Application of Clostridium ghonii spores in combination with pembrolizumab

Abstract

The present invention relates to the application of Clostridium ghonii spores and pembrolizumab in the treatment of cancer. The present invention is the first to discover that the combination of Clostridium ghonii spores and pembrolizumab can significantly improve the therapeutic effect of colon cancer, while reducing the dose of pembrolizumab, with high efficiency and low toxicity. The tumor lysis of Clostridium ghonii can affect TME immunogenicity in various ways, transform TME from an immunosuppressive state to an immunoactive state, and at the same time, regulate immunosuppressive TME and break immune resistance. Under optimal combination conditions, the combination of Clostridium ghonii spores and pembrolizumab completely eliminated about 20% of mouse tumor tissues, expanding the scope of benefit of tumor patients treated with PD-1 antibody, and even showing remarkable therapeutic effects in patients who failed PD-1 antibody treatment.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority from a Chinese patent application filed with the China Patent Office on October 9, 2021, bearing application number CN202111177854.3 and entitled "Application of the combined use of Clostridium ghonii spores and Pd-1 antibody," the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of biomedicine technology, specifically to the combined use of Clostridium ghonii spores and pembrolizumab. [Background technology]

[0003] The tumor microenvironment (TME) is the local, steady-state environment of tumor growth, consisting of tumor cells, stromal cells (including fibroblasts, immune and inflammatory cells, and some vascular endothelial cells), extracellular matrix, and biomolecules infiltrated therein. The TME provides the material foundation necessary for tumor initiation, progression, and invasion, and regulates various biological processes, including tumor metastasis and recurrence. At the same time, the TME can increase tumor drug resistance and radiation resistance, reducing therapeutic efficacy. Immune regulation within the TME plays an important role in tumor initiation and progression and can form local tumor immunosuppression through various mechanisms. How to control the TME and reconstruct a positive immune microenvironment are both key and challenging aspects of antitumor therapy.

[0004] Pembrolizumab is a PD-1 inhibitor. PD-1 is an important immunosuppressive molecule. PD-1 antibodies have demonstrated remarkable antitumor effects in clinical trials, including durable therapeutic effects in patients with late-stage metastasis. Sensitivity to immune checkpoint inhibitors is generally believed to depend on the tumor neoantigen load and the extent and composition of immune cell infiltration in the tumor microenvironment (TME). Unfortunately, many common tumors lack extensive mutations and immune cell infiltration, resulting in insensitivity to immune checkpoint inhibitors and a low response rate, with only 20% of patients responding effectively. Therefore, one of the current research priorities is to develop methods to make these tumors more sensitive to immunotherapy.

[0005] Closridium ghonii (Closridium ghonii) is a strict anaerobe with tumor-deflecting hypoxic properties, and can colonize only in the anoxic areas of tumors. Closridium ghonii oncolysis induces a large number of innate immune cells, including dendritic cells, neutrophils, and macrophages, as well as CD4+. 3+ T, C.D. 4+ T, C.D. 8+ It can recruit specific immune cells, such as T cells, to infiltrate the TME, altering the infiltration rate and composition of immune cells in the TME. It also promotes the enhanced expression of cytokines and chemokines, such as TNF-α, IFN-γ, and IL-6, in tumors. These findings suggest that Closridium ghonii oncolysis affects TME immunogenicity in multiple ways, transforming the TME from an immunosuppressive state to an immunoactive state while simultaneously overcoming immune tolerance. This allows Closridium ghonii to be used in combination with immune checkpoint inhibitors for highly effective antitumor therapy. Summary of the Invention [Problem to be solved by the invention]

[0006] Based on the shortcomings of the prior art, the present invention provides the application of Closridium ghonii spores in combination with pembrolizumab. [Means for solving the problem]

[0007] The technical solution of the present invention is as follows:

