 2 The effect of combining DC vaccine and anti-CTLA-4 antibody treatment on the survival rate of mice with GL261 tumor

CTLA-4 is a protein receptor on T cells, which inhibits and regulates the progress of immune responses and serves as an immune checkpoint. In order to further evaluate the effect of the combined treatment of DC vaccine and anti-CTLA-4 antibody on survival rate, C57BL/6JNar1 mice implanted with mouse brain neuroglioma cells GL261-luc2 were randomly divided into groups, and the following components were injected intraperitoneally: Unstimulated dendritic cells (DC vaccine only), anti-CTLA-4 antibody, hamster IgG isotype control antibody, a combination of DC vaccine and anti-PD-1 antibody, or a combination of DC vaccine and hamster IgG isotype control antibody. Then Kaplan-Meier survival curve was drawn for each group, and the results are shown in Figure 3. According to the data, all mice receiving the combination of DC vaccine and anti-CTLA-4 antibody survived for at least 41 days, while mice receiving other treatments, such as DC vaccine alone or combination treatment of DC vaccine and hamster IgG isotype control antibody Is relatively short. The 120-day survival rate of mice treated with the combination of DC vaccine and anti-CTLA-4 antibody was 62.5%, while the 120-day survival rate of DC vaccine alone was 14.29%.

^a : 當估計值係由設限數據所獲得時，所觀察到的最長存活時間即為設限值。                                                      表4

實施例3 結合DC疫苗和anti-CD25抗體治療對具GL261腫瘤小鼠存活率的增強作用The average survival time and median survival time of the treated mice can be estimated according to the results in FIG. 2. The results are shown in Table 3. In addition, the difference in survival between any of the above two groups was compared based on median survival time or survival rate using a log rank test. As shown in Table 4, compared with the DC vaccine-only treatment (p <0.001), or the combination treatment of the DC vaccine and the hamster IgG isotype control antibody (p = 0.024), the DC vaccine and anti-CTLA-4 antibody combination The treated mice showed a higher survival rate. This result proves that the combination therapy of DC vaccine and anti-CTLA-4 antibody can effectively improve the anti-tumor effect of DC vaccine. table 3

^a : When the estimated value is obtained from the set limit data, the longest observed survival time is the set limit value. Table 4

Example 3 The enhanced effect of combined DC vaccine and anti-CD25 antibody treatment on the survival rate of mice with GL261 tumor

CD25 is the a chain in the IL-2 receptor protein, which affects its affinity for binding to IR-2, and it is present on regulatory T cells. In order to further evaluate the effect of the combined treatment of DC vaccine and anti-CD25 antibody on survival rate, C57BL/6JNar1 mice implanted with mouse brain neuroglioma cells GL261-luc2 were randomly divided into groups, and the following components were injected intraperitoneally: Stimulated dendritic cells (DC vaccine only), anti-CD25 antibody, rat IgG1 isotype control antibody, a combination of DC vaccine and anti-CD25 antibody, or a combination of DC vaccine and rat IgG1 isotype control antibody. After that, Kaplan-Meier survival curves were drawn for each group, and the results are shown in Figure 4. The data shows that all mice treated with a combination of DC vaccine and anti-CD25 antibody survived for at least 39 days, while mice treated with other treatments, such as DC vaccine alone or combination treatment with DC vaccine and rat IgG1 isotype control antibody, were relatively short. The 120-day survival rate of mice treated with a combination of DC vaccine and anti-CD25 antibody was 16.67%.

