Application of the combination of Clistridium gondii spores and pembrolizumab

Abstract

The present invention relates to the application of a combination of Clistridium gondii and pembrolizumab in cancer treatment. The present invention was the first to discover that a combination of Clistridium gondii spores and pembrolizumab can significantly improve the therapeutic effect of colon cancer, reduce the dosage of pembrolizumab, and is highly effective and low toxicity. Tumor lysis by Clistridium gondii affects TME immunogenicity in various ways, causing the TME to change from an immunosuppressive state to an immunoactive state, and breaks immune resistance by regulating immunosuppressive TME. Under conditions of an optimized combination, the combination thoroughly eliminated tumor tissue in approximately 20% of experimental mice, expanded the coverage of patients receiving PD-1 antibody tumor treatment, and further demonstrated a significant therapeutic effect even on patients who had failed PD-1 antibody treatment.

Description

Technology Field

[0001] This application claims priority to a Chinese patent application filed with the China Patent Office on October 9, 2021, application number 202111177854.3, titled "Application of combination of Clostridium gondii spores and PD-1 antibody," the entire contents of which are incorporated into this application by reference.

[0002] The present invention belongs to the field of biomedical technology and, specifically, relates to the application of a combination of Clistridium gondii spores and Pembrolizumab. Background Technology

[0003] The tumor microenvironment (TME) forms a locally stable environment during tumor growth, consisting of tumor cells, stromal cells (including fibroblasts, immune cells, inflammatory cells, and some vascular endothelial cells), the extracellular matrix, and biological molecules infiltrating it. The TME provides the material basis necessary for tumor initiation, progression, and invasion, and regulates various biological behaviors such as metastasis and recurrence. At the same time, the TME can increase oncological drug and radiation resistance and impair therapeutic efficacy. Immunological modulation within the TME plays a crucial role in tumor formation and progression and can establish local immunosuppression against the tumor through various mechanisms. Modulating TME immunotherapeutic strategies and reconstructing a positive immune microenvironment is both a key point and a challenge in antitumor therapy.

[0004] Pembrolizumab is a PD-1 inhibitor drug, PD-1 is an important immunosuppressive molecule, and PD-1 antibodies demonstrate significant antitumor effects in clinical practice, including sustained therapeutic effects for patients with advanced metastatic tumors. Generally, sensitivity to immune checkpoint inhibitors has been considered to be determined by tumor neoantigen load and the degree and composition of immune cell infiltration in the tumor microenvironment (TME). Unfortunately, since many common cancers do not exhibit massive mutations and immune cell infiltration, the tumors are insensitive to immune checkpoint inhibitors, resulting in low response rates and effectiveness in only 20% of patients. Therefore, developing methods to make these tumors more sensitive to immunotherapy is one of the key directions of current research.

[0005] Clostridium gondii is an obligate anaerobic bacterium with tumor hypoxic orientation, capable of colonizing only in hypoxic regions of the tumor. Clostridium gondii tumor lysis involves large quantities of dendritic cells, neutrophils, innate immune cells within macrophages, and CD3 + T, CD4 + T, CD8 + It can recruit specific immune cells such as T1 and infiltrate the TME, altering the degree and composition of immune cell infiltration within the TME. At the same time, it promotes the expression of cytokines and chemokines such as TNF-α, IFN-γ, and IL-6 in the tumor. Consequently, Clistridium gondii tumor lysis affects TME immunogenicity in various ways, changing the TME from an immunosuppressive state to an immune-activated state and breaking immune resistance, thereby enabling a highly effective antitumor effect through the combination of Clistridium gondii and immune checkpoint inhibitors. Prior art literature

[65535] U.S. Patent Application Publication US2015 / 0147315 (May 28, 2015) Janku. F, et al. Journal for ImmunoTherapy of Cancer. BMJ, 2020, Vol.8, Supplement 3, p.A233. The problem to be solved

[0006] Based on the disadvantages of the existing technology, the present invention provides an application of the combination of Clistridium gondii spores and PD-1 antibodies. means of solving the problem

[0007] The means for solving the problem of the present invention are as follows.

