WO2023056962A1 - 戈氏梭菌芽孢联合帕博利珠单抗的应用 - Google Patents

戈氏梭菌芽孢联合帕博利珠单抗的应用 Download PDF

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WO2023056962A1
WO2023056962A1 PCT/CN2022/124020 CN2022124020W WO2023056962A1 WO 2023056962 A1 WO2023056962 A1 WO 2023056962A1 CN 2022124020 W CN2022124020 W CN 2022124020W WO 2023056962 A1 WO2023056962 A1 WO 2023056962A1
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clostridium
pembrolizumab
group
gordii
spores
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PCT/CN2022/124020
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French (fr)
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王勇
朱红
张文华
邢艳秋
王丹
刘园园
王少鹏
郑嘉辉
张蓉
李晓楠
徐兴鲁
姜圣彪
邢立超
郜玉霞
邵石丽
韩停
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山东新创生物科技有限公司
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Priority to CA3228161A priority Critical patent/CA3228161A1/en
Priority to EP22877976.5A priority patent/EP4368204A1/en
Priority to KR1020247012739A priority patent/KR20240063969A/ko
Priority to US18/546,077 priority patent/US20240115627A1/en
Priority to AU2022358832A priority patent/AU2022358832A1/en
Publication of WO2023056962A1 publication Critical patent/WO2023056962A1/zh

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Definitions

  • the invention belongs to the technical field of biomedicine, and in particular relates to the application of Clostridium gordii combined with pembrolizumab (Pembrolizumab).
  • Tumor microenvironment is composed of tumor cells, stromal cells (including fibroblasts, immune, inflammatory cells, and some vascular endothelial cells, etc.) Molecules and so on together constitute the local steady-state environment.
  • TME provides the necessary material basis for tumor occurrence, development, and invasion, and regulates various biological behaviors such as tumor metastasis and recurrence.
  • TME can increase tumor drug resistance and radiation resistance, and reduce the therapeutic effect.
  • the immune regulation in the TME plays an important role in the occurrence and development of tumors, and it can form local tumor immunosuppression through various mechanisms. How to regulate the TME immunotherapy strategy and reshape the positive immune microenvironment is the focus and difficulty of anti-tumor therapy.
  • Pembrolizumab is a PD-1 inhibitor drug
  • PD-1 is an important immunosuppressive molecule
  • PD-1 antibody clinically shows significant anti-tumor effects, including durable treatment for patients with advanced metastasis Effect. It is generally believed that sensitivity to immune checkpoint blockade depends on tumor neoantigen load and the degree and composition of immune cell infiltration in the tumor microenvironment (TME). Unfortunately, most common cancers do not exhibit a large number of mutations and immune cell infiltration, so the tumors are not sensitive to immune checkpoint inhibitors, and the response rate is low, only 20% of patients are effective. Therefore, the development of methods that can make these tumors more sensitive to immunotherapy is also one of the current research directions.
  • Clostridium ghonii is a strictly anaerobic bacterium with tumor-tropic and hypoxic characteristics, and can only colonize in hypoxic areas of tumors.
  • Oncolysis of Clostridium gordii can recruit a large number of immune cells to infiltrate the TME, including innate immune cells including dendritic cells, neutrophils, and macrophages, as well as CD3 + T, CD4 + T, CD8 + T and other specific Immune cells, altering the extent and composition of immune cell infiltration in the TME. At the same time, it promotes the expression of TNF- ⁇ , IFN- ⁇ , IL-6 and other cytokines and chemokines in tumors.
  • Clostridium gordii oncolysis can affect the immunogenicity of TME in various ways, making TME change from an immunosuppressive state to an immune activation state, breaking immune tolerance, and making Clostridium gordii in combination with immune checkpoint inhibitors Efficient anti-tumor becomes possible.
  • the present invention provides the application of Clostridium gordii spore combined with PD-1 antibody.
  • Clostridium gordii spores combined with pembrolizumab in the preparation of pharmaceutical products for treating colon cancer.
  • a medicine for treating colon cancer the active ingredients in the medicine include Clostridium gordoi spores and pembrolizumab.
  • Other preferred bacterial strains are the MW-DCG-HNCv-18 bacterial strain, which is preserved in the Australian National Metrology Institute, with the strain preservation number V12/001485; the MW-DCG-CCv-17 bacterial strain, which is preserved in the Australian National Metrology Institute, Strain preservation number V12/001487 and so on.
  • the Clostridium gordii is in the form of spores.
  • the spore form of Clostridium gordii is freeze-dried powder, and its auxiliary material is 1% sucrose;
  • the pembrolizumab is PD-1 antibody injection.
  • Clostridium gordii spore freeze-dried powder is: freezing stage -40°C for 4h; -35°C for 10min while vacuuming, -30°C for 10min, -25°C for 10min, -20°C for 26h, -15°C for 2h , -10°C 10min, -5°C 10min, 0°C 10min, 10°C 2h, 15°C 10min, 20°C 3h, 27°C 3h by lyophilization.
  • the preferred drug combination of the present invention is 1 ⁇ 10 7 CFU lyophilized powder of Clostridium gordii spores combined with 0.2 mg of pembrolizumab injection at a concentration of 1 mg/mL.
  • the solvent of the pembrolizumab injection is sodium chloride injection with a mass percentage concentration of 0.9%; the solvent of the Clostridium gordii spore lyophilized powder is sterile water for injection and 0.9% chlorine Sodium Hydroxide Injection.
  • Clostridium gordii spore combined with pembrolizumab is used in the order of Clostridium gordii spore first and then pembrolizumab.
  • Clostridium gordii spores combined with pembrolizumab can significantly improve the curative effect against colon cancer, while reducing the dosage of pembrolizumab, with high efficiency and low toxicity.
  • Oncolysis of Clostridium gordii can affect the immunogenicity of TME in various ways, changing TME from an immunosuppressive state to an immune activated state, while regulating the immunosuppressive TME and breaking immune tolerance.
  • the combination of Clostridium gordii spores and pembrolizumab can completely eliminate the tumor tissue of about 20% of the mice, expanding the benefit range of PD-1 antibody therapy for tumor patients, even for PD-1 antibody treatment failure Patients also have outstanding curative effect.