^a : 當估計值係由設限數據所獲得時，所觀察到的最長存活時間即為設限值。                                                      表6

The average survival time and median survival time of the treated mice can be estimated according to the results in FIG. 4, and the results are shown in Table 5. In addition, the difference in survival between any of the above two groups was compared based on median survival time or survival rate using a log rank test. As shown in Table 6, compared to the treatment with DC vaccine alone (p <0.001), or the combination treatment with DC vaccine and rat IgG1 isotype control antibody (p = 0.005), the combination treatment with DC vaccine and anti-CD25 antibody Of mice showed a higher survival rate. This result proves that the combination treatment of DC vaccine and anti-CD25 antibody can effectively improve the anti-tumor effect of DC vaccine. table 5

^a : When the estimated value is obtained from the set limit data, the longest observed survival time is the set limit value. Table 6

實施例4           經放射線處理之抗原於經抗原刺激DC疫苗的抗腫瘤療效之促進作用Examples 1 to 3 have shown DC vaccines and combined treatment strategies using anti-immune checkpoint protein antibodies to block immune checkpoints. According to Table 7, the combination treatment of the present invention greatly improved the survival rate of mice. For example, after tumor cell implantation, the combined treatment of DC vaccine and anti-immune checkpoint protein antibody at 45 days, 60 days, and 120 days, compared with the combination treatment of DC vaccine and isotype antibody, respectively improved 157%~314%, 275%~489% and 183%~978%. Table 7

Example 4 The anti-tumor effect of radiotherapy-treated antigens on antigen-stimulated DC vaccines

In this embodiment, the promotion of the therapeutic effect of the DC vaccine comes from the above-mentioned tumor antigens required for the preparation of dendritic cells after radiation treatment, and then the lysate obtained by the freeze-thaw reaction to further prepare the DC vaccine. To confirm the effect of radiation-treated antigens on the anti-cancer efficacy of DC vaccines, C57BL/6JNar1 mice implanted with mouse brain glioma cells GL261-luc2 were randomly divided into groups, and the following components were injected intraperitoneally: those without antigen stimulation Dendritic cells (DC vaccine only), DC vaccine prepared after stimulation of lysate of brain glioma cells without radiation treatment (ie: DC vaccine stimulated by GBM lysate), stimulation of lysate of brain neuroblastoma cells treated with radiation The DC vaccine prepared afterwards (ie: DC vaccine stimulated by radiation-treated GBM lysate), or blank vector control group (administered saline). After that, Kaplan-Meier survival curves were drawn for each group. The results are shown in FIG. 5, and the average survival time and median survival time of the treated mice were estimated according to the results in FIG. 5. The results are shown in Table 8. According to the data, the median survival time of mice treated with radiation-treated GBM lysate-stimulated DC vaccine was 46±6.547 days, while the DC vaccine treatment without radiation-treated GBM lysate stimulated DC vaccine. Of mice had a median survival time of 32±4.472 days.

^a : 當估計值係由設限數據所獲得時，所觀察到的最長存活時間即為設限值。                                                      表9

實施例5 以經放射線或未經放射線處理抗原所刺激之DC疫苗結合anti-PD-1抗體組合治療的抗腫瘤作用評估The difference in survival between any of the above two groups is compared using a log rank test based on median survival time or survival rate. As shown in Table 9, compared to treatment with DC vaccine prepared after stimulation with non-radiation-treated brain glioma cell lysate (p = 0.035), treatment with radiation-treated brain glioma cell lysate The mice treated with the prepared DC vaccine showed a higher survival rate. This result proves that for the antigen-stimulated DC vaccine, if the tumor antigen used to induce dendritic cells is first treated with radiation, a more effective DC vaccine can be prepared. Table 8

^a : When the estimated value is obtained from the set limit data, the longest observed survival time is the set limit value. Table 9

Example 5 Evaluation of the antitumor effect of combination therapy with DC vaccine stimulated by radioactive or non-radiation-treated antigens combined with anti-PD-1 antibody