[0008] The combination of Clistridium gondii spores and pembrolizumab is an application in the manufacture of pharmaceutical products for the treatment of colon cancer.

[0009] It is a drug for treating colon cancer that contains Clistridium gondii spores and pembrolizumab as active ingredients.

[0010] Preferably in the present invention, the Clistridium gondii is the Clistridium gondii strain MW-DCG-LCv-26 or a strain obtained by domesticating Clistridium gondii, and the Clistridium gondii strain MW-DCG-LCv-26 is stored at the National Institute of Measurement and Research in Australia, and strain storage number V12 / 001486. ​​The other strain is preferably the MW-DCG-HNCv-18 strain, and said strain is stored at the National Institute of Measurement and Research in Australia, and strain storage number V12 / 001485. The MW-DCG-CCv-17 strain is stored at the National Institute of Measurement and Research in Australia, and strain storage number V12 / 001487.

[0011] In the present invention, preferably, the Clistridium gondii is in the form of a spore.

[0012] Preferably in the present invention, the Clistridium gondii spore form is freeze-dried spore powder and the auxiliary agent is 1% sucrose.

[0013] Preferably in the present invention, the pembrolizumab is a PD-1 antibody injection solution.

[0014] More preferably, the freeze-drying method for producing freeze-dried powder of Clistridium gondii spores comprises a freezing step of -40℃ for 4 hours; simultaneously creating a vacuum at -35℃ for 10 minutes, and freeze-drying at -30℃ for 10 minutes, -25℃ for 10 minutes, -20℃ for 26 hours, -15℃ for 2 hours, -10℃ for 10 minutes, -5℃ for 10 minutes, 0℃ for 10 minutes, 10℃ for 2 hours, 15℃ for 10 minutes, 20℃ for 3 hours, and 27℃ for 3 hours.

[0015] Preferably, the drug combination is 1×10 7 It is a combination of CFU of Clistridium gondii spores and 0.2 mg of pembrolizumab solution at a concentration of 1 mg / mL.

[0016] More preferably, the solvent of the above pembrolizumab solution is an injection solution of sodium chloride with a mass percentage concentration of 0.9%, and the solvent of the freeze-dried Clistridium spore powder is sterile water for injection and an injection solution of sodium chloride with a mass percentage concentration of 0.9%.

[0017] More preferably, the order of use of the above-mentioned Clistridium gondispore and pembrolizumab is Clistridium gondispore first, then pembrolizumab. Effects of the invention

[0018] The present invention has discovered for the first time that a combination of Clistridium gondii spores and pembrolizumab can significantly enhance the anti-colon cancer therapeutic effect, while simultaneously lowering the dosage of pembrolizumab and being highly effective with low toxicity. The lysis of Clistridium gondii tumors affects TME immunogenicity in various ways, changing the TME from an immunosuppressive state to an immuno-activated state, and simultaneously modulates immunosuppressive TMEs to break immune resistance. Under optimized combination conditions, the combination of Clistridium gondii spores and pembrolizumab thoroughly eliminates tumor tissue in about 20% of experimental mice, expands the effective range of PD-1 antibodies for treating tumor patients, and has a significant therapeutic effect even in patients for whom PD-1 antibody treatment has failed. Brief explanation of the drawing

