Use of strain in preparation of medicament for treating colon cancer

Abstract

The present application discloses use of a strain in the preparation of a medicament for treating colon cancer, wherein the strain is Lactobacillus rhamnosus, CALM 607, and the accession number of the strain is CGMCC No. 28985. The strain has the potential to improve the response to immunotherapy for MSS colon cancer and can synergize with immunotherapy drugs to achieve interventional treatment of MSS colon cancer.

Description

Application of a strain in the preparation of therapeutic drugs for colon cancer Technical Field

[0001] This application relates to the field of microbial technology, and more specifically, to the use of a strain in the preparation of a therapeutic agent for colon cancer. Background Technology

[0002] MSS colorectal cancer, or microsatellite stable colorectal cancer, is a specific type of colorectal cancer characterized by the absence of mutations in the microsatellite sequences of the patient's tumor genes. Microsatellites are short, repetitive DNA sequences whose stability within the genome is crucial for maintaining normal cellular function.

[0003] The main challenges in treating MSS colorectal cancer currently lie in the following aspects:

[0004] MSS colorectal cancer has a limited response to traditional chemotherapy and radiotherapy. This is because MSS tumors typically have a relatively stable genome, lacking obvious mutations or abnormal expression, making traditional mutation-based targeted therapies ineffective.

[0005] MSS colorectal cancer carries a high risk of recurrence and metastasis. Even with surgical resection and adjuvant therapy, patients may still face the risk of disease recurrence. Furthermore, MSS colorectal cancer is prone to metastasizing to other organs, further complicating treatment.

[0006] The significant individual variability in MSS colorectal cancer makes treatment planning more challenging. Each patient's tumor characteristics, gene expression profile, and immune status may differ, necessitating personalized treatment plans. However, the molecular mechanisms and biological characteristics of MSS colorectal cancer are not yet fully understood, limiting the progress of personalized treatment.

[0007] Immunotherapy for MSS colorectal cancer also faces certain challenges. While immunotherapy has achieved significant therapeutic effects in various tumors, its efficacy in MSS colorectal cancer is less than ideal. This may be because MSS-type tumors lack significant immunogenicity, making it difficult for the immune system to effectively recognize and attack tumor cells. One feasible immunotherapy is immune checkpoint therapy (ICI), which restores the anti-tumor function of immune cells by blocking inhibitory signals (PD-1 / PD-L1; CTLA-4). Currently, immune checkpoint therapy has shown significant efficacy in treating melanoma and non-small cell lung cancer. In the checkmate 142 clinical trial, ICI demonstrated excellent response data, prompting the FDA to approve it as a second-line treatment for patients with MSI-H colorectal cancer. However, MSI-H accounts for only 15% of colorectal cancer patients, while the vast majority of patients are MSS-type. Compared to MSI-type colorectal cancer, the clinical response rate for MSS colorectal cancer is close to zero. Summary of the Invention

[0008] This application addresses the shortcomings of existing methods by proposing the application of a bacterial strain in the preparation of therapeutic drugs for colon cancer, thereby solving the technical problem of insufficient immune checkpoint therapy response rate in related technologies.

[0009] This application provides an example of the use of a strain in the preparation of a therapeutic drug for colon cancer, wherein the strain is Lactobacillus rhamnosus, CALM 607, and the accession number of the strain is CGMCC No. 28985.

[0010] Furthermore, the strain is tolerant to conditions of pH 2.5–3 and to conditions of 0.1% bile salts.

[0011] Alternatively, the strain can be formulated into an oral preparation.

[0012] Alternatively, the colon cancer is microsatellite stable colon cancer.

[0013] Furthermore, the therapeutic drug is used in immune checkpoint therapy.

[0014] Alternatively, the therapeutic agent may also include an αPD-L1 antibody.

[0015] Furthermore, the therapeutic drug can reduce the cell viability of colon cancer cells.

[0016] Furthermore, the therapeutic agent can reduce the tumor volume and / or weight of colon cancer.

[0017] Furthermore, the strain is able to promote the ability of colon cancer cells to infiltrate immune cells.

[0018] Furthermore, the infiltrated immune cells are T cells.

[0019] The beneficial technical effects of the technical solutions provided in this application include:

[0020] (1) The application of a strain in the preparation of a therapeutic drug for colon cancer, the strain being Lactobacillus rhamnosus, CALM 607, accession number CGMCC No.28985, has the potential to improve the immunotherapy response of MSS colon cancer and can work synergistically with immunotherapy drugs to achieve interventional treatment of MSS colon cancer.