In order to further study the difference of anti-tumor effect of DC vaccine stimulated by radioactive or non-radiation-treated antigen combined with anti-PD-1 antibody combination therapy, C57BL implanted with mouse brain glioma cells GL261-luc2 / 6JNar1 mice were randomly divided into groups and injected intraperitoneally with the following components: dendritic cells without antigen stimulation (DC vaccine only), anti-PD-1 antibody, and DC vaccine without GBM lysate stimulation without radiation treatment (hereinafter referred to as DCv) ), DC vaccine stimulated by radiation-treated GBM lysate (hereinafter referred to as IR-DCv), combination of DC vaccine stimulated by radiation-treated GBM lysate and anti-PD-1 antibody, stimulated by radiation-treated GBM lysate The combination of DC vaccine and anti-PD-1 antibody, or blank vector control group (administered saline). After that, Kaplan-Meier survival curves were drawn for each group. The results are shown in FIG. 6, and the average survival time and median survival time of the treated mice were estimated based on the results in FIG. According to the data, the median survival time of mice treated with anti-PD-1 antibody was 31±1.309 days, and they were treated with a combination of DC vaccine and anti-PD-1 antibody stimulated by GBM lysate without radiation treatment. Of mice had a median survival time of 31±2.858 days, and all mice treated with the combination of radiation-treated GBM lysate-stimulated DC vaccine and anti-PD-1 antibody survived for at least 58 days. The day survival rate is as high as 88.89%.

^a : 當估計值係由設限數據所獲得時，所觀察到的最長存活時間即為設限值。                                                 表11

實施例6 以經放射線或未經放射線處理抗原所刺激之DC疫苗結合anti-CTLA-4抗體組合治療的抗腫瘤作用評估The difference in survival between any of the above two groups is compared using a log rank test based on median survival time or survival rate. As shown in Table 11, the DC vaccine stimulated with radiation-treated GBM lysate compared with anti-PD-1 DC vaccine and anti-PD-1 antibody combination treatment (p <0.0001), and the DC vaccine stimulated with radiation-treated GBM lysate and anti-PD The mice treated with the PD-1 antibody combination showed a higher survival rate. This result proves that in the treatment strategy of combination treatment with antigen-stimulated DC vaccine and anti-PD-1 antibody, the procedure of treating tumor antigens with radiation treatment plays a key role in improving the anti-tumor efficacy. Table 10

^a : When the estimated value is obtained from the set limit data, the longest observed survival time is the set limit value. Table 11

Example 6 Evaluation of the antitumor effect of combination treatment with anti-CTLA-4 antibody and DC vaccine stimulated by radioactive or non-radioactive antigens

In order to further study the difference of anti-tumor effect produced by the combination of anti-CTLA-4 antibody and DC vaccine stimulated by radioactive or non-radiation-treated antigens, C57BL implanted with mouse brain neuroglioma cells GL261-luc2 / 6JNar1 mice were randomly divided into groups and injected intraperitoneally with the following components: dendritic cells without antigen stimulation (DC vaccine only), anti-CTLA-4 antibody, and DC vaccine without GBM lysate stimulation without radiation treatment (hereinafter referred to as DCv) ). Radiation-treated GBM lysate stimulated DC vaccine (hereinafter referred to as IR-DCv), combination of radiation-treated GBM lysate stimulated DC vaccine and anti-CTLA-4 antibody, radiation-treated GBM lysate stimulated The combination of the DC vaccine and anti-CTLA-4 antibody, or the blank vector control group (administered saline). The Kaplan-Meier survival curve was then drawn for each group. The results are shown in FIG. 7, and the average survival time and median survival time of the treated mice were estimated based on the results in FIG. 7. The results are shown in Table 12. According to the data, the median survival time of mice treated with anti-CTLA-4 antibody was 28±1.837 days, and they were treated with a combination of DC vaccine stimulated with GBM lysate and anti-CTLA-4 antibody without radiation treatment. Of mice had a median survival time of 32±1.837 days, and all mice treated with a combination of DC vaccine stimulated with radiation-treated GBM lysate and anti-CTLA-4 antibody survived for at least 41 days. The daily survival rate is 62.5%.