[0019] Figure 1 shows the tumor weight (a) and tumor inhibition rate (b) of each group of a model in which experimental mice containing CT26.WT colon cancer tumors were treated with Clistridium gondii spores in Example 1, and the data are indicated as P<0.05 when comparing * with the Control group and P<0.01 when comparing ** with the Control group. Figure 2 shows the tumor weight (a) and tumor inhibition rate (b) of each group of a model in which experimental mice containing MC38 colon cancer tumors were treated with a combination of Clistridium gondii spores and Pembrolizumab in Example 2, and the data are indicated as *P<0.05 and **P<0.01. Figure 3 shows the change in tumor volume of each group in a model in which experimental mice containing MC38 colon cancer tumors were treated with a combination of Clistridium gondii spores and Pembrolizumab in Example 2. The data is indicated as P<0.05 when comparing * with the Control group, P<0.01 when comparing ** with the Control group, P<0.05 for the C.ghonii+Pembrolizumab group when comparing # with the C.ghonii group, and P<0.01 for the C.ghonii+Pembrolizumab group when comparing ## with the C.ghonii group. Figure 4 shows the tumor volume and tumor inhibition rate at the end of the experiment in a model in which experimental mice containing MC38 colon cancer tumors were treated with a combination of Clistridium gondii spores and Pembrolizumab in Example 2, and the data are indicated as *P<0.05 and **P<0.01. Figure 5 shows the tumor volume and tumor inhibition rates at each round of a model in which experimental mice containing MC38 colon cancer tumors were treated with a combination of Clistridium gondii spores and Pembrolizumab in Example 2, where Figure a represents experimental round 1, Figure b represents experimental round 2, and Figure c represents experimental round 3. Figure 6 shows photographs of the tumor morphology of each group in a model in which experimental mice containing MC38 colon cancer tumors were treated with a combination of Clistridium gondii spores and Pembrolizumab in Example 2. Figure 7 shows the tumor weight of each group in a model in which experimental mice containing MC38 colon cancer tumors were treated by administering a combination of Clistridium gondii spores and Pembrolizumab in different sequences in Example 3. Figure 8 shows the change in tumor volume of each group of a model in which experimental mice containing MC38 colon cancer tumors were treated by administering Clistridium gondii spores and Pembrolizumab in combination in different orders in Example 3. Specific details for implementing the invention

[0020] The present invention will be described in detail below in conjunction with specific embodiments. The embodiments described below are merely examples to help people understand the means for solving some problems of the present invention, and such embodiments are intended only to explain the present invention and do not limit the scope of protection of the present invention, which is defined by the claims.

[0021] Example 1: Therapeutic effect of Clistridium gondii spores on an experimental mouse model containing colon cancer tumors

[0022] Materials: Freeze-dried powder of Clistridium gondii spores for main use, strain MW-DCG-LCv-26 strain, the strain is stored at the National Metrology Institute (NMI) in Australia, strain storage number V12 / 001486, and was researched and manufactured by Shandong Xinchuang Biotechnology Co., Ltd. The freeze-dried powder of Clistridium gondii spores for injectable use uses Clistridium gondii spores as the active ingredient and 1% sucrose as an adjuvant, and is manufactured according to a freeze-drying procedure of -40℃ for 4 hours; -35℃ for 10 minutes simultaneously under vacuum, -30℃ for 10 minutes, -25℃ for 10 minutes, -20℃ for 26 hours, -15℃ for 2 hours, -10℃ for 10 minutes, -5℃ for 10 minutes, 0℃ for 10 minutes, 10℃ for 2 hours, 15℃ for 10 minutes, 20℃ for 3 hours, and 27℃ for 3 hours, 1X10 8 It was manufactured to the specifications of CFU / unit. The lot number of the control freeze-dried powder is 201803001F, and it was researched and manufactured by Shandong Xinchang Bio Company Limited; it was prepared using 1 mL of 1% sucrose solution through the above freeze-dried powder manufacturing procedure. The lot number of the 0.9% sodium chloride injection solution is 1803122161, and Cisen Pharmaceutical CO.,LTD.( It is sold at ), and the lot number of the sterile water for injection is 1704242163 and it is sold by Cisen Pharmaceutical CO., LTD. The number of the CT26.WT colon cancer cells is 3131C0001000800037 and it is sold by the Cell Resource Center of the Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, and the animal certificate numbers for the BALB / c female laboratory mice are No. 11401300088557 (120 mice), No. 11401300089721 (60 mice), and No. 11401300089721 (60 mice), and it is sold by Beijing Hfk Bioscience Co., Ltd.