[0008] Application of Clostridium ghonii spores in combination with pembrolizumab in the production of a drug for the treatment of colon cancer. A drug for treating colon cancer, wherein the active ingredients in the drug include Clostridium ghonii spores and pembrolizumab. In the present invention, the Clostridium ghonii bacterium is preferably the Clostridium ghonii MW-DCG-LCv-26 strain (stored at the Australian National Metrology Institute, strain number V12 / 001486). Other preferred strains include the MW-DCG-HNCv-18 strain (stored at the Australian National Metrology Institute, strain number V12 / 001485) and the MW-DCG-CCv-17 strain (stored at the Australian National Metrology Institute, strain number V12 / 001487). In the present invention, preferably, the Clostridium ghonii bacterium is in the form of a spore. In the present invention, preferably, the Clostridium ghonii spore form is a freeze-dried powder, and the additive therefor is 1% sucrose. In the present invention, preferably, the pembrolizumab is a PD-1 antibody injection. More preferably, the freeze-drying process for the freeze-dried powder of Clostridium ghonii spores comprises the following freeze-drying steps: -40°C for 4 hours, -35°C for 10 minutes with simultaneous vacuum application, -30°C for 10 minutes, -25°C for 10 minutes, -20°C for 26 hours, -15°C for 2 hours, -10°C for 10 minutes, -5°C for 10 minutes, 0°C for 10 minutes, 10°C for 2 hours, 15°C for 10 minutes, 20°C for 3 hours, and 27°C for 3 hours. The preferred drug combination of the present invention is 1×10 7 It is a combination of 0.2 mg of pembrolizumab injection at a concentration of 1 mg / mL per CFU Clostridium ghonii spore lyophilized powder. More preferably, the solvent for the pembrolizumab injection is a sodium chloride injection solution with a mass percentage concentration of 0.9%, and the solvent for the Clostridium ghonii spore freeze-dried powder is sterile water for injection and a sodium chloride injection solution with a mass percentage concentration of 0.9%. More preferably, the order of administration of the combination of Clostridium ghonii spores and pembrolizumab is first gothic Clostridium spores followed by pembrolizumab. [Effects of the Invention]

[0009] This invention is the first to discover that the combination of Clostridium ghonii spores and pembrolizumab can significantly enhance the therapeutic effect of colon cancer, while at the same time reducing the dose of pembrolizumab, with high efficiency and low toxicity. It affects TME immunogenicity in many ways, transforming the TME from an immunosuppressive state to an immunoactive state, while regulating the immunosuppressive TME and overcoming immune tolerance. Under optimal combination conditions, the combination of Clostridium ghonii spores and pembrolizumab completely eliminated approximately 20% of tumor tissue in mice, expanding the range of benefit for tumor patients treated with PD-1 antibodies and even showing significant therapeutic effects in patients who had failed PD-1 antibody treatment. [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 shows the tumor weight (a) and tumor inhibition rate (b) of each group in the CT26.WT colon cancer tumor-burdened mouse model treated with Clostridium ghonii spores in Example 1. Data indicate * P<0.05 compared to the control group, and ** P<0.01 compared to the control group. [Figure 2]Figure 2 shows the tumor weight (a) and tumor weight tumor inhibition rate (b) of each group in the MC38 colon cancer tumor-burdened mouse model treated with a combination of Clostridium ghonii spores and pembrolizumab in Example 2. The treatment data indicate *P<0.05, **P<0.01. [Figure 3] Figure 2 shows the changes in tumor volume in each group of the MC38 colon cancer tumor-burdened mouse model treated with a combination of Clostridium ghonii spores and pembrolizumab in Example 2. The treatment data indicate: * P<0.05 compared to the control group; ** P<0.01 compared to the control group; # P<0.05 for the C. ghoni + pembrolizumab group compared to the C. ghonii group; and ## P<0.01 for the C. ghoni + pembrolizumab group compared to the C. ghonii group. [Figure 4] In Example 2, the tumor inhibition rate of tumor volume at the end point of the experiment in an MC38 colon cancer tumor-burdened mouse model was measured by treatment with a combination of Clostridium ghonii spores and pembrolizumab. The data indicates *P<0.05, **P<0.01. [Figure 5] Figure 2 shows the tumor inhibition rate of tumor volume for each lot in an MC38 colon cancer tumor-burdened mouse model treated with a combination of Clostridium ghonii spores and pembrolizumab in Example 2, where Figure a is experimental lot 1, Figure b is experimental lot 2, and Figure c is experimental lot 3. [Figure 6] 1 shows photographs of tumor morphology in each group of a mouse model of MC38 colon cancer tumors administered in combination with Clostridium ghonii spores and pembrolizumab in Example 2. [Figure 7] 1 shows the tumor weights of the MC38 colon cancer tumor-burdened mouse model groups treated with different administration sequences of Clostridium ghonii spores and pembrolizumab in Example 3. [Figure 8] 1 shows the changes in tumor volume in each group of MC38 colon cancer tumor-burdened mouse model treated with different administration sequences of Clostridium ghonii spores and pembrolizumab in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention will be described in more detail below with reference to specific examples. The described examples are only a part of the enumerated technical aspects for the public's understanding, and are only used to explain the present invention, rather than limiting the protection scope of the present invention, which is defined by the claims.