  • Fig. 3 In Example 2, Clostridium gordii spores combined with Pembrolizumab treated the tumor volume changes in each group of the MC38 colon cancer tumor-bearing mouse model; the data are expressed as: *P ⁇ 0.05 compared with the Control group, **P ⁇ 0.05 compared with the Control group 0.01; #Compared with C.ghonii group, C.ghonii+Pembrolizumab group P ⁇ 0.05, ##Compared with C.ghonii group, C.ghonii+Pembrolizumab group P ⁇ 0.01;
  • FIG. 5 In Example 2, Clostridium gordii spores combined with Pembrolizumab treated MC38 colon cancer tumor-bearing mouse model with tumor volume inhibition rate of each batch;
  • Figure a Experimental batch 1
  • Figure b Experimental batch 2
  • Figure c Experimental batch 3;
  • Figure 7 Example 3 Clostridium gordii spores combined with pembrolizumab in different administration orders to treat the tumor weight of each group in the MC38 colon cancer tumor-bearing mouse model;
  • Fig. 8 The changes in tumor volume in each group of the MC38 colon cancer tumor-bearing mouse model treated with different administration sequences of Clostridium gordii spores combined with Pembrolizumab in Example 3.
  • Embodiment 1 Clostridium gordii spore treatment effect on colon cancer tumor-bearing mouse model
  • Clostridium gordii spore freeze-dried powder for injection uses Clostridium gordii spores as the active ingredient and 1% sucrose as the excipient, after -40°C for 4h; -35°C for 10min while vacuuming; , -20°C 26h, -15°C 2h, -10°C 10min, -5°C 10min, 0°C 10min, 10°C 2h, 15°C 10min, 20°C 3h, 27°C 3h freeze-drying procedures, the specification is 1 ⁇ 10 8 CFU/tube; reference substance freeze-dried powder, batch number: 201803001F, developed by Shandong Xinchuang Biotechnology Co., Ltd., prepared with 1mL 1% sucrose solution through the above freeze-drying procedure; 0.9% sodium chloride injection, batch number: 1803122161, Chenxin Pharmaceutical Co., Ltd.; Sterile Water for Injection, batch number: 1704242163, available from Chenxin Pharmaceutical Co., Ltd.; CT26
  • CT26.WT cells were resuscitated and passaged to the required number of cells, and a cell suspension with a concentration of 7.5 ⁇ 10 6 cells/mL was prepared for inoculation, and the cell viability was above 90%, which was used to establish colon cancer subcutaneous transplantation in BALB/c mice
  • 0.2 mL of cell suspension was inoculated subcutaneously in the right forelimb of mice, and experimental animals with a tumor volume of about 0.30 cm 3 were selected for the test in about 10 days; the qualified mice with tumor formation were randomly divided into 4 groups by lottery: the control group ( Control), low-dose treatment group (L C.ghonii), middle-dose treatment group (M C.ghonii), high-dose treatment group (H C.ghonii), 8 in each group; After reconstitution with 0.1mL sterilized water for injection, prepare the concentration of 5 ⁇ 10 7 cfu/mL, 1 ⁇ 10 8 cfu/mL, 2 ⁇ 10 8 cfu
  • the tumor weight of each treatment group was smaller than that of the Control group, and the M C. ghonii group had the best anti-tumor effect (a in Figure 1).
  • Each dose treatment group showed an inhibitory effect on tumor growth, and the tumor weight inhibition rate of the MC.ghonii group in experimental batches 1 and 3 was the best, and the tumor weight inhibition rate of the L C.ghonii group in experimental batch 2 best effect.
  • the tumor weight inhibition rate of the H C.ghonii group was significantly different from that of the Control group (p ⁇ 0.05), and the tumor weight of the L C.ghonii group and the M C.ghonii group Compared with the Control group, the tumor inhibition rate had a very significant difference (p ⁇ 0.01), as shown in b in Figure 1 .
  • the lyophilized powder of Clostridium gordoi spores can inhibit the growth of colon cancer, and the research shows that the MC.ghonii group (1 ⁇ 10 7 cfu/time) is better than other treatment groups. Therefore, the preferred dose of C.ghonii in combination with pembrolizumab is 1 ⁇ 10 7 cfu/time.
  • Example 2 The therapeutic effect of Clostridium gordii spores combined with Pembrolizumab on the PD-1 humanized transgenic colon cancer tumor-bearing mouse model
  • Clostridium gordoi spore freeze-dried powder for injection developed by Shandong Xinchuang Biotechnology Co., Ltd.
  • the preparation method is the same as the material in Example 1; Pembrolizumab injection, batch number: S001188, specification: 100mg/4mL, is available from Merck & Co., USA; the reference substance freeze-dried powder, batch number 201910002F, 201803001F, was developed and prepared by Shandong Xinchuang Biotechnology Co., Ltd.
  • Example 1 The method is the same as the material in Example 1; 0.9% sodium chloride injection, batch number: 1809282161, is available from Chenxin Pharmaceutical Co., Ltd.; sterile water for injection, batch number: 1902212162, is available from Chenxin Pharmaceutical Co., Ltd.; MC38 Colon cancer cells, product number T1917, available from Abm Biotechnology Co., Ltd.; C57BL/6PD-1 humanized genetically engineered mice, animal certificate number: No.20170010002500 (90), No.312024300008757 (35), No. .20170010001319 (50 pieces), available from Shanghai Southern Model Biotechnology Co., Ltd.
  • mice were recovered and passaged to the required number of cells, and a cell suspension with a concentration of 7.5 ⁇ 10 6 cells/mL was prepared for inoculation. The cell viability was above 90% to establish the colon of C57BL/6PD-1 humanized genetically engineered mice.
  • mice were subcutaneously inoculated with 0.2 mL of cell suspension in the right forelimb, and experimental animals with a tumor volume greater than 0.15 cm 3 were selected for the test in about 10 days; the animals that met the requirements were screened out by random and divided into three groups by lottery.
  • control group Control
  • Clostridium gordoi spore treatment group C.ghonii
  • Pembrolizumab group Clostridium gordoi spore combined with pembrolizumab group (C.ghonii+Pembrolizumab), no less than 5 rats in each group
  • Clostridium spore freeze-dried powder was first reconstituted with 0.1mL sterile water for injection, and then prepared a suspension with a concentration of 1 ⁇ 10 8 cfu/mL with 0.9% by mass percent sodium chloride injection, and injected 0.1mL into the tumor.