^a : 當估計值係由設限數據所獲得時，所觀察到的最長存活時間即為設限值。                                                     表13

The difference in survival between any of the above two groups is compared using a log rank test based on median survival time or survival rate. As shown in Table 13, compared to the combination treatment of DC vaccine stimulated with GBM lysate and anti-CTLA-4 antibody without radiation treatment (p <0.0001), the DC vaccine stimulated with radiation-treated GBM lysate and anti -CTLA-4 antibody combination-treated mice showed a higher survival rate. This result proves that in the treatment strategy of combination treatment with antigen-stimulated DC vaccine and anti-CTLA-4 antibody, the procedure of treating tumor antigens with radiation treatment plays a key role in improving the anti-tumor efficacy. Table 12

^a : When the estimated value is obtained from the set limit data, the longest observed survival time is the set limit value. Table 13

In summary, the present invention uses a combination of drugs for the treatment of brain tumors, including antibodies against immune checkpoint proteins, such as anti-PD-1, anti-CTLA-4 and anti-CD25 antibodies, combined with radiation-treated tumors Antigens enhance the anti-tumor efficacy of DC vaccines, allowing tumor-bearing mice to significantly prolong their survival period, which is superior to the efficacy of previous treatment strategies. Therefore, the combination of the present invention including a DC vaccine and an anti-T cell immune checkpoint protein antibody can be combined with the excellent search and identification of tumor antigens of the DC vaccine, and the anti-T cell immune checkpoint protein antibody's inhibitory adjustment properties The blocking effect produced by the immune checkpoint prevents the tumor cells from evading the role of the immune system and completely eliminates the tumor cells. And in particular, the DC vaccine of the present invention, the tumor antigen used in the preparation process, is first treated by radiation, so that the prepared DC vaccine is treated with anti-T cell immune checkpoint protein antibody in the present invention Under the circumstances, it can further significantly improve the anti-tumor efficacy.

no

FIG. 1 is a schematic diagram of the timeline flow of administering DC vaccine and immune checkpoint protein antibody to mice transplanted with GL261-luc2 cells according to an embodiment of the present invention. Fig. 2 is an embodiment of the present invention in which mice with GL261 tumors were administered with DC vaccine without antigen stimulation (ie: DC vaccine only), anti-PD-1 antibody, rat IgG2a isotype control antibody, and DC vaccine Kaplan-Meier survival curve of anti-PD-1 antibody, or DC vaccine and rat IgG2a isotype control antibody. 3 is an example of the present invention in mice with GL261 tumors were administered without antigen-stimulated DC vaccine (ie: DC vaccine only), anti-CTLA-4 antibody, hamster IgG isotype control antibody, combined DC vaccine and Kaplan-Meier survival curve of anti-CTLA-4 antibody, or a combination of DC vaccine and hamster IgG isotype control antibody. FIG. 4 is an embodiment of the present invention in which mice with GL261 tumors were administered with DC vaccine without antigen stimulation (ie: DC vaccine only), anti-CD25 antibody, rat IgG1 isotype control antibody, combined DC vaccine and anti -Kaplan-Meier survival curve of CD25 antibody, or a DC vaccine and rat IgG1 isotype control antibody. FIG. 5 shows the DC prepared by stimulating DC vaccine without antigen stimulation (ie DC vaccine only) and non-radiation-treated brain glioma cell lysate in mice with GL261 tumors according to an embodiment of the present invention Vaccine (ie: DC vaccine stimulated by GBM lysate), DC vaccine prepared after stimulation of radiation-treated brain glioma cell lysate (ie: DC vaccine stimulated by radiation-treated GBM lysate), or blank vector control group Kaplan-Meier survival curve. 6 is an embodiment of the present invention is an embodiment of the present invention, respectively, in mice with GL261 tumors were administered without antigen-stimulated DC vaccine (ie: DC vaccine only), anti-PD-1 antibody, without radiation treatment DC vaccine prepared after stimulation of brain glioma cell lysate (ie: DC vaccine stimulated by GBM lysate, referred to as DCv), DC vaccine prepared after stimulation of radiation-treated brain glioma cell lysate (ie: radiation treatment GBM lysate-stimulated DC vaccine, referred to as IR-DCv), combined with GBM lysate-stimulated DC vaccine (DCv) and anti-PD-1 antibody, combined with radiation-treated GBM lysate-stimulated DC vaccine (IR-DCv) Kaplan-Meier survival curve with anti-PD-1 antibody or blank vector control group. 7 is an embodiment of the present invention is an embodiment of the present invention, respectively, in mice with GL261 tumors were administered without antigen-stimulated DC vaccine (ie: DC vaccine only), anti-CTLA-4 antibody, without radiation treatment DC vaccine prepared after stimulation of brain glioma cell lysate (ie: DC vaccine stimulated by GBM lysate, referred to as DCv), DC vaccine prepared after stimulation of radiation-treated brain glioma cell lysate (ie: radiation treatment GBM lysate-stimulated DC vaccine, referred to as IR-DCv), combined with GBM lysate-stimulated DC vaccine (DCv) and anti-CTLA-41 antibody, combined with radiation-treated GBM lysate-stimulated DC vaccine (IR-DCv) Kaplan-Meier survival curve with anti-CTLA-4 antibody or blank vector control group.