[0023] Method: CT26.WT cells were subcultured to the required number of cells until the concentration reached 7.5×10 6 An inoculation cell suspension with cells / mL is prepared, and a cell viability of over 90% is used to establish a BALB / c experimental mouse colon cancer subcutaneous tumor graft model; 0.2 mL of the cell suspension is subcutaneously inoculated into the right forelimb of an experimental mouse, and after about 10 days, the tumor volume is approximately 0.30 cm² 2 Experimental animals were selected and used in the experiment. Experimental mice that passed the tumor formation test were randomly divided into four groups by lottery. The control group (Control), low-dose treatment group (LC. ghonii), medium-dose treatment group (MC. ghonii), and high-dose treatment group (HC. ghonii) were divided into groups of eight mice each. After redissolving the freeze-dried powder of Clistridium gondii spores in 0.1 mL of sterile water for injection, the concentrations were each 5×10⁻¹⁰ with a sodium chloride injection solution having a mass percentage concentration of 0.9%. 7 cfu / mL, 1X10 8 Prepare a suspension of cfu / mL and 2×10×cfu / mL, inject 0.1mL into the tumor, and administer once every other day for a total of 5 times, with the dosage being 5×10 each time. 6 cfu / times, 1x10 7 cfu / times, 2x10 7 The dose is cfu / dose, and the control concentration is the same as that of the high-dose treatment group. After starting treatment, clinical symptoms such as animal behavior, death, or morbidity are observed daily. All surviving animals are dissected on the second day after the last administration to weigh the tumors, and the tumor inhibition rate is calculated based on the tumor weight. Tumor Weight Tumor Inhibition Rate (IR) TW %) = (Control group average tumor weight - Experiment group average tumor weight) / Control group average tumor weight × 100%. Independent replicate experiments were conducted three times each using the above experimental method.

[0024] Results: During the experiment period, there were no deaths or morbidities in any of the animals during the third experiment. After the experiment ended, the average tumor weight of the tumor-containing experimental mice in each group and the tumor inhibition rate of the treatment group in the third experiment are shown in Tables 1 and 2.

[0025]

[0026]

[0027] Compared to the control group, the tumor weight of each treatment group was smaller than that of the control group, and the MC.ghonii group showed the best anti-tumor effect (Fig. 1a). Inhibitory effects on tumor growth were observed in all dose treatment groups; furthermore, the MC.ghonii group showed the best tumor weight inhibition rate between the first and third trials, while the LC.ghonii group showed the best tumor weight inhibition rate in the second trial. As indicated in Table 1b, statistical analysis based on the results of the third trial showed a significant difference in the tumor weight inhibition rate of the HC.ghonii group compared to the control group (p<0.05), and a very significant difference in the tumor weight inhibition rates of the LC.ghonii and MC.ghonii groups compared to the control group (p<0.01).

[0028] In summary, freeze-dried powder of Clistridium gondii spores has an inhibitory effect on colon cancer growth, and according to research, MC.ghonii group (1X10 7 cfu / dose) is more effective than other treatment groups. Therefore, the preferred dose for the C. Ghonii group during combined administration with Pembrolizumab is 1×10 7 It is cfu / times.

[0029] Example 2: Therapeutic effect of the combination of Clistridium gondii spores and Pembrolizumab on an experimental mouse model containing PD-1 humanized genetically modified colon cancer tumors

[0030] Materials: Freeze-dried powder of injectable Clistridium gondii spores, researched and manufactured by Shandong Xinchang Bio Company Limited. The manufacturing method is the same as that of the materials in Example 1. The lot number of Pembrolizumab injection solution is S001188, the specification is 100 mg / 4 mL, and it is sold by Merck & Co., Inc. in the USA. The lot numbers of the control freeze-dried powder are 201910002F and 201803001F, researched and manufactured by Shandong Xinchang Bio Company Limited, and the manufacturing method is the same as that of the materials in Example 1. The lot number of 0.9% sodium chloride injection solution is 1809282161 and it is sold by Cisen Pharmaceutical CO., LTD., and the lot number of sterile water for injection is 1902212162 and it is sold by Cisen Pharmaceutical CO., LTD. The cargo management number for MC38 colon cancer cells is T1917, and they are sold by Abm Biocompany Limited. The animal certification numbers for the C57BL / 6PD-1 humanized gene process experimental mice are No. 20170010002500 (90 mice), No. 312024300008757 (35 mice), and No. 20170010001319 (50 mice), and they are sold by the Shanghai Model Organisms Center, Inc.