  • the C.ghonii group and the C.ghonii+Pembrolizumab group were injected with 1 ⁇ 10 7 cfu/time of spores, the dose of the Control group and the Pembrolizumab group was 0 cfu/time, once every other day, for a total of 6 times; Dilute the Pembrolizumab injection to a final concentration of 1mg/mL working solution, inject 0.2mL intraperitoneally, inject 0.2mg/time of Pembrolizumab in the Pembrolizumab group and C.ghonii+Pembrolizumab group, and inject 0.2mg/time in the Control group and C.ghonii group. mL0.9% sodium chloride injection, administered 2 times a week, 4 times in total.
  • Clostridium gordii spores were given priority and then PD-1 antibody was administered.
  • the order of administration was as follows: Clostridium gordii spores were injected every other day from day 1; pembrolizumab was injected on days 3, 6, 9 and 13 respectively.
  • Observation and evaluation indicators after the start of administration, observe the animal behavior, death or dying situation every day; observe the tumor volume every 1-2 days, and calculate the tumor inhibition rate based on the tumor volume measured at the end of the experiment; all surviving animals were given The tumors were dissected on the second day after the administration, and the tumor weight was weighed, and the tumor inhibition rate was calculated based on the tumor weight.
  • Cure rate (%) number of cured animals in each group/total number of experimental animals in each group ⁇ 100% (the time node is the end of the experiment).
  • the average tumor weights of tumor-bearing mice in each group of the three batches of experiments are shown in Table 3.
  • the average 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, which was significantly lower than that of 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, P ⁇ 0.05) (a in Figure 2).
  • the tumor inhibition rate was C.ghonii+Pembrolizumab group>Pembrolizumab group>C.ghonii group (b in Figure 2).
  • the tumor inhibition rates of the C.ghonii+pembrolizumab group were increased by about 20%, 20%, and 25% compared with the pembrolizumab alone group (Table 4).
  • the tumor inhibition rate was calculated by tumor volume, and the tumor inhibition rate of the C.ghonii+Pembrolizumab group was greater than that of the other groups during the three batches of experiments ( Figure 4).
  • pembrolizumab On the 7th day after the administration in the 3 batches of experiments, pembrolizumab was administered twice. After the pembrolizumab dose was 0.4 mg, the tumor inhibition rates of the C.ghonii+pembrolizumab group were 51.29% (greater than 50%) and 49.78% (approx. 50%), 59.33% (greater than 50%), while the tumor inhibition rates of the pembrolizumab group alone were 25.67%, 7.68%, and 35.10%, respectively.
  • the Pembrolizumab group alone was given 3 times, and after the Pembrolizumab dose was 0.6 mg, the tumor inhibition rate reached the tumor inhibition rate when the C.ghonii+Pembrolizumab group was given 2 times of Pembrolizumab (dose 0.4 mg), which were 69.31% respectively , 52.83%, 53.08% ( Figure 5). It can be seen that the C.ghonii+Pembrolizumab group significantly improved the anti-tumor effect of Pembrolizumab, and the dose of Pembrolizumab required for the C.ghonii+Pembrolizumab group to obtain the same therapeutic effect was reduced by 50%, with high efficiency and low toxicity.
  • mice in the three batches of C.ghonii+Pembrolizumab groups had tumors completely eliminated (Table 5), and no tumor growth was seen at the end of the experiment, while the Pembrolizumab group Neither group nor C.ghonii group showed complete disappearance of tumor, and the cure rate was 0%.
  • This may be related to the germination of Clostridium gordii spores in the hypoxic area of the tumor, which affects the immunogenicity of the TME in various ways, changing the TME from an immunosuppressive state to an immune active state, while regulating the immunosuppressive TME and breaking immune tolerance.
  • Clostridium gordii is expected to be an excellent "sensitizer" for immunotherapy of tumor patients.
  • the morphology of the tumors in each group is shown in Figure 6.
  • Example 3 Effect of Clostridium gordii spores and pembrolizumab administration sequence on therapeutic effect of PD-1 humanized transgenic colon cancer tumor-bearing mouse model
  • Clostridium gordoi spore freeze-dried powder for injection developed by Shandong Xinchuang Biotechnology Co., Ltd.
  • the preparation method is the same as the material in Example 1; Pembrolizumab injection, batch number: S006648, specification: 100mg/4mL, available from Merck, USA ; reference substance lyophilized powder, batch number 201910002F, developed by Shandong Xinchuang Biotechnology Co., Ltd., the preparation method is the same as the material in Example 1; 0.9% sodium chloride injection, batch number: J18070104, sold by Shandong Hualu Pharmaceutical Co., Ltd.; Bacteria water for injection, batch number: 1902212162, available from Chenxin Pharmaceutical Co., Ltd.; MC38 colon cancer cells, item number T1917, available from Abm Biotechnology Co., Ltd.; C57BL/6PD-1 humanized genetic engineering mice, animal certificate No.: No.20170010002500 (90 pieces), available from Shanghai Southern Model Biotechnology Co., Ltd.
  • Clostridium spore group (C.ghonii), C pembrolizumab group, D first pembrolizumab then C.ghonii group, E first C.ghonii then pembrolizumab group, F C.ghonii+pembrolizumab simultaneous group, 8 rats in each group; C.ghonii
  • the dosages are 0cfu/time, 1 ⁇ 10 7 cfu/time, 0cfu/time, 1 ⁇ 10 7 cfu/time, 1 ⁇ 10 7 cfu/time and 1 ⁇ 10 7 cfu/time respectively, and the dosage volume is 0.1 mL/time, administered once every other day; 1 mg/mL pembrolizumab injection was intraperitoneally administered 0.2 mL, the dose was 0.2 mg/time, administered once every other day.
  • the administration process of the first stage is shown in Table 6.
  • the administration of the first stage was completed, and after 6 days of observation, the administration process of the first stage was repeated in the second stage.
  • Observation and evaluation indicators After the start of administration, observe animal behavior, death or near-death conditions every day, observe tumor volume every 1-2 days, and calculate the tumor inhibition rate based on the tumor volume measured at the end of the experiment. All surviving animals were dissected on day 8 after the last administration of the second period. The tumor weight was weighed, and the tumor inhibition rate was calculated based on the tumor weight.