Claims (18)

A use of a dendritic cell vaccine and an anti-T cell immune checkpoint protein antibody in the preparation of a drug for treating brain tumors, wherein the dendritic cell vaccine includes dendritic cells stimulated by radioactive antigen. The use as described in item 2 of the patent application scope, wherein the dendritic cell vaccine includes dendritic cells capable of presenting glioma antigens, and the dendritic cells are treated with radiation-treated cerebral glioma cells The lysate is further co-cultured with a bone marrow cell and differentiated. The use as described in item 2 of the patent application scope, wherein the brain glioma cells are treated with X-rays at a dose of 10-30 Gy. The use as described in Item 2 of the patent application scope, wherein the lysate of the brain glioma cells is obtained by processing the brain glioma cells in a freeze-thaw cycle. The use as described in Item 2 of the patent application range, wherein the dendritic cell line is obtained by differentiating the bone marrow cells with lipopolysaccharide. The use as described in item 1 of the patent application scope, wherein the anti-T cell immune checkpoint protein anti-system anti-PD1 antibody, anti-CTLA-4 antibody or anti-CD25 antibody. The use as described in item 6 of the patent application scope, wherein the administration dose of the anti-PD1 antibody or the anti-CD25 antibody is one dose of 200 μg every two days for a total of four doses. The use as described in item 6 of the patent application range, wherein the anti-CTLA-4 antibody is administered at a dose of 100 μg every three days for a total of 3 doses. The use according to any one of items 1 to 8 of the patent application range, wherein the dendritic cell vaccine includes at least 1×10 ⁶ dendritic cells. A combined drug combination for treating brain tumors includes: a dendritic cell vaccine and an anti-T cell immune checkpoint protein antibody, wherein the dendritic cell vaccine includes dendritic cells stimulated by radioactive antigens. The combined drug combination as described in item 10 of the patent application scope, wherein the dendritic cell vaccine includes dendritic cells capable of presenting glioma antigen, and the dendritic cell line is a neuroblastoma treated with radiation The lysate of the cells is further obtained by co-cultivating and differentiation with a bone marrow cell. The combined drug combination as described in item 11 of the patent application scope, wherein the brain glioma cells are treated with X-rays at a dose of 10-30 Gy. The combined drug combination as described in item 11 of the patent application scope, wherein the lysate of the brain glioma cells is obtained by processing the brain glioma cells in a freeze-thaw cycle. The combined drug combination as described in item 11 of the patent application scope, wherein the dendritic cell line is obtained by differentiating the bone marrow cells with lipopolysaccharide. The combined drug combination as described in item 10 of the patent application range, wherein the anti-T cell immune checkpoint protein anti-system anti-PD1 antibody, anti-CTLA-4 antibody or anti-CD25 antibody. The combined drug combination as described in item 15 of the patent application scope, wherein the dose of the anti-PD1 antibody or the anti-CD25 antibody is at least 200 μg. The combined drug combination as described in item 15 of the patent application scope, wherein the dose of the anti-CTLA-4 antibody is at least 100 μg. The combined drug combination according to any one of items 10 to 17 of the patent application range, wherein the dendritic cell vaccine includes at least 1×10 ⁶ dendritic cells.

Images