[0031] Methods: MC38 cells were subcultured to the required number of cells until the concentration was 7.5 x 10 6 An inoculation cell suspension with cells / mL was prepared and used to establish a C57BL / 6PD-1 humanized gene-processed subcutaneous colon cancer model in laboratory mice with a cell viability of over 90%, and 0.2 mL of the cell suspension was subcutaneously inoculated into the right forelimb of a laboratory mouse, and after about 10 days, the tumor volume was 0.15 cm² 3Larger experimental animals were selected and used in the experiment. Experimental mice meeting the criteria were selected according to the principle of randomization and divided into four groups by lot. The control group, the C. ghonii spore treatment group, the Pembrolizumab group, and the combined group of C. ghonii spores and Pembrolizumab (C. ghonii + Pembrolizumab) were divided into at least 5 animals per group. After redissolving the freeze-dried C. ghonii spore powder in 0.1 mL of sterile water for injection, a sodium chloride injection solution with a mass percentage concentration of 0.9% and a solution with a concentration of 1 x 10 8 A suspension with cfu / mL is prepared and 0.1 mL is injected into the tumor, and for the C. ghonii group and the C. ghonii+Pembrolizumab group, spores are 1×10⁶ 7 cfu / dose is administered by injection, and the dose for the Control group and Pembrolizumab group is 0 cfu / dose, administered once every other day for a total of 6 doses. Pembrolizumab injection solution is diluted with 0.9% sodium chloride injection solution to a final concentration of 1 mg / mL working solution and 0.2 mL is injected into the peritoneal cavity. The Pembrolizumab group and the C.ghonii+Pembrolizumab group receive 0.2 mg / dose of Pembrolizumab, while the Control group and the C.ghonii group receive 0.2 mL of 0.9% sodium chloride injection solution, administered twice a week for a total of 4 doses.

[0032] First, Clistridium gondii spores were administered, followed by PD-1 antibodies, and the order of administration was as follows. Clistridium gondii spores were injected every other day starting from day 1, and Pembrolizumab was injected on days 3, 6, 9, and 13, respectively.

[0033] Observation and Evaluation Criteria: After the start of drug administration, the animals' behavior, death, or necrotic state is observed daily. Tumor volume is observed at intervals of 1-2 days, and the tumor inhibition rate is calculated based on the tumor volume measured at the end of the experiment. All surviving animals are dissected on the 2nd day after the last drug administration to weigh the tumors, and the tumor inhibition rate is calculated based on the tumor weight.

[0034] Tumor Weight Tumor Inhibition Rate (IR) TW %) = (Control group average tumor weight - Experiment group average tumor weight) / Control group average tumor weight × 100%.

[0035] Tumor Volume Tumor Inhibition Rate (IR) TV %) = 1 - (average tumor volume at the start of the experimental group - average tumor volume at the end of the experimental group) / (average tumor volume at the start of the control group - average tumor volume at the end of the control group) × 100%.

[0036] Healing rate (%) = Number of healed animals in each group / Total number of experimental animals in each group × 100% (time period is at the end of the experiment).

[0037] Independent repeated experiments were conducted three times each using the above experimental method.

[0038] Results: During the experiment period, one experimental mouse died in the control group during the first experiment, and there were no deaths or dying animals in each group during the other experiments.

[0039] 1. Tumor weight and tumor inhibition rate of experimental mice containing tumors in each group

[0040] The average tumor weight of the experimental mice containing tumors in each group during the third experiment is shown in Table 3. The tumor weight of each treatment group was smaller than that of the Control group, and the tumor weight of the C.ghonii + Pembrolizumab group was smaller than that of any single drug treatment group, showing a very significant difference when compared to the Control group (p<0.01). The average tumor weight of the C.ghonii + Pembrolizumab group was also significantly smaller than that of the C.ghonii alone group and the Pembrolizumab alone group (p<0.05) (Fig. 2a).