  • Cure rate (%) number of cured animals in each group/total number of experimental animals in each group ⁇ 100% (the time node is the end of the experiment).
  • the results of tumor weight detection in each group are shown in Table 7 and Figure 7.
  • the tumor inhibition rate was calculated according to the tumor weight, and the results showed that the tumor inhibition rates were 37.39% and 76.20% in the C.ghonii group, pembrolizumab group, pembrolizumab first then C.ghonii group, C.ghonii first then pembrolizumab group, and C.ghonii+pembrolizumab group respectively , 87.26%, 92.71% and 92.19%, each treatment group showed a significant inhibitory effect on tumor growth, see Table 8.
  • the tumor inhibition rate was C.ghonii first and then Pembrolizumab group>C.ghonii+Pembrolizumab simultaneous group>Pembrolizumab first and then C.ghonii group>Pembrolizumab group>Control group, see Table 9.
  • the tumor cure rate of the mice in the C.ghonii first followed by Pembrolizumab group was significantly higher than that of other groups, which was twice the cure rate of the first Pembrolizumab followed by C.ghonii group and the simultaneous C.ghonii+Pembrolizumab group. This may be due to the fact that intratumoral administration of Clostridium gordoi spores effectively and indiscriminately dissolves tumor tissue and destroys the TME. At the same time, bacterial oncolysis also recruits immune cells to infiltrate the TME, changing the degree and composition of immune cell infiltration in the TME.
  • Clostridium gordii spores for treatment and then combined with immunotherapy can enhance the anti-tumor effect, reduce the dosage of immune drugs, and have high efficiency and low toxicity.

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Abstract

本发明涉及戈氏梭菌联合帕博利珠单抗(Pembrolizumab)在癌症治疗中的应用。本发明首次发现戈氏梭菌芽孢联合帕博利珠单抗可以高抗结肠癌的疗效，同时降低帕博利珠单抗的使用剂量，高效低毒。戈氏梭菌溶瘤可通过多种方式影响TME免疫原性，使TME从一个免疫抑制状态变成免疫激活状态，同时调节免疫抑制性TME，打破免疫耐受。在最佳组合条件下戈氏梭菌芽孢与帕博利珠单抗联合约有20％小鼠肿瘤组织彻底清除，将PD-1抗体治疗肿瘤患者的受益范围扩大，甚至对PD-1抗体治疗失败患者也有疗效。

Description

戈氏梭菌芽孢联合帕博利珠单抗的应用
本申请要求于2021年10月09日提交中国专利局、申请号为CN202111177854.3、发明名称为“戈氏梭菌芽孢联合PD-1抗体的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物医药技术领域，具体涉及戈氏梭菌联合帕博利珠单抗(Pembrolizumab)的应用。
背景技术
肿瘤微环境(Tumor microenvironment，TME)是在肿瘤生长过程中，由肿瘤细胞、基质细胞(包括成纤维细胞、免疫、炎性细胞以及一些血管内皮细胞等)和细胞外基质以及浸润在其中的生物分子等共同构成局部稳态环境。TME为肿瘤发生、发展、侵袭等提供了必要物质基础，调控肿瘤转移、复发等多种生物学行为。同时TME可以增加肿瘤耐药性和耐辐射性，降低治疗效果。TME内的免疫调节在肿瘤发生发展中具有重要功能，其可通过多种机制，形成肿瘤局部免疫抑制。如何调控TME免疫治疗策略，重塑积极的免疫微环境是抗肿瘤治疗的重点和难点。
帕博利珠单抗(Pembrolizumab)是一种PD-1抑制剂药物，PD-1是一种重要的免疫抑制分子，PD-1抗体临床表现出显著抗肿瘤效果，包括对晚期转移患者的持久治疗效果。一般认为，对免疫检查点阻断剂的敏感性取决于肿瘤新生抗原负荷以及肿瘤微环境(TME)中免疫细胞浸润程度和组成。不幸的是，多数常见癌症并未表现出大量的突变和免疫细胞浸润，因此肿瘤对免疫检查点抑制剂不敏感，响应率低，仅20％患者使用有效。因此，开发能使这些肿瘤对免疫治疗更敏感方法也是目前重点研究方向之一。