[0021] (2) The application of a strain of this application in the preparation of a therapeutic drug for colon cancer, wherein the strain is derived from probiotics in healthy individuals and has strong acid resistance and bile salt resistance, and has the potential to be prepared into an oral drug, which can alleviate the patient's treatment discomfort.

[0022] Additional aspects and advantages of this application will be set forth in part in the description which follows, and will become apparent from the description or may be learned by practice of this application. Attached Figure Description

[0023] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein:

[0024] Figure 1 is a colony culture diagram of the strain provided in the embodiments of this application;

[0025] Figure 2 is a schematic diagram of the optical microscope morphology of the strain provided in the embodiment of this application;

[0026] Figure 3 is a growth curve of the strain provided in the embodiments of this application;

[0027] Figure 4 shows the statistical results of the survival rate of the strains provided in the embodiments of this application under different pH conditions;

[0028] Figure 5 shows the statistical results of the survival rate of the strains provided in the embodiments of this application under 0.1% bile salt conditions;

[0029] Figure 6 shows the statistical results of the strains provided in the embodiments of this application in mice undergoing immune checkpoint therapy for colon cancer;

[0030] Figure 7 shows the statistical results of the ability of the strains provided in the embodiments of this application to affect the infiltration of cancer cells of colon cancer into immune cells;

[0031] Figure 8 shows the statistical results of the strains provided in the embodiments of this application in the intervention of immune checkpoint therapy for colorectal cancer organoids. Detailed Implementation

[0032] The embodiments of this application are described below with reference to the accompanying drawings. It should be understood that the embodiments described below with reference to the accompanying drawings are exemplary descriptions for explaining the technical solutions of the embodiments of this application, and do not constitute a limitation on the technical solutions of the embodiments of this application.

[0033] Those skilled in the art will understand that, unless specifically stated otherwise, the terms "described" and "the" as used herein may also include plural forms. It should be further understood that the term "comprising" as used in the specification of this application means the presence of the stated features, integers, steps, or operations, but does not exclude implementation as other features, information, data, steps, operations, elements, components, and / or combinations thereof supported by the art. The term "and / or" as used herein refers to at least one of the items defined by the term; for example, "A and / or B" can be implemented as "A," or as "B," or as "A and B."

[0034] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.

[0035] This application provides the use of a strain in the preparation of a therapeutic drug for colon cancer. Specifically, the strain is Lactobacillus rhamnosus, CALM 607, with accession number CGMCC No. 28985. It was deposited on November 13, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0036] This strain was discovered during research on the NOD2 signaling pathway and was eventually isolated. It originated from the vulvar flora of healthy women and was confirmed by 16S and whole-genome sequencing to be a novel strain of Lactobacillus rhamnosus, named CALM 607, and subsequently referred to as LR607.

[0037] NOD2 (Nucleotide-binding oligomerization domain-containing protein 2), also known as CARD15, is a pattern recognition receptor primarily involved in the regulation of inflammatory and immune responses. NOD2 is mainly expressed in monocytes, macrophages, and dendritic cells, and can recognize intracellular bacterial components, such as bacterial peptidoglycan. When NOD2 recognizes these bacterial components, it activates downstream signaling pathways, leading to the activation of transcription factors such as NF-κB, thereby triggering a series of inflammatory responses. This response is a natural defense mechanism of the body against bacterial infection, helping to clear pathogens and promote tissue repair.

[0038] The fact that the strain described in this application can be recognized by NOD2 indicates that the strain can trigger an immune response, particularly in the digestive tract.

[0039] This application involves screening and isolating novel bacterial strains from healthy human bodies and verifying their basic characteristics. Specifically:

[0040] 1. Growth curve identification

[0041] Inoculation: Take 100 μL of bacterial culture with OD=1 and inoculate it into 10 ml of MRS liquid medium and mix well.

[0042] Culture: The inoculated culture tubes were placed in an anaerobic incubator and cultured at 37°C. The tubes were removed after 0, 3, 6, 9, 12, 15, 18, 21 and 24 hours of culture.

[0043] Detection: The OD values ​​of the bacterial culture at each time point were recorded using a 600nm wavelength ultraviolet spectrophotometer, and the culture was zeroed using uninoculated MRS medium.

[0044] Plot the growth curve: Plot the growth curve of the bacterial strain with the OD value of the bacterial suspension on the ordinate and the culture time on the abscissa.