[0041]

[0042] According to the statistical analysis of the results from the third trial, each treatment group showed an inhibitory effect on tumor growth, with a tumor inhibition rate of over 30%, and the tumor inhibition rates were in the order of C.ghonii+Pembrolizumab group > Pembrolizumab group > C.ghonii group (Fig. 2b). After administering Pembrolizumab at the same dose, the tumor inhibition rates of the C.Ghonii+Pembrolizumab group improved by approximately 20%, 20%, and 25%, respectively, compared to the Pembrolizumab monotherapy group (Table 4).

[0043]

[0044] 2. Average tumor volume and tumor inhibition rate of tumor-bearing experimental mice in each group

[0045] When the groups were divided before the start of the experiment, there was no significant difference in the average tumor volume of each group in each round (P>0.05). When compared to the terminal Control group administered to experimental mice containing tumors in the third round of the experiment, the tumor volumes of the C.ghonii group, Pembrolizumab group, and C.ghonii+Pembrolizumab group were significantly smaller than those of the Control group (p=0.001, p=0.000053, p=0.000). The tumor volume of the C.ghonii+Pembrolizumab group was significantly smaller than that of the C.ghonii group and Pembrolizumab group. Additionally, on day 10, there was a significant difference in the average tumor volume between the combined group and the Control group, at which time the tumor volume of the C.ghonii+Pembrolizumab group was about 1 time smaller than that of the Pembrolizumab group (Fig. 3).

[0046] When the tumor inhibition rate was calculated based on tumor volume, the tumor inhibition rate of the C.ghonii+Pembrolizumab group was higher than that of the other groups during the third experimental period (Fig. 4).

[0047] On the 7th day after administration during the 3rd trial, two doses of Pembrolizumab were completed, and after administering 0.4 mg of Pembrolizumab, the tumor inhibition rates of the C.ghonii+Pembrolizumab group were 51.29% (greater than 50%), 49.78% (about 50%), and 59.33% (greater than 50%), respectively, while the tumor inhibition rates of the Pembrolizumab monotherapy group were 25.67%, 7.68%, and 35.10%, respectively. The tumor inhibition rate reached the level of the C.ghonii+Pembrolizumab group when Pembrolizumab (0.4 mg) was administered twice, with rates of 69.31%, 52.83%, and 53.08%, respectively (Fig. 5), only after administering 0.6 mg of Pembrolizumab to the monotherapy group three times. From this, it can be seen that the C.ghonii+Pembrolizumab group significantly enhances the antitumor effect of Pembrolizumab and is a highly effective, low-toxicity treatment that reduces the amount of Pembrolizumab required by the C.ghonii+Pembrolizumab group by 50% to achieve the same therapeutic effect.

[0048] 3. Healing rate

[0049] In the third experiment, tumors in approximately 20% (1 / 6, 1 / 5, and 1 / 6) of the experimental mice in the C. ghonii + Pembrolizumab group were completely eliminated (Fig. 5), and no tumor growth was observed until the end of the experiment. In contrast, in the Pembrolizumab and C. ghonii groups, there were no instances of complete tumor disappearance, and the cure rate was 0%. This may be because Clistridium gondii spores germinate in the hypoxic regions of the tumor and influence TME immunogenicity in various ways, shifting the TME from an immunosuppressive state to an immuno-activated state while simultaneously regulating the immunosuppressive TME to break immune tolerance. After combination with Pembrolizumab, the therapeutic effect of the PD-1 antibody was further amplified, and approximately 20% of the experimental mice were cured. Clistridium gondii is expected to serve as an excellent 'sensitizer' for the immunotherapy of tumor patients. The tumor morphology of each group is shown in Fig. 6.

[0050]

[0051] Example 3: Effect of the Dosage Order of Clistridium gondii Spores and Pembrolizumab on the Therapeutic Effect in an Experimental Mouse Model Containing PD-1 Humanized Gene-Modified Colon Cancer Cells