戈氏梭菌(Clostridium ghonii)为严格厌氧菌，具有趋肿瘤低氧特性，仅能定植在肿瘤乏氧区。戈氏梭菌溶瘤可募集大量免疫细胞向TME浸润，包括树突状细胞、中性粒细胞、巨噬细胞在内的先天免疫细胞及CD3 +T、CD4 +T、CD8 +T等特异性免疫细胞，改变TME中免疫细胞浸润程度和组成。同时促进肿瘤中TNF-α、IFN-γ、IL-6等细胞因子和趋化因子表达增强。可见，戈氏梭菌溶瘤可以通过多种方式影响TME免疫原性，使TME从一个免疫抑制状态变成免疫激 活状态，打破免疫耐受，使得戈氏梭菌与免疫检查点抑制剂联用高效抗肿瘤成为可能。
发明内容
本发明根据现有技术的不足，提供戈氏梭菌芽孢联合PD-1抗体的应用。
本发明技术方案如下：
戈氏梭菌芽孢联合帕博利珠单抗(Pembrolizumab)在制备治疗结肠癌医药制品中的应用。
一种治疗结肠癌的药物，药物中的药效成分包括戈氏梭菌芽孢和帕博利珠单抗。
根据本发明优选的，所述的戈氏梭菌为戈氏梭菌MW-DCG-LCv-26菌株或者戈氏梭菌驯化后获得的菌株；戈氏梭菌MW-DCG-LCv-26菌株保藏于澳大利亚国家计量研究院，菌株编号为V12/001486。其它优选的菌株为MW-DCG-HNCv-18菌株，该菌株保藏于澳大利亚国家计量研究院，菌株保藏号V12/001485；MW-DCG-CCv-17菌株，该菌株保藏于澳大利亚国家计量研究院，菌株保藏号V12/001487等。
根据本发明优选的，所述的戈氏梭菌为芽孢形式。
本发明优选的，所述戈氏梭菌的芽孢形式为冻干粉剂，其辅料为1％蔗糖；
本发明优选的，所述帕博利珠单抗为PD-1抗体注射液。
进一步优选的戈氏梭菌芽孢冻干粉剂冻干工艺为：冻结阶段-40℃4h；-35℃10min同时抽真空、-30℃10min、-25℃10min、-20℃26h、-15℃2h、-10℃10min、-5℃10min、0℃10min、10℃2h、15℃10min、20℃3h、27℃3h冻干制备。
本发明优选的药物组合是1×10 7CFU戈氏梭菌芽孢冻干粉联合浓度1mg/mL的帕博利珠单抗注射液0.2mg。
进一步优选的，所述帕博利珠单抗注射液的溶媒为质量百分比浓度0.9％的氯化钠注射液；戈氏梭菌芽孢冻干粉的溶媒为灭菌注射用水和质量百分比浓度0.9％氯化钠注射液。
进一步优选的，所述戈氏梭菌芽孢联合帕博利珠单抗使用顺序为先戈氏梭菌芽孢后帕博利珠单抗。
有益效果
本发明首次发现戈氏梭菌芽孢联合帕博利珠单抗可以显著提高抗结肠癌的 疗效，同时降低帕博利珠单抗的使用剂量，高效低毒。戈氏梭菌溶瘤可通过多种方式影响TME免疫原性，使TME从一个免疫抑制状态变成免疫激活状态，同时调节免疫抑制性TME，打破免疫耐受。在最佳组合条件下戈氏梭菌芽孢与帕博利珠单抗联合约有20％小鼠肿瘤组织彻底清除，将PD-1抗体治疗肿瘤患者的受益范围扩大，甚至对PD-1抗体治疗失败患者也有突出疗效。
附图说明
图1实施例1中戈氏梭菌芽孢治疗CT26.WT结肠癌荷瘤鼠模型各组肿瘤重量(a)、抑瘤率(b)；数据表示为：*与Control组相比P＜0.05，**与Control组相比P＜0.01；
图2实施例2中戈氏梭菌芽孢联合Pembrolizumab治疗MC38结肠癌荷瘤鼠模型各组肿瘤重量(a)、肿瘤重量抑瘤率(b)；数据表示为：*P＜0.05，**P＜0.01；
图3实施例2中戈氏梭菌芽孢联合Pembrolizumab治疗MC38结肠癌荷瘤鼠模型各组肿瘤体积变化；数据表示为：*与Control组相比P＜0.05，**与Control组相比P＜0.01；#与C.ghonii组相比，C.ghonii+Pembrolizumab组P＜0.05，##与C.ghonii组相比，C.ghonii+Pembrolizumab组P＜0.01；
图4实施例2中戈氏梭菌芽孢联合Pembrolizumab治疗MC38结肠癌荷瘤鼠模型实验终点肿瘤体积抑瘤率；数据表示为：*P＜0.05，**P＜0.01；
图5实施例2中戈氏梭菌芽孢联合Pembrolizumab治疗MC38结肠癌荷瘤鼠模型各批肿瘤体积抑瘤率；a图实验批次1，b图实验批次2，c图实验批次3；
图6实施例2中戈氏梭菌芽孢联合Pembrolizumab治疗MC38结肠癌荷瘤鼠模型各组肿瘤瘤体形态照片；
图7实施例3中戈氏梭菌芽孢联合Pembrolizumab不同给药顺序治疗MC38结肠癌荷瘤鼠模型各组肿瘤重量；
图8实施例3中戈氏梭菌芽孢联合Pembrolizumab不同给药顺序治疗MC38结肠癌荷瘤鼠模型各组肿瘤体积变化。
具体实施方式
下面将结合具体实施例对本发明做进一步的详细说明，所描述的实施例仅仅是本发明为了使公众便于理解所列举的一部分技术方案，这些实施例仅用于说明本发明而不用于限制本发明的保护范围，本发明的保护范围由权利要求限 定。
实施例1戈氏梭菌芽孢对结肠癌荷瘤鼠模型治疗效果
材料：注射用戈氏梭菌芽孢冻干粉，菌株为MW-DCG-LCv-26菌株，该菌株保藏于澳大利亚国家计量研究院，菌株保藏号V12/001486，山东新创生物科技有限公司研制。注射用戈氏梭菌芽孢冻干粉以戈氏梭菌芽孢为活性成分，以1％蔗糖为辅料，经过-40℃4h；-35℃10min同时抽真空、-30℃10min、-25℃10min、-20℃26h、-15℃2h、-10℃10min、-5℃10min、0℃10min、10℃2h、15℃10min、20℃3h、27℃3h冻干程序制备，规格为1×10 8CFU/支；对照品冻干粉，批号：201803001F，山东新创生物科技有限公司研制，以1mL1％蔗糖溶液经过上述冻干程序制备；0.9％氯化钠注射液，批号：1803122161，辰欣药业股份有限公司有售；灭菌注射用水，批号：1704242163，辰欣药业股份有限公司有售；CT26.WT结肠癌细胞，编号：3131C0001000800037，中国科学院上海生命科学研究院细胞资源中心有售；BALB/c雌性小鼠，动物合格证号：No.11401300088557(120只)，No.11401300089721(60只)，No.11401300089721(60只)，北京华阜康生物科技股份有限公司有售。