[0012] Example 1: Therapeutic effect of Clostridium ghonii spores on a colon cancer tumor-burdened mouse model

[0013] Materials: Clostridium ghonii spore lyophilized powder for injection. The strain was MW-DCG-LCv-26, a strain kept at the Australian National Metrology Institute with the strain reserve number V12 / 001486, and developed by Shandong Xinchuang Bioscience and Technology Co., Ltd. The Clostridium ghonii spore lyophilized powder for injection was prepared with Clostridium ghonii spores as the active ingredient and 1% sucrose as an additive. The lyophilized powder was prepared by the following lyophilization program: -40°C for 4 hours, -35°C for 10 minutes under vacuum, -30°C for 10 minutes, -25°C for 10 minutes, -20°C for 26 hours, -15°C for 2 hours, -10°C for 10 minutes, -5°C for 10 minutes, 0°C for 10 minutes, 10°C for 2 hours, 15°C for 10 minutes, 20°C for 3 hours, and 27°C for 3 hours; the specifications were 1 x 10 8CFU / unit, control freeze-dried powder, lot number: 201803001F, developed by Shandong Xinchuang Bioscience and Technology Co., Ltd., prepared by the above freeze-drying process with 1 mL of 1% sucrose solution; 0.9% sodium chloride injection, lot number: 1803122161, sold by Chenxin Pharmaceutical Co., Ltd.; sterile water for injection, lot number: 1704242163, sold by Chenxin Pharmaceutical Co., Ltd.; C T26.WT colon cancer cells, number: 3131C0001000800037, were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; BALB / c female mice, animal qualification certificate numbers: No. 11401300088557 (120 mice), No. 11401300089721 (60 mice), No. 11401300089721 (60 mice), were purchased from Beijing Huafukang Bioscience and Technology Co., Ltd.