[0052] Materials: Freeze-dried powder of Clistridium gondii spores for injection, researched and manufactured by Shandong Xinchang Bio Company Limited, and the manufacturing method is the same as that of the materials in Example 1. The lot number of Pembrolizumab injection solution is S006648, the specification is 100 mg / 4 mL, and it is sold by Merck & Co., Inc. in the USA. The lot number of the control free-dried powder is 201910002F, researched and manufactured by Shandong Xinchang Bio Company Limited, and the manufacturing method is the same as that of the materials in Example 1. The lot number of 0.9% sodium chloride injection solution is J18070104 and it is sold by Shandong Hualu Pharmaceutical Co., Ltd., and the lot number of sterile water for injection is 1902212162 and it is sold by Cisen Pharmaceutical CO., LTD. The cargo management number for MC38 colon cancer cells is T1917, and they are sold by Abm Biocompany Limited. The animal certificate number for C57BL / 6PD-1 humanized genetic engineering laboratory mice is No. 20170010002500 (90 mice), and they are sold by the Shanghai Model Organisms Center, Inc.

[0053] Method: The method for constructing a subcutaneous tumor model of MC38 colon cancer in C57BL / 6PD-1 humanized genetically engineered mice is the same as in Example 2. The mice successfully modeled were randomly divided by lottery into A (Control), B (C. ghonii), C (Pembrolizumab), C (Pembrolizumab), C. ghonii group with D followed by Pembrolizumab, C. ghonii group with E followed by C. ghonii, and F (C. ghonii + Pembrolizumab simultaneous group), with 8 mice in each group. The dosage of C. ghonii was 0 cfu / dose and 1 x 10, respectively. 7 cfu / times, 0 cfu / times, 1 x 10 7 cfu / times, 1x10 7 cfu / times and 1x10 7The dose is cfu / dose, and it is administered at daily intervals with a dosage of 0.1 mL / dose. 0.2 mL of 1 mg / mL Pembrolizumab injection is administered intraperitoneally, and a dose of 0.2 mg / dose is administered once daily. The administration procedure for the first stage is as shown in Table 6.

[0054]

[0055] After the first stage of medication is completed and observed for 6 days, the second stage repeats the medication process of the first stage.

[0056] Observation and Evaluation Criteria: After the start of drug administration, the animals' behavior, death, or necrotic state is observed daily. Tumor volume is observed at intervals of 1-2 days, and the tumor inhibition rate is calculated based on the tumor volume measured at the end of the experiment. All surviving animals are dissected on the 8th day after the completion of the final drug administration of Phase 2. The tumor is weighed, and the tumor inhibition rate is calculated based on the tumor weight.

[0057] Tumor Weight Tumor Inhibition Rate (IR) TW %) = (Control group average tumor weight - Experiment group average tumor weight) / Control group average tumor weight × 100%.

[0058] Healing rate (%) = Number of healed animals in each group / Total number of experimental animals in each group × 100% (Time period is at the end of the experiment).

[0059] Results: During the experiment, four animals in the Control group died due to tumors growing too large or ulcers, while no animals died or were critically ill in the other groups.

[0060] 1. Tumor weight and tumor suppression rate

[0061] Compared to the control group, the tumor weights of the C.ghonii group, Pembrolizumab group, C.ghonii group after Pembrolizumab first, Pembrolizumab group after C.ghonii first, and C.ghonii+Pembrolizumab group were all significantly smaller than those of the control group (p=0.005, p=0.000, p=0.000, p=0.000, p=0.000). The results of the tumor weight tests for each group are shown in Table 7 and Figure 7.

[0062]

[0063] Note: Compared to the control group, **p<0.01.

[0064] As a result of calculating the tumor inhibition rate based on tumor weight, the tumor inhibition rates for the C.ghonii group, Pembrolizumab group, C.ghonii group after Pembrolizumab first, Pembrolizumab group after C.ghonii first, and C.ghonii+Pembrolizumab group were 37.39%, 76.20%, 87.26%, 92.71%, and 92.19%, respectively, and as shown in Table 8, all treatment groups showed a clear inhibitory effect on tumor growth.