方法：CT26.WT细胞复苏传代至所需细胞数量，制备浓度为7.5×10 6个/mL的接种用细胞悬液，细胞活率在90％以上用于建立BALB/c小鼠结肠癌皮下移植瘤模型，小鼠右前肢皮下接种细胞悬液0.2mL，10天左右选择肿瘤体积约0.30cm 3的实验动物用于试验；成瘤合格的小鼠以抽签法随机分为4组：对照组(Control)、低剂量治疗组(L C.ghonii)、中剂量治疗组(M C.ghonii)、高剂量治疗组(H C.ghonii)，每组8只；戈氏梭菌芽孢冻干粉先用0.1mL灭菌注射用水复溶后，再用质量百分比浓度0.9％氯化钠注射液配制浓度分别为5×10 7cfu/mL、1×10 8cfu/mL、2×10 8cfu/mL的混悬液，瘤内注射0.1mL，隔天给药1次，共给药5次，给药剂量分别为5×10 6cfu/次、1×10 7cfu/次、2×10 7cfu/次，对照组的对照品浓度与高剂量治疗组相同；开始给药后，每天观察动物行为、死亡或濒死等临床症状，所有存活动物于末次给药后第2天进行解剖，称量肿瘤重量，并以肿瘤重量计算抑瘤率，肿瘤重量抑瘤率(IR TW％)＝(对照组平均瘤重-实验组平均瘤重)/对照组平均瘤重×100％。以上述实验方法，分别进行3批独立重复实验。
结果：实验期间，3批实验所有动物均未见濒死/死亡情况。实验结束，3 批实验各组荷瘤鼠平均肿瘤重量及治疗组抑瘤率如表1、表2所示。
表1各组荷瘤鼠平均肿瘤重量(g)
实验批次 Control组 L C.ghonii组 M C.ghonii组 H C.ghonii组
1 2.85±0.59 1.98±0.76 1.84±0.67 2.45±0.34
2 4.15±0.51 2.00±0.61 2.23±0.78 2.50±0.71
3 2.69±0.52 1.73±0.56 1.72±0.42 1.93±0.39
表2治疗组肿瘤重量抑瘤率(％)
实验批次 L C.ghonii组 M C.ghonii组 H C.ghonii组
1 30.40 35.58 14.12
2 51.90 46.17 39.79
3 35.46 35.97 28.06
与Control组相比，每批治疗组肿瘤重量均小于Control组，以M C.ghonii组抗肿瘤效果最好(图1中a)。各剂量治疗组均表现出对肿瘤生长的抑制作用，且实验批次1和3中MC.ghonii组肿瘤重量抑瘤率效果最好，实验批次2中L C.ghonii组肿瘤重量抑瘤率效果最好。根据3批次实验结果进行统计学分析发现，H C.ghonii组肿瘤重量抑瘤率与Control组相比具有显著性差异(p＜0.05)，L C.ghonii组和M C.ghonii组肿瘤重量抑瘤率与Control组相比具有极显著差异(p＜0.01)，图1中b所示。
综上所述，戈氏梭菌芽孢冻干粉对结肠癌生长具有抑制作用，且研究表明MC.ghonii组(1×10 7cfu/次)优于其它治疗组效果。因此，在接下来与Pembrolizumab组合用药中C.ghonii优选剂量1×10 7cfu/次。
实施例2戈氏梭菌芽孢联合Pembrolizumab对PD-1人源化转基因结肠癌荷瘤鼠模型治疗效果
材料：注射用戈氏梭菌芽孢冻干粉，山东新创生物科技有限公司研制。制备方法同实施例1中材料；Pembrolizumab注射液，批号：S001188，规格：100mg/4mL，美国默沙东公司有售；对照品冻干粉，批号201910002F，201803001F，山东新创生物科技有限公司研制，制备方法同实施例1中材料；0.9％氯化钠注射液，批号：1809282161，辰欣药业股份有限公司有售；灭菌注射用水，批号：1902212162，辰欣药业股份有限公司有售；MC38结肠癌细胞，货号T1917，Abm生物科技有限公司有售；C57BL/6PD-1人源化基因工程小鼠，动物合格证号：No.20170010002500(90只)，No.312024300008757(35只)，No.20170010001319(50只)，上海南方模式生物科技股份有限公司有售。
方法：MC38细胞复苏传代至所需细胞数量，制备浓度为7.5×10 6个/mL的接种用细胞悬液，细胞活率在90％以上建立C57BL/6PD-1人源化基因工程小鼠结肠癌皮下移植瘤模型，小鼠右前肢皮下接种0.2mL细胞悬液，10天左右选择肿瘤体积大于0.15cm 3的实验动物用于试验；采用随机原则将筛选出符合要求的动物以抽签法分为4组：对照组(Control)、戈氏梭菌芽孢治疗组(C.ghonii)、Pembrolizumab组和戈氏梭菌芽孢联合Pembrolizumab组(C.ghonii+Pembrolizumab)，每组不少于5只；戈氏梭菌芽孢冻干粉先用0.1mL灭菌注射用水复溶后，再用质量百分比0.9％氯化钠注射液配制浓度1×10 8cfu/mL的混悬液，瘤内注射0.1mL，C.ghonii组和C.ghonii+Pembrolizumab组注射芽孢1×10 7cfu/次，Control组和Pembrolizumab组剂量为0cfu/次，隔1天给药1次，共给药6次；以0.9％氯化钠注射液将Pembrolizumab注射液稀释成终浓度为1mg/mL工作液，腹腔注射0.2mL，Pembrolizumab组和C.ghonii+Pembrolizumab组注射Pembrolizumab为0.2mg/次，Control组和C.ghonii组注射0.2mL0.9％氯化钠注射液，每周给药2次，共4次。
优先给予戈氏梭菌芽孢再给予PD-1抗体，给药顺序如下：戈氏梭菌芽孢于第1天起，隔一天注射一次；Pembrolizumab于第3、6、9和13天分别进行注射。
观测及评价指标：开始给药后，每天观察动物行为、死亡或濒死情况；每隔1-2天观测肿瘤体积，并以实验末次测量的肿瘤体积计算抑瘤率；所有存活动物于末次给药第2天后进行解剖，称量肿瘤重量，并以肿瘤重量计算抑瘤率。
肿瘤重量抑瘤率(IR TW％)＝(对照组平均瘤重-实验组平均瘤重)/对照组平均瘤重×100％；
肿瘤体积抑瘤率(IR TV％)＝1-(实验组开始平均肿瘤体积-实验组结束平均肿瘤体积)/(对照组开始平均肿瘤体积-对照组结束平均肿瘤体积)×100％；
治愈率(％)＝每组治愈的动物数/每组总实验动物数×100％(时间节点为试验结束)。
以上述实验方法，分别进行3批独立重复实验。
结果：实验期间，实验批次1中对照组1只小鼠死亡，其他各批各组未出现死亡或濒死情况。
1、各组荷瘤鼠平均肿瘤重量与抑瘤率