[0014] Methods: CT26.WT cells were resuscitated and passaged to the required cell number at a concentration of 7.5 × 10 6 A cell suspension for inoculation was prepared at a cell concentration of 100 cells / mL. The cell survival rate was 90% or higher, and this was used to construct a subcutaneously transplanted tumor model of BALB / c colon cancer in mice. 0.2 mL of the cell suspension was inoculated subcutaneously into the right forelimb of the mouse, and a tumor volume of approximately 0.30 cm was obtained in about 10 days. 3 Experimental animals were selected for the test, and mice that passed tumor formation were randomly divided into four groups: a control group (Control), a low-dose treatment group (LC.ghonii), a medium-dose treatment group (MC.ghonii), and a high-dose treatment group (HC.ghonii), each of which consisted of eight mice. The freeze-dried powder of Clostridium ghonii spores was first reconstituted in 0.1 mL of sterile water for injection, and then diluted to a concentration of 5 × 10 with 0.9% sodium chloride injection by mass. 7 cfu / mL, 1 × 10 8 cfu / mL, 2 × 10 8 A suspension of 5 × 10 cfu / mL was prepared and injected into the tumor at 0.1 mL, once every other day for a total of 5 doses. 6 cfu / time, 1 × 10 7 cfu / time, 2 × 10 7The control group had the same concentration of control drug as the high-dose treatment group. After the start of administration, animals were observed daily for behavior and clinical signs such as death or moribundity. All surviving animals were dissected two days after the final administration and tumor weights were measured. The tumor inhibition rate was calculated based on tumor weight: tumor weight tumor inhibition rate (IRTW%) = (control group average tumor weight - experimental group average tumor weight) / control group average tumor weight × 100%. Using the above experimental method, three independent experiments were repeated.

[0015] Results: None of the animals in the three experimental batches were moribund or dead during the experiment. At the end of the experiment, the average tumor weights of tumor-bearing mice in each experimental group in the three experimental batches and the tumor inhibition rates of the treatment groups are shown in Tables 1 and 2.

[0016] [Table 1]

[0017] [Table 2]

[0018] The tumor weights of the treatment groups for each lot were smaller than those of the control group, and the antitumor effect of the MC. ghonii group was the highest (Fig. 1a). The tumor weight inhibition rate of the MC.ghonii group in experimental lots 1 and 3 was the best, while that of the LC.ghonii group in experimental lot 2 was the best. Statistical analysis based on the experimental results of the three lots revealed that the tumor weight inhibition rate of the HC.ghonii group was significantly different from that of the control group (p<0.05), and that the tumor weight inhibition rates of the LC.ghonii and MC.ghonii groups were significantly different from that of the control group (p<0.01), as shown in Figure 1b.

[0019] In conclusion, freeze-dried powder of Clostridium ghonii spores has an inhibitory effect on the growth of colon cancer, and research has shown that the MC. ghonii group (1 × 107 cfu / dose) is superior to the efficacy of other treatment groups. Therefore, the preferred dose of C. ghonii in the following combination with pembrolizumab is 1 × 10 7 cfu / time.

[0020] Example 2: Therapeutic effect of Clostridium ghonii spores in combination with Pembrolizumab on a PD-1 humanized gene-transfected colon cancer tumor-burdened mouse model

[0021] Materials: Clostridium ghonii spore lyophilized powder for injection, developed by Shandong Xinchuang Biotechnology Co., Ltd. and manufactured using the same method as in Example 1; Pembrolizumab injection, lot number: S001188, specification: 100 mg / 4 mL, sold by Merck, USA; control lyophilized powder, lot numbers: 201910002F, 201803001F, developed by Shandong Xinchuang Biotechnology Co., Ltd. and manufactured using the same method as in Example 1; 0.9% sodium chloride injection, lot number: 1809282161, sold by Chenxin Pharmaceutical Co., Ltd.; sterile water for injection , Lot number: 190221262, sold by Chenxin Pharmaceutical Co., Ltd.; MC38 colon cancer cells, Product number: T1917, sold by Abm Biotechnology Co., Ltd.; C57BL / 6PD-1 humanized genetically engineered mice, Animal Qualification Certificate Number: No. 20170010002500 (90 mice), No. 312024300008757 (35 mice), No. 201700100013119 (50 mice), sold by Shanghai Nanfang Model Biological Science and Technology Co., Ltd.

