Application of shigella flexneri and related serotype and preparation thereof in preparation of anti-tumor immune activation and treatment drugs

Abstract

The invention relates to shigella flexneri as well as related serotype and application of a preparation of the shigella flexneri in preparation of anti-tumor immune activation and treatment medicines. The shigella flexneri and the preparation thereof disclosed by the invention can improve a tumor immune microenvironment by activating anti-tumor immune response of an organism so as to inhibit tumor growth, and are suitable for prevention and / or treatment of various tumors; the strain and the preparation thereof can be independently used and can also be combined with existing tumor therapies such as chemotherapy, radiotherapy and immunotherapy to synergistically enhance the anti-tumor curative effect; the invention provides a new microbial resource and strategy for tumor immunotherapy, and has an important clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of oncology technology, and specifically relates to the application of Shigella flexneri, its related serotypes and preparations in the preparation of antitumor immune-activating and therapeutic drugs. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive tract worldwide, ranking third in incidence and second only to lung cancer in mortality. In recent years, immune checkpoint inhibitors (such as anti-PD-1 therapy) have shown significant efficacy in various cancers, but this therapy is currently only effective for CRC patients with high microsatellite instability (MSI-H). Clinically, approximately 85% of CRC patients present with microsatellite stable (MSS) CRC, which responds poorly to existing immunotherapies and has a poor prognosis. Therefore, finding new strategies to overcome immunotherapy resistance in MSS-type CRC has become a critical issue that urgently needs to be addressed in this field.

[0003] Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates that form in chronically inflammatory tissues such as tumors. Studies have shown that the presence of TLS in various malignant tumors is closely related to prolonged patient survival, enhanced immune cell infiltration, and improved response to immune checkpoint inhibitor therapy. In colorectal cancer, the formation of TLS is considered an important marker of anti-tumor immune activation and a predictor of good prognosis. However, the formation mechanism of TLS in the tumor microenvironment remains unclear, especially the key regulatory factors driving its maturation, which limits the development of TLS-induced immunotherapeutic strategies.

[0004] In recent years, the role of gut and tumor tissue-resident microbiota in regulating tumor immunity and treatment response has received increasing attention. Previous studies have suggested that specific microorganisms can influence tumor progression by modulating immune cell function; however, whether and how they participate in the induction and maturation of tumor lesions that lead to tumor thromboembolism (TLS), especially in immunotherapy-resistant MSS-type CRC, remains unclear. Currently, no key microbial strains have been identified that can specifically induce TLS formation and enhance the effects of immunotherapy. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide the application of Shigella flexneri, its related serotypes and preparations in the preparation of anti-tumor immune activation and therapeutic drugs. The strain and its preparations can effectively inhibit tumor growth by remodeling the tumor immune microenvironment, especially by inducing the formation of functional TLS, and can be used in combination with existing therapies (such as chemotherapy, radiotherapy and immunotherapy) to exert a synergistic effect.

[0006] In a first aspect, the present invention provides the application of Shigella flexneri in the preparation of antitumor immune-activating and therapeutic drugs.

[0007] Preferably, the Shigella flexneri includes strains with accession numbers Bio-52755 or CMCC(B) 51572.

[0008] Preferably, the serotype of Shigella flexneri includes one or more of 1b, 1c, 2a, 2b, 3a, 3b, 4a, 4b, 5a, and 5b. More preferably, the serotype of Shigella flexneri is 1b.

[0009] Furthermore, the strain or its associated serotype can activate antitumor immunity and / or induce the formation of tertiary lymphoid structures.

[0010] Preferably, the tumor includes one or more of colorectal cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, small bowel cancer, and cervical cancer. More preferably, the tumor includes various digestive tract tumors, including intestinal cancer, gastric cancer, and small bowel cancer. Further, the colorectal cancer is microsatellite stable colorectal cancer.

[0011] Secondly, the present invention provides a formulation comprising the aforementioned Shigella flexneri, the formulation comprising one or more of inactivated bacterial cells, metabolites, fermentation broth, and supernatant.

[0012] Preferably, the amount of bacteria contained in a unit dose of the preparation is 1×10^6 CFU to 1×10^11 CFU.

[0013] Thirdly, the present invention provides a pharmaceutical composition comprising the aforementioned Shigella flexneri.

[0014] Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0015] Preferably, the excipients are selected from one or more of diluents, dispersants, excipients, stabilizers, lubricants, and disintegrants.