[0065]

[0066] 2. Tumor Volume and Tumor Suppression Rate

[0067] Before treatment, there was no significant difference in tumor volume among the experimental mice containing tumors in each group (p > 0.05). At the end of treatment, when compared to the control group, the tumor volumes of the C. ghonii group, Pembrolizumab group, group with Pembrolizumab followed by C. ghonii, group with C. ghonii followed by Pembrolizumab, and the C. ghonii + Pembrolizumab simultaneous group were significantly smaller than those of the control group (p=0.002, p=0.000, p=0.000, p=0.000, p=0.000, p=0.000, Fig. 8). As shown in Table 9, the tumor inhibition rates were: group with Pembrolizumab followed by C. ghonii > group with C. ghonii + Pembrolizumab simultaneous group > group with Pembrolizumab followed by C. ghonii > Pembrolizumab group > C. ghonii group.

[0068]

[0069] 3. Healing Rate

[0070] The experimental period and the healing rates of each group are as shown in Table 10.

[0071]

[0072] The tumor cure rate in experimental mice in the group administered Pembrolizumab after C. ghonii was significantly higher than in other groups, and was twice the cure rate of the group administered Pembrolizumab after C. ghonii and the group administered C. ghonii + Pembrolizumab simultaneously. This may be attributed to the fact that the Clistridium gondii spores administered first into the tumor effectively lyse tumor tissue indiscriminately and destroy the TME; simultaneously, after the bacteria lyse the tumor, they attract immune cells and infiltrate the TME, thereby altering the degree and composition of immune cell infiltration within the TME, and that the products of cell division are large amounts of CD8 +This involves recruiting T cells and applying them to PD-1 antibody therapy following oncolytic bacteria to bypass immunosuppression during immunotherapy and initiate a large-scale T-cell attack against tumor cells, thereby exerting a 'dual' anticancer effect. Therefore, when using oncolytic bacteria and immunosuppressants in combination, the order of administration is also critical to the therapeutic effect on the tumor.

[0073] In summary, using Clistridium gondii spore therapy first and then combining it with immunotherapy enhances the antitumor effect, reduces the dosage of immunotherapy drugs, and is highly effective and low toxicity.

[0074] Although the above embodiments describe the present invention in detail, they are only some embodiments of the invention and not all embodiments. Anyone can obtain other embodiments based on these embodiments without requiring creativity, and all such embodiments fall within the scope of protection of the present invention.

[0075] Name of Depositing Institution: NATIONAL MEASUREMENT INSTITUTE Trustee Number: V12 / 001485 Date of Deposit: 2012-04-18 Name of Depositing Institution: NATIONAL MEASUREMENT INSTITUTE Trustee Number: V12 / 001486 Date of Deposit: 2012-04-18 Name of Depositing Institution: NATIONAL MEASUREMENT INSTITUTE Trustee Number: V12 / 001487 Date of Deposit: 2012-04-18

Claims

Claim 1 A pharmaceutical composition for treating colon cancer, wherein the active ingredients of the pharmaceutical composition comprise Clistridium gondii spores and pembrolizumab, wherein the Clistridium gondii is the Clistridium gondii MW-DCG-LCv-26 strain, the Clistridium gondii MW-DCG-LCv-26 strain is stored at the Australian National Laboratory of Measurement and Measurement and has strain number V12 / 001486, and the order of administration among the active ingredients is Clistridium gondii first, followed by pembrolizumab. Claim 2 A pharmaceutical composition according to claim 1, characterized in that the Clistridium gondii spore in the drug is an injectable Clistridium gondii spore freeze-dried powder, and the adjuvant of the injectable Clistridium gondii spore freeze-dried powder is 1% sucrose. Claim 3 A pharmaceutical composition according to claim 1, characterized in that the pembrolizumab is a PD-1 antibody injection solution. Claim 4 In paragraph 1, 1X10 7 A pharmaceutical composition characterized by combining CFU of CFU of Clistridium gondii spores with 0.2 mg of pembrolizumab solution at a concentration of 1 mg / mL. Claim 5 A pharmaceutical composition according to claim 4, characterized in that the solvent of the pembrolizumab solution is a sodium chloride injection solution having a mass percentage concentration of 0.9%. Claim 6 A pharmaceutical composition according to claim 4, wherein the Clistridium gondii spore is freeze-dried powder of Clistridium gondii spore for injection, and the solvent of the freeze-dried powder of Clistridium gondii spore for injection is sterile water for injection and a sodium chloride injection solution having a mass percentage concentration of 0.9%. Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete

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