[0022] Method: MC38 cells were resuscitated and passaged to the required cell number, and the concentration was 7.5 × 10 6 A cell suspension containing 0.2 mL of cells was prepared for inoculation. A C57BL / 6PD-1 humanized colon cancer subcutaneous tumor model was constructed with a cell viability of 90% or higher. 0.2 mL of the cell suspension was inoculated subcutaneously into the right forelimb of the mouse. The tumor volume reached 0.15 cm within approximately 10 days. 3Larger experimental animals were selected for the test, and animals meeting the requirements were randomly selected and divided into four groups by lottery: a control group (Control), a Clostridium ghonii spore treatment group (C. ghonii), a Pembrolizumab group, and a Clostridium ghonii spore and Pembrolizumab combination group (C. ghonii + Pembrolizumab). Each group contained at least five animals. The Clostridium ghonii spore lyophilized powder was first reconstituted in 0.1 mL of sterile water for injection, and then diluted to a concentration of 1 x 10 with 0.9% sodium chloride injection by mass. 8 A suspension of cfu / mL was prepared and 0.1 mL was injected into the tumor. 1 × 10 spores were injected into the C. ghonii group and the C. ghonii + Pembrolizumab group. 7 The control and pembrolizumab groups received a dose of 0 cfu / injection, administered once every other day for a total of six doses. Pembrolizumab injection was diluted with 0.9% sodium chloride injection to a final working solution of 1 mg / mL and administered intraperitoneally at 0.2 mL. The pembrolizumab and C. ghonii + pembrolizumab groups received 0.2 mg of pembrolizumab / injection, and the control and C. ghonii groups received 0.2 mL of 0.9% sodium chloride injection twice weekly for a total of four doses.

[0023] Clostridium ghonii spores were administered first, followed by PD-1 antibodies, in the following order: Clostridium ghonii spores were injected once every other day starting from day 1, and pembrolizumab was injected on days 3, 6, 9, and 13, respectively.

[0024] Observation and evaluation index: After the start of administration, the animals' behavior and death or moribund status were observed daily, and the tumor volume was observed every 1-2 days. The tumor volume measured at the end of the experiment was used to calculate the tumor inhibition rate. All surviving animals were dissected two days after the final administration, and the tumor weight was weighed. The tumor inhibition rate was calculated based on the tumor weight. Tumor weight Tumor inhibition rate (IR TW%) = (mean tumor weight in the control group − mean tumor weight in the experimental group) / mean tumor weight in the control group × 100%; Tumor volume tumor inhibition rate (IR TV %) = 1 - (mean tumor volume at the beginning of the experimental group - mean tumor volume at the end of the experimental group) / (mean tumor volume at the beginning of the control group - mean tumor volume at the end of the control group) × 100%; Cure rate (%) = number of cured animals in each group / number of experimental animal groups in each group × 100% (time node is end of test).

[0025] Each independent experiment was repeated three times using the above experimental method. Results: During the experiment, one mouse died in the control group of experimental lot 1, but no deaths or moribund events occurred in any of the other groups in each lot.

[0026] 1. Average tumor weight and tumor inhibition rate of tumor-bearing mice in each group

[0027] The mean tumor weights of tumor-bearing mice in each group in the 3-rod test are shown in Table 3. The mean tumor weights of all treatment groups were smaller than those of the control group, and the tumor weights of the C. ghonii + Pembrolizumab group were smaller than those of either drug-only treatment group, showing a significantly greater decrease than that of the control group (P<0.01). The mean tumor weights of the C. ghonii + Pembrolizumab group were also significantly smaller than those of the C. ghonii alone group and the Pembrolizumab alone group (P<0.05, P<0.05) (Figure 2a).

[0028] [Table 3]

[0029] Statistical analysis based on the results of the three experimental batches revealed that each treatment group exhibited an inhibitory effect on tumor growth, with tumor inhibition rates of over 30%. The tumor inhibition rates were expressed as follows: C. ghonii + pembrolizumab group > pembrolizumab group > C. ghonii group (Figure 2b). After administration of the same dose of pembrolizumab, the C. ghonii + pembrolizumab group had tumor inhibition rates approximately 20%, 20%, and 25% higher than the pembrolizumab alone group (Table 4).