[0045] As shown in Figures 1 and 2, the main characteristics of Lactobacillus rhamnosus, CALM 607 are as follows: the colonies are white and round with neat edges, and the surface is smooth, moist, and glossy; LR607 is a Gram-negative, rod-shaped bacterium. Figure 3 shows the 24-hour growth curve of Lactobacillus rhamnosus, CALM 607. This strain enters the logarithmic growth phase at 6 hours and reaches the plateau phase at 18 hours.

[0046] 2. Acid resistance test:

[0047] To prepare artificial gastric juice: Take 0.82 ml of dilute hydrochloric acid, add 40 ml of distilled water and 0.5 g of pepsin. Shake well to dissolve the pepsin, and bring the volume to 50 ml. Then adjust the pH to 2.5 or 3 with 1N NaOH. Filter through a 0.22 μm filter to obtain the artificial gastric juice.

[0048] Cell preparation: Centrifuge the bacterial culture, discard the supernatant, resuspend in physiological saline, centrifuge again, and discard the supernatant.

[0049] Bacteria were resuspended in artificial gastric fluid and diluted to OD=1. The viable bacterial counts were measured at 0, 1, 2 and 3 h (bacteria were resuspended in physiological saline at 0 h).

[0050] Viable cell counts were performed using a 10-fold serial dilution method: 100 μL of bacterial culture was added to 900 μL of physiological saline and gradually diluted to the appropriate concentration. 20 μL of the bacterial culture from the 5th and 6th dilutions were then plated and spotted onto agar plates. The plates were incubated at 37°C for 48 hours, and colonies were counted.

[0051] As shown in Figure 4, Lactobacillus rhamnosus, CALM 607 has high acid resistance and can grow normally in artificial gastric juice at pH 3 or pH 2.5.

[0052] 3. Bile salt tolerance test:

[0053] Preparation of bile salt solutions: Take bovine bile salts, dissolve them in MRS medium, and prepare solutions with concentrations of 0.1% and 0.3% respectively. Filter to remove bacteria.

[0054] Bacterial preparation: Centrifuge the bacterial culture, discard the supernatant, resuspend in physiological saline, centrifuge again, and discard the supernatant. Use the traditional Lactobacillus rhamnosus (LGG, ATCC53103) as a control.

[0055] Bacteria were resuspended in bile salt solution, and the bacterial suspension was diluted to OD=1. The viable bacterial counts were measured at 0, 2 and 4 h (bacteria were resuspended in MRS medium at 0 h).

[0056] Viable cell counts were performed using a 10-fold serial dilution method: 100 μL of bacterial culture was added to 900 μL of physiological saline and gradually diluted to the appropriate concentration. 20 μL of the bacterial culture from the 5th and 6th dilutions were then plated and spotted onto agar plates. The plates were incubated at 37°C for 48 hours, and colonies were counted.

[0057] As shown in Figure 5, Lactobacillus rhamnosus, CALM 607 has better bile salt tolerance than the traditional LGG, and can survive and grow in 0.1% bile powder.

[0058] Its strains are derived from probiotics in healthy individuals and possess strong acid and bile salt resistance, making it a promising candidate for oral medication.

[0059] After understanding the basic characteristics of LR607, we further verified the effect of this strain on MSS colon cancer.

[0060] This application utilizes a mouse MSS-type subcutaneous tumor-bearing model of colon cancer, as detailed below:

[0061] 1. Preparations before mold making:

[0062] Seven-week-old BALB / c mice were administered a quadruple antibiotic mixture at 200 μL for five consecutive days (Day 5): 10 mg / ml vancomycin, 20 mg / ml ampicillin, 20 mg / ml metronidazole, and 20 mg / ml neomycin.

[0063] LR607 culture. LR607 was revived and cultured in MRS liquid medium, and the concentration was adjusted to 10⁹ CFU. After gavage in BALB / c mice, LR607 was colonized by gavage. Each mouse was gavaged with 200 μL of 10⁹ CFU of LR607 until the end of the experiment.

[0064] 2. Cell preparation: Mouse colon cancer cells CT26 were cultured at 37°C and 5% CO2 in RPM-1640 medium containing 10% fetal bovine serum and 1% penicillin plus streptomycin.

[0065] 3. Inoculation of CT26 cells: After BALB / c mice were continuously administered LR607 cells by gavage for 7 days (Day 12), CT26 cells were inoculated on day 8. CT26 cells were digested with trypsin, washed twice, resuspended in PBS, and placed on ice. Mice were anesthetized with 3% isoflurane and the hair on the right hind limb was shaved. CT26 cells were inoculated into the right hind limb of each mouse at a rate of 1 × 10⁶ cells per mouse. 6 The inoculation volume is 100 μL. The mice's recovery status needs to be observed on the day of inoculation.