3批实验各组荷瘤鼠平均肿瘤重量如表3所示。各治疗组平均肿瘤重量均小于Control组，且C.ghonii+Pembrolizumab组肿瘤重量均小于任何一种药物单独治疗组，与Control组相比，极显著降低(P＜0.01)。C.ghonii+Pembrolizumab组平均肿瘤重量也显著小于单独C.ghonii组和单独Pembrolizumab组(P＜0.05，P＜0.05)(图2中a)。
表3各组荷瘤鼠平均肿瘤重量(g)
实验批次 Control组 C.ghonii组 Pembrolizumab组 C.ghonii+Pembrolizumab组
1 3.46±1.33 2.24±0.74 0.83±0.74 0.18±0.19
2 7.97±2.46 5.42±1.70 1.92±1.42 0.56±0.45
3 6.41±1.46 3.60±1.09 2.48±1.40 0.86±0.79
根据3批次实验结果进行统计学分析发现各治疗组表现出对肿瘤生长的抑制作用，抑瘤率均在30％以上，抑瘤率表现为C.ghonii+Pembrolizumab组＞Pembrolizumab组＞C.ghonii组(图2中b)。Pembrolizumab给药相同剂量后，C.ghonii+Pembrolizumab组比单独Pembrolizumab组抑瘤率分别提高了约20％，20％，25％(表4)。
表4治疗组肿瘤重量抑瘤率(％)
实验批次 C.ghonii组 Pembrolizumab组 C.ghonii+Pembrolizumab组
1 35.33 76.04 94.76
2 32.03 75.95 92.95
3 43.82 61.27 86.61
2、各组荷瘤鼠平均肿瘤体积与抑瘤率
实验开始前分组时各批次各组平均肿瘤体积均无显著性差异(P＞0.05)。批次3实验中荷瘤鼠给药末期，与Control组相比，C.ghonii组、Pembrolizumab组和C.ghonii+Pembrolizumab肿瘤体积显著小于Control组(p＝0.001，p＝0.000053，p＝0.000)。C.ghonii+Pembrolizumab组肿瘤体积明显小于C.ghonii组和Pembrolizumab组，且在第10天联合组与Control组平均肿瘤体积出现显著性差异，而此时C.ghonii+Pembrolizumab组肿瘤体积比Pembrolizumab组小近1倍(图3)。
以肿瘤体积计算抑瘤率，3批试验期间C.ghonii+Pembrolizumab组抑瘤率均大于其它各组抑瘤率(图4)。
3批次实验中给药后第7天，Pembrolizumab完成给药2次，Pembrolizumab给药剂量0.4mg后，C.ghonii+Pembrolizumab组抑瘤率分别为51.29％(大于 50％)，49.78％(约50％)，59.33％(大于50％)，而此时单独Pembrolizumab组抑瘤率分别为25.67％，7.68％，35.10％。单独Pembrolizumab组给药3次，Pembrolizumab给药剂量0.6mg后，其抑瘤率才达到C.ghonii+Pembrolizumab组给药2次Pembrolizumab(给药剂量0.4mg)时的抑瘤率，分别为69.31％，52.83％，53.08％(图5)。可见，C.ghonii+Pembrolizumab组显著提高了Pembrolizumab抗肿瘤效果，在获得同样治疗效果C.ghonii+Pembrolizumab组所需Pembrolizumab使用剂量降低50％，高效低毒。
3、治愈率
3批实验C.ghonii+Pembrolizumab组分别有约20％(1/6、1/5、1/6)的小鼠肿瘤完全消除(表5)，至实验终点也未见肿瘤生长，而Pembrolizumab组和C.ghonii组均未出现肿瘤彻底消失情况，治愈率0％。这可能与戈氏梭菌芽孢在肿瘤乏氧区萌发，通过多种方式影响TME免疫原性，使TME从一个免疫抑制状态变成免疫激活状态，同时调节免疫抑制性TME，打破免疫耐受。联合Pembrolizumab后可将PD-1抗体治疗效果进一步放大，约20％小鼠被治愈。戈氏梭菌有望成为肿瘤病人免疫治疗优秀的“增敏剂”。各组肿瘤瘤体形态见图6。
表5治愈率(％)
实验批次 Control组 C.ghonii组 Pembrolizumab组 C.ghonii+Pembrolizumab组
1 0 0 0 16.7
2 0 0 0 20.0
3 0 0 0 16.7
实施例3戈氏梭菌芽孢和Pembrolizumab给药顺序对PD-1人源化转基因结肠癌荷瘤鼠模型治疗效果的影响
材料：注射用戈氏梭菌芽孢冻干粉，山东新创生物科技有限公司研制，制备方法同实施例1中材料；Pembrolizumab注射液，批号：S006648，规格：100mg/4mL，美国默沙东公司有售；对照品冻干粉，批号201910002F，山东新创生物科技有限公司研制，制备方法同实施例1中材料；0.9％氯化钠注射液，批号：J18070104，山东华鲁制药有限公司有售；灭菌注射用水，批号：1902212162，辰欣药业股份有限公司有售；MC38结肠癌细胞，货号T1917，Abm生物科技有限公司有售；C57BL/6PD-1人源化基因工程小鼠，动物合格证号：No.20170010002500(90只)，上海南方模式生物科技股份有限公司有售。
方法：C57BL/6PD-1人源化基因工程小鼠MC38结肠癌皮下移植瘤模型建 立方法同实施例2；以抽签法将建模成功的小鼠随机分为A对照组(Control)、B戈氏梭菌芽孢组(C.ghonii)、C Pembrolizumab组、D先Pembrolizumab后C.ghonii组、E先C.ghonii后Pembrolizumab组、F C.ghonii+Pembrolizumab同时组，每组8只；C.ghonii给药剂量分别为0cfu/次、1×10 7cfu/次、0cfu/次、1×10 7cfu/次、1×10 7cfu/次和1×10 7cfu/次，给药容量为0.1mL/次，隔1天给药1次；1mg/mL Pembrolizumab注射液腹腔给药0.2mL，剂量为0.2mg/次，隔1天给药1次。第一阶段给药过程如表6所示。
表6第一阶段给药过程
Figure PCTCN2022124020-appb-000001
第一阶段给药结束，观察6天后，第二阶段重复第一阶段的给药过程。
观测及评价指标：开始给药后，每天观察动物行为、死亡或濒死情况，每隔1-2天观测肿瘤体积，并以实验末次测量的肿瘤体积计算抑瘤率。所有存活动物于第二阶段末次给药后第8天进行解剖。称量肿瘤重量，并以肿瘤重量计算抑瘤率。