[0030] [Table 4]

[0031] 2. Average tumor volume and tumor inhibition rate of tumor-bearing mice in each group

[0032] When the groups were divided before the start of the experiment, there was no significant difference in the mean tumor volume between the groups within each lot (P > 0.05). At the end of the tumor-burdened mice administration period in the lot 3 study, the tumor volumes in the C. ghonii group, the Pembrolizumab group, and the C. ghonii + Pembrolizumab group were significantly smaller than those in the control group (p = 0.001, p = 0.000053, p = 0.000). The tumor volume in the C. ghonii + Pembrolizumab group was significantly smaller than those in the C. ghonii and Pembrolizumab groups. On day 10, a significant difference was observed between the mean tumor volumes of the combination group and the control group; however, the tumor volume in the C. ghonii + Pembrolizumab group was nearly one-fold smaller than that of the Pembrolizumab group (Figure 3).

[0033] The tumor inhibition rate was calculated based on tumor volume. During the three-rod test, the tumor inhibition rates in the C. ghonii + Pembrolizumab group were all greater than those in the other groups (Figure 4).

[0034] In three batches of pembrolizumab, after two 0.4 mg doses of pembrolizumab were administered on the seventh day, the tumor inhibition rates in the C. ghonii + pembrolizumab group were 51.29% (greater than 50%), 49.78% (approximately 50%), and 59.33% (greater than 50%), respectively, while the tumor inhibition rates in the pembrolizumab monotherapy group were 25.67%, 7.68%, and 35.10%, respectively. After three 0.6 mg doses of pembrolizumab in the pembrolizumab monotherapy group, the tumor inhibition rates reached the tumor inhibition rates of the C. ghonii + pembrolizumab group when 0.4 mg pembrolizumab was administered twice, which were 69.31%, 52.83%, and 53.08%, respectively (Figure 5). As can be seen from the above, the C. ghonii + Pembrolizumab group significantly enhanced the antitumor effect of Pembrolizumab, and the amount of Pembrolizumab required to achieve the same therapeutic effect was 50% lower in the C. ghonii + Pembrolizumab group, demonstrating high efficiency and low toxicity.

[0035] 3, cure rate

[0036] In the three-rod experiment, approximately 20% of mice (1 / 6, 1 / 5, and 1 / 6) in the C. ghonii + pembrolizumab group experienced complete tumor regression (Table 5), with no tumor growth observed by the end of the experiment. However, neither the pembrolizumab nor the C. ghonii group experienced complete tumor regression, resulting in a 0% cure rate. This suggests that Clostridium ghonii spores germinate in hypoxic areas of tumors and affect TME immunogenicity in multiple ways, potentially transforming the TME from an immunosuppressive state to an immunoactive state while modulating the immunosuppressive TME and overcoming immune tolerance. When combined with pembrolizumab, the therapeutic effect of PD-1 antibodies was further amplified, resulting in approximately 20% of mice being cured. Clostridium ghonii is expected to be an effective "sensitizer" for immunotherapy in tumor patients. The tumor morphology in each group is shown in Figure 6.

[0037] [Table 5]

[0038] Example 3: Effect of the administration order of Clostridium ghonii spores and Pembrolizumab on the therapeutic effect of a PD-1-humanized transfected colon cancer tumor-burdened mouse model

[0039] Materials: Clostridium ghonii spore lyophilized powder for injection, developed by Shandong Xinchuang Biotechnology Co., Ltd. and manufactured using the same method as in Example 1; Pembrolizumab injection, lot number: S006648, specification: 100 mg / 4 mL, sold by Merck, USA; control lyophilized powder, lot number: 201910002F, developed by Shandong Xinchuang Biotechnology Co., Ltd. and manufactured using the same method as in Example 1; 0.9% sodium chloride injection, lot number: J 18070104, sold by Shandong Hualu Pharmaceutical Co., Ltd.; Sterile water for injection, batch number: 190221262, sold by Chenxin Pharmaceutical Co., Ltd.; MC38 colon cancer cells, product number T1917, sold by Abm Biotechnology Co., Ltd.; C57BL / 6PD-1 humanized genetically engineered mice, animal qualification certificate number: No. 20170010002500 (90 mice), sold by Shanghai Nanfang Model Biological Science and Technology Co., Ltd.