[0066] 4. Tumor volume measurement: When the tumor is approximately 25-100 mm in each experiment. 3 After defining the tumor size (Day 18), tumor volume was measured every two days. The length and width of each tumor were measured using digital calipers, and the tumor volume was calculated as length × width. 2 ×0.5, where the width is the smaller of the two measurements.

[0067] 5. Drug intervention: When the tumor is approximately 25-100mm in each experiment. 3 After the range was cleared (Day 18), αPD-L1 antibody intervention was started, once every 4 days, for a total of 3 times.

[0068] 6. Mouse treatment: Mice were euthanized on Day 37, with tumor volume controlled to be below 2000 mmHg during this period. 3 If the tumor ulcerates or grows too rapidly, the mice will be euthanized in advance. The tumor tissue, blood, and feces of the mice will be preserved for future use.

[0069] To evaluate the therapeutic effect of LR607, tumor growth curves and tumor growth data were used to assess the effect of inhibiting tumor growth.

[0070] Figure 6 shows that Lactobacillus rhamnosus, CALM 607 promotes immune checkpoint therapy in MSS colon cancer mice: (A) LR607 synergistically inhibits the absolute volume of subcutaneous tumor growth in mice with αPD-L1, with no significant difference between the intervention groups; (B) LR607 synergistically inhibits the relative volume of subcutaneous tumor growth in mice with αPD-L1, with a significant difference between the LR607 synergistically inhibiting αPD-L1 and the control group, while other intervention groups showed no significant difference; (C) LR607 synergistically reduces tumor weight in mice, with a significant difference between the LR607 synergistically inhibiting αPD-L1 and the control group, while other intervention groups showed no significant difference; (D) Tumor size images show that LR607 intervention alone, αPD-L1 intervention alone, and LR607 synergistically inhibiting αPD-L1 all reduced tumor volume, with the LR607 synergistically inhibiting αPD-L1 intervention group showing a more significant reduction in tumor volume. In the figure, * indicates a p-value less than 0.05.

[0071] Furthermore, flow cytometry was used to assess the effect of LR607 on the ability of tumor-infiltrating immune cells. Tumor-infiltrating immune cells refer to the interactions and relationships between tumor cells and surrounding immune cells during their growth and spread. This process involves the infiltration of immune cells by tumor cells, i.e., tumor cells invade and affect the function and distribution of immune cells. Specific implementation plan: Fresh tumor tissue was digested into a single-cell suspension using a digestive solution containing collagenase IV, DNase II, and hyaluronidase, and then incubated with erythrocyte lysis buffer at room temperature for 5 min to remove erythrocytes. After washing the cells twice with PBS, flow cytometry staining was performed, with staining markers including live, CD45, CD3, and CD8.

[0072] As shown in Figure 7, there was a significant difference between the LR607 synergistic αPD-L1 intervention group and the control group, while there were no significant differences between other intervention groups and the control group. In the figure, * indicates a p-value less than 0.05. Flow cytometry results showed that the combined use of LR607 and αPD-L1 could enhance the ability of MSS-type tumors to infiltrate CD8+ T cells.

[0073] Furthermore, this application utilizes tumor tissue from MSS colon cancer patients to construct organoids and verify their therapeutic effects. The specific implementation plan includes the following:

[0074] Organoid construction: Biopsies from colorectal cancer patients were collected in 5 ml of ice-cold PBS containing penicillin-streptomycin. The tissue samples were gently washed and minced into 1-2 mm pieces. 2 After thinning, the fresh tumor tissue was digested for 30 minutes with 10 mL of a digestive solution containing collagenase IV, DNase II, and hyaluronidase to form a single-cell suspension. The suspension was then digested on ice for 30 minutes. The dissociated cells were passed through a 100 μm cell filter and resuspended in ice-cold PBS. The centrifuged cells were then resuspended in matrix gel and seeded into 24-well agar plates. After curing at 37°C for 20 minutes in a 5% CO2 incubator, commercial organoid culture medium (with the addition of IL-2) was added, and the medium was changed every three days.

[0075] Within one month of organoid formation, LR607 culture supernatant, ImmunoCult Hu CD3 / CD28 T-Cell Act, and αPD-L1 antibody were added and co-cultured for one week in the presence of tumor-infiltrating immune cells. The organoid area was assessed on day 3 and day 7, respectively. After one week of co-culture, the organoid proliferation was detected using a CCK8 kit.