[0016] Preferably, the pharmaceutical composition further includes other therapeutic agents selected from one or more of chemotherapy drugs, immune checkpoint inhibitors, immune cell therapy drugs, ferroptosis inducers, KRAS inhibitors, and radiosensitizers.

[0017] Specifically, the chemotherapy drugs include, but are not limited to: 5-fluorouracil, oxaliplatin, irinotecan, cisplatin, paclitaxel, doxorubicin, mitomycin, epirubicin, camptothecin, and etoposide.

[0018] Specifically, the immune checkpoint inhibitors include, but are not limited to, antibodies or small molecule inhibitors targeting PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, 4-1BB, OX40, and GITR.

[0019] Specifically, the immune cell therapy drugs include, but are not limited to: CAR-T, TCR-T, and TILs.

[0020] Specifically, the radiosensitizer is a photosensitizer, including one or more of borodipyrrole, dihydroporphyrin, or Bengal red; or a photothermal agent, including one or more of indocyanine green, neoindocyanine green, or gold nanoparticle rods.

[0021] Preferably, the dosage form of the pharmaceutical composition includes non-gastrointestinal and / or gastrointestinal dosage forms. For example, non-gastrointestinal dosage forms may be injections (such as intravenous injection, intratumoral injection), suppositories, etc.; gastrointestinal dosage forms may be capsules, tablets, granules, powders, oral liquids, suspensions, etc.

[0022] The Shigella strain involved in this invention is classified and named Shigella flexneri serotype 1b, and its information is as follows:

[0023] Accession number: Bio-52755; CMCC(B) 51572.

[0024] Collection classification name: Shigella flexneri.

[0025] Address of depositary institutions: BioBW Collection Center and China Medical Bacterial Culture Collection Center.

[0026] The strain described is merely an experimental example; technicians can infer from conventional knowledge that other Shigella flexneri strains will have the same effect.

[0027] Beneficial effects

[0028] (1) This invention first discovered and verified the function of Shigella flexneri as a TLS-specific microbial inducer: Through clinical sample analysis and in vivo experiments, it was first confirmed that Shigella flexneri can specifically induce the formation of functional tertiary lymphoid structures (TLS) in the tumor microenvironment, providing a new perspective for understanding the regulation of tumor immunity by microorganisms.

[0029] (2) A revolutionary strategy for overcoming immunotherapy resistance: For microsatellite stable colorectal cancer (MSS-CRC) for which there is currently no effective treatment, the Shigella flexneri and its preparations provided by this invention can reshape the “cold tumor” immune microenvironment and transform it into a “hot tumor”, thereby overcoming the inherent resistance to immune checkpoint inhibitors such as PD-1 / PD-L1, which has significant clinical translational value.

[0030] (3) Provides multi-level prevention and treatment applications: The Shigella flexneri and its preparations of the present invention can not only be used to treat existing tumors, but also show significant preventive effects in the Apc KO spontaneous tumor model, indicating that they have application potential in both tumor prevention and intervention stages.

[0031] (4) Clear safety and transformation prospects: The Shigella flexneri strain used in this invention is a known standard strain with clear biological characteristics. Experiments show that it has good antitumor activity at effective doses, while the virulence genes related to pathogenicity may be missing or inactivated, laying the foundation for its development into a safe live bacterial preparation or bacterial component drug.

[0032] (5) Diverse application forms and strong compatibility: The strains of the present invention can be used in various forms such as live bacteria, inactivated bacteria, metabolites, and supernatants, and can be easily combined with existing chemotherapy, radiotherapy, immunotherapy and other methods to form a synergistic treatment plan, thus broadening the clinical application scenarios.

[0033] (6) In summary, the Shigella flexneri and its preparations provided by the present invention, as a novel microbial immunotherapy agent, have broad application prospects and significant socio-economic benefits in the prevention and treatment of tumors, especially in reversing immunotherapy resistance. Attached Figure Description

[0034] Figure 1 To identify and analyze the intratumoral microbiota of colorectal cancer patients; A represents the taxonomic phylogenetic diagram, and the linear discriminant analysis effect size (LEfSe) analysis of the tumor microbiota in the high TLS tumor group and the low TLS tumor group; B represents the linear discriminant analysis (LDA) score of bacteria with significant differences in abundance between the high TLS tumor group and the low TLS tumor group; C represents the proportion of bacteria with significant differences in abundance between the high TLS tumor group and the low TLS tumor group.

[0035] Figure 2 AB represents the colonization of Escherichia coli and Shigella in colorectal cancer tissue.

[0036] Figure 3 AC represents the therapeutic effects of Escherichia coli and Shigella on colorectal cancer.