[0040] Methods: The method for constructing a C57BL / 6PD-1 humanized genetically engineered mouse MC38 colon cancer subcutaneous tumor model was the same as in Example 2. Mice that were successfully modeled by lottery were randomly divided into A control group, B Clostridium ghonii spore group (C. ghonii), C Pembrolizumab group, D Pembrolizumab first followed by C. ghonii group, E C. ghonii first followed by Pembrolizumab group, and F C. ghonii + Pembrolizumab combination group. Each group consisted of 8 mice, and the C. ghonii doses were 0 cfu / dose, 1 x 10 7 cfu / time, 0 cfu / time, 1 × 10 7 cfu / time, 1 × 107 cfu / time and 1 × 10 7 cfu / dose, the administration volume was 0.1 mL / dose, once every other day, and 0.2 mL of 1 mg / mL Pembrolizumab injection was administered intraperitoneally at a dose of 0.2 mg / dose, once every other day. The administration schedule for the first phase is shown in Table 6.

[0041] [Table 6]

[0042] After the first administration was completed, the animals were observed for 6 days, and then the first administration process was repeated for the second administration.

[0043] Observation and evaluation index: After the start of administration, animals were observed daily for behavior and death or moribundity. Tumor volume was monitored every 1-2 days. The tumor volume measured at the end of the experiment was used to calculate the tumor inhibition rate. All surviving animals were sacrificed 8 days after the final administration of Phase 2. Tumor weight was weighed, and the tumor inhibition rate was calculated based on the tumor weight. Tumor weight tumor inhibition rate (IRTW%) = (mean tumor weight of control group - mean tumor weight of experimental group) / mean tumor weight of control group × 100%; Cure rate (%) = number of cured animals in each group / number of experimental animal groups in each group × 100% (time node is end of test).

[0044] Results: During the experiment, four animals died in the control group due to oversized tumors or ulcers, while no animals died or became moribund in the other groups.

[0045] 1. Tumor weight and tumor inhibition rate

[0046] The tumor weights in the C. ghonii group, Pembrolizumab group, Pembrolizumab-first group, C. ghonii-first group, Pembrolizumab-first group, and C. ghonii + Pembrolizumab group were all significantly smaller than those in the control group (p = 0.005, p = 0.000, p = 0.000, p = 0.000, p = 0.000, p = 0.000). The tumor weight measurements for each group are shown in Table 7 and Figure 7.

[0047] [Table 7]

[0048] The tumor inhibition rate was calculated according to tumor weight. The tumor inhibition rates of the C. ghonii group, Pembrolizumab group, Pembrolizumab first followed by C. ghonii group, C. ghonii first followed by Pembrolizumab group, and C. ghonii + Pembrolizumab group were 37.39%, 76.20%, 87.26%, 92.71%, and 92.19%, respectively. Each treatment group showed a significant inhibitory effect on tumor growth (see Table 8).

[0049] [Table 8]

[0050] 2. Tumor volume and tumor inhibition rate

[0051] There was no significant difference in tumor volume between the groups before treatment (p>0.05). At the end of treatment, tumor volumes in the C. ghonii group, Pembrolizumab group, Pembrolizumab-first C. ghonii group, C. ghonii-first Pembrolizumab group, and C. ghonii + Pembrolizumab combination group were all significantly smaller than the control group (p=0.002, p=0.000, p=0.000, p=0.000, p=0.000, p=0.000, Figure 8). The tumor inhibition rate was expressed as follows: C. ghonii-first Pembrolizumab group > C. ghonii + Pembrolizumab combination group > Pembrolizumab-first C. ghonii group > Pembrolizumab group > Control group (see Table 9).