肿瘤重量抑瘤率(IR TW％)＝(对照组平均瘤重-实验组平均瘤重)/对照组平均瘤重×100％；
治愈率(％)＝每组治愈的动物数/每组总实验动物数×100％(时间节点为试验结束)。
结果：实验期间，Control组因肿瘤过大或破溃出现4只动物死亡，其它各组未出现动物死亡或濒死情况。
1、肿瘤重量与抑瘤率
与Control组相比，C.ghonii组、Pembrolizumab组、先Pembrolizumab后C.ghonii组、先C.ghonii后Pembrolizumab组、C.ghonii+Pembrolizumab组肿瘤重量均显著小于Control组(p＝0.005，p＝0.000，p＝0.000，p＝0.000，p＝0.000)。各组肿瘤重量检测结果见表7，图7。
表7肿瘤重量
Figure PCTCN2022124020-appb-000002
备注：与Control组相比，**p<0.01。
根据肿瘤重量计算抑瘤率，结果显示C.ghonii组、Pembrolizumab组、先Pembrolizumab后C.ghonii组、先C.ghonii后Pembrolizumab组、C.ghonii+Pembrolizumab组抑瘤率分别为37.39％、76.20％、87.26％、92.71％和92.19％，各治疗组均表现出对肿瘤生长的明显抑制作用，见表8。
表8抑瘤率
Figure PCTCN2022124020-appb-000003
2、肿瘤体积与抑瘤率
治疗前，各组荷瘤鼠肿瘤体积均无显著差异(p>0.05)。给药末期，与Control组相比，C.ghonii组、Pembrolizumab组、先Pembrolizumab后C.ghonii组、先C.ghonii后Pembrolizumab组、C.ghonii+Pembrolizumab同时组肿瘤体积均显著小于Control组(p＝0.002，p＝0.000，p＝0.000，p＝0.000，p＝0.000，图8)。抑瘤率表现为先C.ghonii后Pembrolizumab组>C.ghonii+Pembrolizumab同时组>先Pembrolizumab后C.ghonii组>Pembrolizumab组>Control组，见表9。
表9抑瘤率
Figure PCTCN2022124020-appb-000004
3、治愈率
试验期间，各组的治愈率见表10。
表10治愈率
Figure PCTCN2022124020-appb-000005
Figure PCTCN2022124020-appb-000006
先C.ghonii后Pembrolizumab组小鼠肿瘤治愈率明显高于其他组，为先Pembrolizumab后C.ghonii组和C.ghonii+Pembrolizumab同时组治愈率的2倍。这可能是由于先瘤内给予戈氏梭菌芽孢有效而不加区分溶解肿瘤组织，破坏TME，同时细菌溶瘤后也会募集免疫细胞向TME的浸润，改变了TME中免疫细胞浸润程度和组成，包括细胞裂解产物会吸引大量的CD8 +T细胞聚集，在溶瘤细菌之后应用PD-1抗体治疗，避免免疫治疗中的免疫抑制，发动T细胞对肿瘤细胞的大规模攻击，从而发挥“双重”抗癌功效。因此溶瘤细菌与免疫抑制剂联合使用时给药顺序对于肿瘤的治疗效果也十分关键。
综上，优先使用戈氏梭菌芽孢治疗再与免疫疗法联用可以增强抗肿瘤功效，降低免疫药物的使用剂量，高效低毒。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。

Claims (15)

  1. 戈氏梭菌芽孢联合帕博利珠单抗在制备治疗结肠癌的医药制品中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述戈氏梭菌芽孢和帕博利珠单抗的使用顺序为先戈氏梭菌芽孢后帕博利珠单抗。
  3. 根据权利要求2所述的应用，其特征在于，所述戈氏梭菌芽孢为注射用戈氏梭菌芽孢冻干粉。
  4. 一种治疗结肠癌的药物，所述药物中的药效成分包括戈氏梭菌芽孢和帕博利珠单抗。
  5. 如权利要求4所述的药物，其特征在于，所述的戈氏梭菌为戈氏梭菌MW-DCG-LCv-26菌株或者戈氏梭菌驯化后获得的菌株；所述戈氏梭菌MW-DCG-LCv-26菌株保藏于澳大利亚国家计量研究院，菌株编号为V12/001486。
  6. 如权利要求4所述的药物，其特征在于，所述戈氏梭菌驯化后获得的菌株为MW-DCG-HNCv-18菌株，所述MW-DCG-HNCv-18菌株保藏于澳大利亚国家计量研究院，菌株保藏号V12/001485。
  7. 如权利要求4所述的药物，其特征在于，所述戈氏梭菌驯化后获得的菌株为MW-DCG-CCv-17菌株，所述MW-DCG-CCv-17菌株保藏于澳大利亚国家计量研究院，菌株保藏号V12/001487。
  8. 如权利要求4所述的药物，其特征在于，所述药物中的戈氏梭菌芽孢为注射用戈氏梭菌芽孢冻干粉，所述注射用芽孢冻干粉中的辅料为1％蔗糖。
  9. 如权利要求8所述的药物，其特征在于，所述注射用戈氏梭菌冻干粉的冻干工艺为：冻结阶段-40℃ 4h；-35℃ 10min同时抽真空、-30℃ 10min、-25℃ 10min、-20℃ 26h、-15℃ 2h、-10℃ 10min、-5℃ 10min、0℃ 10min、10℃ 2h、15℃ 10min、20℃ 3h、27℃ 3h冻干制备。
  10. 如权利要求3所述的药物，其特征在于，所述帕博利珠单抗为PD-1抗体注射液。
  11. 如权利要求3所述的药物，其特征在于，所述每1×10 7CFU戈氏梭菌芽孢联合浓度1mg/mL的帕博利珠单抗溶液0.2mg。
  12. 如权利要求11所述的药物，其特征在于，所述帕博利珠单抗溶液的溶 媒为质量百分比浓度0.9％的氯化钠注射液。
  13. 如权利要求11所述的药物，其特征在于，所述戈氏梭菌芽孢为注射用戈氏梭菌芽孢冻干粉，所述注射用戈氏梭菌芽孢冻干粉的溶媒为灭菌注射用水和质量百分浓度0.9％氯化钠注射液。
  14. 权利要求3～13任一项所述的药物在治疗结肠癌中的应用。
  15. 如权利要求14所述的应用，其特征在于，所述药物中戈氏梭菌芽孢和帕博利珠单抗的使用顺序为先戈氏梭菌芽孢后帕博利珠单抗。
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