[0037] Figure 4To induce the formation of tertiary lymphoid structures in colorectal cancer in mice using Shigella flexneri.

[0038] Figure 5 AB represents the therapeutic effects of Escherichia coli and Shigella on colorectal cancer.

[0039] Figure 6 AC represents the preventive effect of Escherichia coli and Shigella against colorectal cancer. Detailed Implementation

[0040] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0041] The reagents and biological materials used in this invention are all commercially available.

[0042] Example 1

[0043] Identification and composition of intratumoral flora in colorectal cancer patients

[0044] To investigate the effects of microbial colonization within tumor tissue on the formation of tertiary lymphoid structures (TLS) and antitumor immunity, a microbiome-pathology association analysis based on a cohort of colorectal cancer patients was conducted.

[0045] This study included 72 patients with colorectal cancer who underwent radical surgery, and tumor tissue was collected postoperatively. All tumor tissue sections were stained with hematoxylin and eosin (HE) and quantitatively assessed using the published Thromboplasty (TLS) histological scoring system. Patients were divided into high TLS and low TLS groups based on the median score. There were no statistically significant differences between the two groups in terms of sex, age, and tumor differentiation.

[0046] After extracting total DNA from tissue samples, amplicon sequencing of the V3-V4 region of the 16S rRNA gene was performed using the Illumina platform. Raw data underwent Fastp quality control, and were then aligned with human sequences using BWA for decontamination. Sequence denoising and clustering were performed using QIIME2 to generate an ASV feature table, and species annotation was performed based on the SILVA database. Differential species identification was performed using linear discriminant analysis effect size (LEfSe) (LDA score > 3.0).

[0047] LEFSe analysis showed that the abundance of Escherichia-Shigella and Escherichia coli was significantly increased in the high TLS tumor group. Figure 1A and B). The Wilcoxon test further confirmed that the relative abundance of Escherichia-Shigella in the high TLS group was significantly higher than that in the low TLS group (A and B). Figure 1 C).

[0048] Example 2

[0049] Fluorescence in situ hybridization identification of Escherichia coli and Shigella in colorectal cancer tissue

[0050] To clarify the colonization of *E. coli* and *Shigella* in tumors, this embodiment employs fluorescence in situ hybridization (FISH) to specifically detect and locate *E. coli* and *Shigella* in paraffin-embedded tissue sections from colorectal cancer. The specific steps are as follows: Paraffin-embedded tissue sections with a thickness of 4 µm were dewaxed with xylene and subjected to a series of dehydration and hydration treatments using graded ethanol. Subsequently, the sections were permeabilized with a 20 µg / mL proteinase K solution at 37°C for 30 minutes. Specific DNA probes for *E. coli* (sequence: 5'-GCATAAGCGTCGCTGCCG-3') and specific DNA probes for *Shigella* (sequence: 5'-CGCTTTACGCCCAGTAATTCCGATTAACGC-3') were diluted to a working concentration of 400 nM, mixed with formamide-containing hybridization buffer, and then added dropwise to the tissue sections. After covering with coverslips, the sections were placed in a humidified chamber and hybridized overnight at 37°C to 45°C in the dark. After hybridization, the tissues were thoroughly washed to remove unbound probes, and then counterstained with DAPI. The slides were then mounted with anti-fluorescence quenching mounting medium. All slides were imaged and analyzed using a PANNORAMIC PRACTICE II panoramic slide scanning system. Results showed that the specific fluorescence signal intensity of *E. coli* and *Shigella* in the high TLS group was significantly higher than that in the low TLS group. Figure 2 ).

[0051] Example 3

[0052] Validation of the therapeutic effects of Shigella flexneri on tumors in vivo.

[0053] To evaluate the antitumor effects of Shigella and Escherichia coli in vivo, a colorectal cancer xenograft model was established in immunocompetent BALB / c mice. All animal experiments followed the ARRIVE guidelines and were approved by the Experimental Animal Management and Use Committee of Shanghai Yishang Biotechnology Co., Ltd. Shigella flexneri (CMCC(B) 51572), Shigella sonnei (ATCC 29930), and Escherichia coli (ATCC 25922) used in the experiments were obtained commercially and preserved through aerobic culture on LB agar plates at 30°C. Six- to eight-week-old male SPF-grade BALB / c mice were selected and housed in a specific pathogen-free environment. Infections were caused by subcutaneous inoculation of 5 × 10⁵ bacteria on the back of the mice. 6 A xenograft model was constructed using CT26 colon cancer cells. One week after inoculation, tumor-bearing mice were randomly divided into experimental and control groups. Starting from day 8 post-tumor inoculation, the experimental group received multiple injections of bacterial suspension (total dose 10) around the tumor. 8 Mice were injected with CFU / 50 μL PBS, while the control group received an equal volume of PBS. Tumor length and width were measured and volume calculated every 3 days during the intervention. After the intervention, the tumors were dissected and weighed. Results showed that, compared to the control group, mice receiving peritumoral injections of Shigella flexneri or Escherichia coli exhibited significantly inhibited tumor growth, characterized by slower tumor volume growth and a reduced final tumor weight, confirming the antitumor efficacy of this strain in vivo. Figure 3 ).