[0052] [Table 9]

[0053] 3, cure rate

[0054] The cure rates for each group during the study are shown in Table 10.

[0055] [Table 10]

[0056] The tumor cure rate in mice in the C. ghonii-first group followed by Pembrolizumab was significantly higher than in the other groups, and was twice as high as that in the Pembrolizumab-first group followed by C. ghonii and the C. ghonii + Pembrolizumab combination group. This indicates that the prior administration of Clostridium ghonii spores into the tumor is effective, destroying the TME without distinguishing between tumor tissues, and recruiting immune cells to infiltrate the TME even after bacterial tumor lysis, changing the degree and composition of immune cell infiltration into the TME, resulting in a large number of CD8+ cells in the lysate. +This involves T cell attraction and aggregation, and treatment with oncolytic bacteria followed by PD-1 antibodies may avoid immunosuppression during immunotherapy and initiate a massive attack by T cells on tumor cells, potentially achieving a "dual" anti-cancer effect. Therefore, the order of administration of oncolytic bacteria and immunosuppressants is very important for the efficacy of tumor treatment.

[0057] Therefore, the combination of Clostridium ghonii spore therapy and immunotherapy is preferred to enhance the antitumor effect, reduce the amount of immunotherapy drugs used, and is highly efficient and low toxic.

[0058] Although the present invention has been described in detail through the above examples, these are only some examples of the present invention, not all examples, and other embodiments can be obtained according to these examples without inventiveness, and all of these examples belong to the protection scope of the present invention.

Claims

1. A drug combination for treating colon cancer, the combination comprising Clostridium ghonii bacterial spores and pembrolizumab as active ingredients; The Clostridium ghonii spores are freeze-dried powder of Clostridium ghonii spores for injection; The pembrolizumab is a PD-1 antibody injection, the combination is a combination of the lyophilized powder of Clostridium ghonii spores for injection and the PD-1 antibody injection solution, The order of administration of the lyophilized Clostridium ghonii spore powder for injection and the PD-1 antibody injection solution is first the lyophilized Clostridium ghonii spore powder for injection, and then the PD-1 antibody injection solution.

2. 2. The pharmaceutical combination according to claim 1, wherein the Clostridium ghonii is Clostridium ghonii MW-DCG-LCv-26 strain (stored in the Australian National Metrology Institute, strain number V12 / 001486) or a conditioned strain of Clostridium ghonii.

3. The pharmaceutical combination according to claim 2, wherein the adapted strain of Clostridium ghonii is MW-DCG-HNCv-18 strain (stored in the Australian National Metrology Institute, strain collection number V12 / 001485).

4. The pharmaceutical combination according to claim 2, wherein the adapted strain of Clostridium ghonii is MW-DCG-CCv-17 strain (stored in the National Institute of Metrology, Australia, strain collection number V12 / 001487).

5. 2. The pharmaceutical combination of claim 1, wherein the additive in the Clostridium ghonii spore lyophilized powder for injection is 1% sucrose.

6. The drug combination of claim 1, wherein 0.2 mg of pembrolizumab solution at a concentration of 1 mg / mL is used in combination per 1 x 107 CFU of Clostridium ghonii spores.

7. The drug combination according to claim 6, wherein the solvent of the pembrolizumab solution is 0.9% by mass sodium chloride injection.

8. 7. The drug combination according to claim 6, wherein the Clostridium ghonii spores are lyophilized powder of Clostridium ghonii spores for injection, and the solvent of the lyophilized powder of Clostridium ghonii spores for injection is sterilized water for injection and 0.9% sodium chloride injection solution by mass percentage.

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