[0076] As shown in Figure 8, the experimental results show that (A) representative images (scale bar = 100 μm) of organoids from colorectal cancer patients after 3 and 7 days of culture following intervention with αPD-L1 antibody combined with LR607. It is evident that the visibility, volume, and number of cancer cells decreased after intervention with αPD-L1 antibody combined with LR607. (B) Surface area of ​​organoids cultured for 3 days (104 mm²). 2 (c) The LR607 synergistic αPD-L1 intervention group showed a significant difference compared to the control group, while other intervention groups showed no significant difference compared to the control group; (c) Surface area of ​​organoids cultured to day 7 (104 mm²) 2(A) Significant differences were found between the LR607 synergistic αPD-L1 intervention group and the control group, while no significant differences were found between other intervention groups and the control group. (D) Organoid viability was detected by CKK8 assay on day 7. LR607 intervention alone, αPD-L1 intervention alone, and LR607 synergistic αPD-L1 intervention all reduced tumor cell viability, with the LR607 synergistic αPD-L1 intervention group showing a more significant decrease in tumor cell viability. In the figure, * indicates p-value less than 0.05, ** indicates p-value less than 0.01, and **** indicates p-value less than 0.0001. The supernatant of LR607 effectively inhibited tumor cell proliferation and killed tumor cells. Lactobacillus rhamnosus, CALM 607 promoted immune checkpoint therapy of organoids derived from MSS colon cancer patients.

[0077] Therefore, the strains screened in this application can promote immune checkpoint therapy for MSS colorectal cancer, and are expected to overcome the low response rate of immune checkpoint therapy in MSS colorectal cancer. The Lactobacillus rhamnosus, CALM607, identified in this application, can be used as an adjuvant drug for colorectal cancer immunotherapy. Specifically,

[0078] This application provides an example of the application of a strain in the preparation of a therapeutic drug for colon cancer, wherein the strain is *Lactobacillus rhamnosus*, CALM 607, with accession number CGMCC No. 28985. Further, the strain tolerates conditions of pH 2.5–3 and 0.1% bile salts. Optionally, the strain can be formulated into an oral preparation. The colon cancer is microsatellite stable colon cancer. The therapeutic drug is used in immune checkpoint therapy. Optionally, the therapeutic drug further includes an αPD-L1 antibody. The therapeutic drug can reduce the cell viability of colon cancer cells. The therapeutic drug can reduce the tumor volume and / or reduce the tumor weight of colon cancer. The strain can promote the ability of colon cancer cells to infiltrate immune cells. The infiltrated immune cells are T cells.

[0079] Those skilled in the art will understand that the steps, measures, and solutions in the various operations, methods, and processes discussed in this application can be alternated, modified, combined, or deleted. Furthermore, other steps, measures, and solutions in the various operations, methods, and processes discussed in this application can also be alternated, modified, rearranged, decomposed, combined, or deleted. Furthermore, steps, measures, and solutions in related technologies that are similar to those disclosed in this application can also be alternated, modified, rearranged, decomposed, combined, or deleted.

[0080] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0081] In the description of this specification, specific features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

[0082] The above description is only a partial implementation of this application. It should be noted that for those skilled in the art, other similar implementation methods based on the technical concept of this application, without departing from the technical concept of this application, also fall within the protection scope of the embodiments of this application.

Claims

1. Use of a strain in the manufacture of a medicament for the treatment of colon cancer, characterized in that, The strain is Lactobacillus rhamnosus, CALM 607, and its accession number is CGMCC No. 28985.

2. Use according to claim 1, wherein The strain is tolerant to conditions with a pH of 2.5 to 3 and to conditions with 0.1% bile salts.

3. Use according to claim 2, wherein the compound is ###0002### The strain can be formulated into oral preparations.

4. The application as described in claim 1, characterized in that, The colon cancer mentioned is microsatellite stable colon cancer.

5. The application as described in claim 4, characterized in that, The therapeutic drug is used in immune checkpoint therapy.

6. The application as described in claim 5, characterized in that, The therapeutic agents also include αPD-L1 antibodies.

7. The application as described in claim 4 or 5, characterized in that, The therapeutic drug can reduce the viability of colon cancer cells.

8. The application as described in claim 4 or 5, characterized in that, The therapeutic drug can reduce the size and / or weight of colon cancer tumors.

9. The application as described in claim 4, characterized in that, The strain can promote the ability of colon cancer cells to infiltrate immune cells.

10. The application as described in claim 9, characterized in that, The infiltrated immune cells are T cells.

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