[0054] Example 4

[0055] Analysis of the activation of mouse immune response by induction of tertiary lymphoid structure formation by Shigella flexneri in vivo

[0056] This embodiment aims to investigate the effects of Escherichia coli and Shigella flexneri colonization on the remodeling of the tumor microenvironment in a mouse model of colorectal cancer through systematic histopathological analysis, particularly on the maturation of tertiary lymphoid structures, cell proliferation, and apoptosis.

[0057] The experimental methods are as follows: After euthanizing mice at the experimental endpoint, tumor tissue was collected, fixed in 4% paraformaldehyde, and routinely embedded in paraffin. 4 μm thick serial sections were prepared and subjected to hematoxylin-eosin staining, multiplex immunofluorescence staining, TUNEL apoptosis detection, and Ki67 immunohistochemical staining. Multiplex immunofluorescence staining was performed according to a published protocol, the simplified procedure being: sections were dewaxed, hydrated, and subjected to antigen retrieval at 98°C for 20 minutes in Tris-EDTA buffer (pH 9.0). After blocking endogenous peroxidase, sequential staining was performed using a multiplex immunofluorescence kit. Each round of staining included: overnight incubation with primary antibodies (e.g., CD3, CD20, CD23) at 4°C, incubation with HRP-labeled secondary antibodies at room temperature for 60 minutes, washing with PBS, and reaction with a tyramine signal-amplifying fluorescent probe for 10 minutes. After each round of labeling, the antibody complex was dissociated by microwave treatment (98°C, 10 minutes) to prepare for the next round of target labeling. After all labeling was completed, cell nuclei were counterstained with DAPI, and slides were mounted with anti-fluorescence quenching mounting medium. All slice images were acquired and quantitatively analyzed using the PANNORAMIC PRACTICE II panoramic slice scanning system.

[0058] Experimental results confirmed that *E. coli* treatment mainly induced the formation of clusters of T and B lymphocytes at the tumor invasion front; while *Shigella flexneri* treatment drove the formation of a more mature tertiary lymphoid structure, characterized by clear T / B cell septa and expression of the germinal center marker CD23. Further functional analysis showed that bacterial colony implantation did not significantly alter the proliferative activity of tumor cells (Ki67 positivity rate), but *Shigella flexneri* specifically and significantly promoted tumor cell apoptosis (enhanced TUNEL positive signal). Figure 4 ).

[0059] Example 5

[0060] Validation of the therapeutic effect of oral Shigella flexneri on in situ colorectal tumors.

[0061] This embodiment established an orthotopic cecal xenograft model in immunocompetent BALB / c mice and used oral gavage to more realistically simulate the physiological microenvironment of colorectal cancer patients to evaluate the antitumor effects of Shigella and Escherichia coli in vivo. All animal experiments followed the ARRIVE guidelines and were ethically reviewed by the Laboratory Animal Management and Use Committee of Shanghai Yishang Biotechnology Co., Ltd. Six- to eight-week-old male SPF-grade Balb / c mice were selected. The model was established surgically: after anesthesia and abdominal disinfection, a 1 cm incision was made in the midline of the lower abdomen to expose the cecum. A suspension of CT26 colorectal cancer cells (e.g., 5 × 10⁻⁶ cells) was injected using a microsyringe. 6(50 μL of 1000 cells) was slowly injected into the subserosa of the cecal wall, where a tiny vacuole was visibly formed. After injection, gentle pressure was applied for a few moments to prevent leakage, and then the cecum was returned to the abdominal cavity, with the muscle and skin sutured layer by layer. One week after inoculation, mice underwent a 3-day antibiotic pretreatment to reduce the influence of the native flora: after fasting and withholding water for 4-6 hours each morning, streptomycin solution (250 mg / mL, 0.1 mL / mouse) was administered orally by gavage. After pretreatment, mice were randomly divided into four groups: PBS control group, Escherichia coli treatment group, Shigella flexneri treatment group, and Shigella sonnei treatment group. Starting from day 7 post-inoculation, the corresponding interventions were initiated: the treatment groups were administered the corresponding bacterial suspension (10 × 10⁻⁶ cells / mL) by gavage. 8 The dose was 0.1 mL / mouse (PFU / mL), while the control group received an equal volume of PBS. Interventions were administered on days 7, 10, 13, 16, and 19, for a total of 5 times. Mouse weight and general condition were monitored throughout the experiment. All mice were sacrificed on day 21 post-inoculation, and cecal tumor tissue was dissected and separated for tumor weight, volume calculation, and subsequent histological analysis. Results showed that, compared to the control group, mice receiving Shigella flexneri orally via gavage exhibited significantly inhibited tumor growth, as evidenced by reduced tumor volume and weight, confirming the antitumor efficacy of this strain in vivo. Figure 5 ).

[0062] Example 6

[0063] Validation of the preventive intervention effect of oral Shigella flexneri on spontaneous intestinal tumors.

[0064] This embodiment aims to evaluate the preventive intervention potential of Shigella and Escherichia coli in the tumorigenesis stage using a genetically engineered spontaneous intestinal tumor model. Commercially purchased Apc KO mice (C57BL / 6JCya-Apcem2, product number: C001511, Cyagen Biosciences) were used. This strain is based on C57BL / 6JCya and was constructed by knocking out the mutant cluster region (MCR) in the Apc gene using gene editing technology. Homozygous mice of this strain are lethal, while heterozygous (Apc KO) mice spontaneously develop intestinal adenomas. Eight-week-old Apc KO heterozygous mice were used. First, a 3-day antibiotic pretreatment was administered to reduce the influence of the native intestinal flora: mice were fasted and deprived of water for 4-6 hours each morning, and then orally administered streptomycin solution (concentration 250 mg / mL, gavage volume 0.1 mL / mouse). After pretreatment, mice were randomly divided into four groups: PBS control group, Escherichia coli treatment group, Shigella flexneri treatment group, and Shigella sonnei treatment group. Intervention began at 9 weeks of age, with the treatment group receiving oral administration of the corresponding bacterial suspension (concentration 10 × 10⁻⁶). 8PFU / mL, gavage volume 0.1 mL / mouse; control group received an equal volume of PBS. Intervention was administered at weeks 9, 11, and 13, twice weekly for a total of 6 weeks. Mouse weight and general clinical status were continuously monitored throughout the experiment. All mice were sacrificed at 18 weeks of age, and intestinal tumor tissue (focusing on the cecum and colon) was dissected and isolated. Tumor weight and volume were measured, and samples were collected for subsequent histopathological (e.g., HE staining) and immunohistochemical analysis to assess tumor burden, morphology, and changes in the immune microenvironment. Results showed that, compared to the control group, mice receiving oral gavage of Shigella flexneri exhibited significantly inhibited tumor growth, characterized by a reduction in tumor number and size, confirming the preventative intervention potential of this strain during tumorigenesis. Figure 6 ).

Claims

1. Application of Shigella flexneri in the preparation of antitumor immune-activating and therapeutic drugs.

2. The application according to claim 1, characterized in that, The Shigella flexneri strains include those with accession numbers Bio-52755 or CMCC(B) 51572.

3. The application according to claim 1, characterized in that, The serotypes of Shigella flexneri include one or more of 1b, 1c, 2a, 2b, 3a, 3b, 4a, 4b, 5a, and 5b.

4. The application according to claim 1, characterized in that, The tumors include one or more of the following: colorectal cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, stomach cancer, small intestine cancer, and cervical cancer.

5. A formulation comprising Shigella flexneri as described in any one of claims 1 to 4, characterized in that, The preparation includes one or more of the following: inactivated bacteria, metabolites, fermentation broth, and supernatant.

6. A pharmaceutical composition comprising Shigella flexneri as described in any one of claims 1 to 4.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

8. The pharmaceutical composition according to claim 7, characterized in that, The excipients are selected from one or more of the following: diluents, dispersants, excipients, stabilizers, lubricants, and disintegrants.

9. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition further includes other therapeutic agents selected from one or more of chemotherapy drugs, immune checkpoint inhibitors, immune cell therapy drugs, ferroptosis inducers, KRAS inhibitors, and radiosensitizers.

10. The pharmaceutical composition according to claim 6, characterized in that, The dosage forms of the pharmaceutical composition include non-gastrointestinal and / or gastrointestinal dosage forms.

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