An antitumor preparation based on respiratory tract symbiotic bacteria, and a preparation method and application thereof

Abstract

This invention relates to an antitumor agent based on respiratory tract symbiotic bacteria, its preparation method, and its application. Addressing the current clinical issue of the unclear role of respiratory flora in lung cancer patients' progression, we provide the first evidence that the respiratory tract symbiotic *Streptococcus osticulata* possesses antitumor activity. When *Streptococcus osticulata* is delivered back into the respiratory tract, it can stimulate an antitumor immune response, and its efficacy can be further enhanced by adenosylcobalamin. Currently, there is a lack of non-invasive, non-invasive inhaled treatments for lung cancer. The antitumor agent of this invention can be combined with novel non-invasive inhaled bacterial-metabolite immunotherapy to achieve a long-term chronic disease management model, representing a safe and transferable treatment approach. In addition to achieving effective lung tumor control when used alone, this therapy can synergistically enhance the efficacy of immunotherapy, inducing complete tumor regression in 50% of tumor-bearing mice.

Description

Technical Field

[0001] This invention relates to antitumor methods and technologies, and more specifically, to an antitumor preparation based on respiratory symbiotic bacteria, its preparation method, and its application. Background Technology

[0002] Lung cancer, a malignant tumor with leading incidence and mortality rates worldwide, remains a major challenge in clinical treatment due to its complex pathobiological characteristics and high heterogeneity. While molecular targeted therapy, immune checkpoint inhibitors, radiotherapy, chemotherapy, and their combinations have brought some survival benefits to some patients, efficacy is still limited by factors such as drug resistance, systemic toxicity, and suppression of the local immune microenvironment. Current clinical treatments mostly rely on invasive drug delivery or systemic exposure, making it difficult to achieve precise control of lung lesions. Therefore, there is an urgent need for a locally acting, long-term, and highly adherent treatment modality, similar to the mature inhaled therapy modality used in asthma and chronic obstructive pulmonary disease. Inhaled therapy delivers drugs directly to the lungs, achieving higher safety, more precise targeting, and better efficacy. However, non-invasive inhaled therapy for lung cancer has not yet developed into a widely applicable and mature protocol, and there is a lack of effective inhaled biological agents for local immune modulation or microenvironment modification. Existing research mainly focuses on nanodelivery materials or local chemotherapeutic drugs, while exploration of inhaled interventions for lung cancer utilizing the inherent ecological characteristics of the respiratory tract is very limited, and significant gaps remain in related technological pathways.

[0003] In recent years, the crucial role of respiratory microbiota in lung homeostasis and tumor development has been increasingly recognized. Multiple studies have shown significant differences between the respiratory microbiota of lung cancer patients and healthy individuals. The respiratory microbiota plays a dual role in shaping the tumor immune microenvironment. On the one hand, certain respiratory microorganisms and their metabolites can promote tumor-associated inflammation, inhibit immune cell infiltration, or enhance tumor cell survival, indirectly or directly promoting tumor development and progression. On the other hand, respiratory commensal flora may also participate in maintaining respiratory immune homeostasis, promoting antigen-presenting cell function, and enhancing local anti-tumor immunity, thereby exerting a protective effect against tumor growth. Existing studies have utilized microbiome analysis to reveal characteristic microbiota changes in lung cancer patients, but how to further translate these microecological differences into therapeutic targets or intervention strategies remains a lack of systematic research. In particular, in vivo inhalation-based applications based on commensal flora still have significant research gaps regarding safety, immune regulation mechanisms, and modes of action. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an antitumor agent based on respiratory symbiotic bacteria, its preparation method, and its application.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0006] A method for preparing an antitumor agent based on respiratory symbiotic bacteria includes the following steps:

[0007] An antitumor agent was obtained by preparing oral streptococcal bacterial solution.

[0008] Furthermore, adenosylcobalamin was also added to the oral streptococcal bacterial solution.

[0009] Furthermore, the concentration of the oral streptococcal bacterial solution is (1.5-2.5) × 10⁻⁶. 6 CFU / mL.

[0010] Furthermore, the concentration of adenosylcobalamin in the oral streptococcal bacterial solution is 0.6-1.0 mg / mL.

[0011] An antitumor preparation, characterized in that it is prepared by the above method.

[0012] The above-mentioned antitumor agents are used in the preparation of lung cancer treatment drugs.

[0013] The beneficial effects of this invention are as follows: In response to the current clinical problem that the role of respiratory flora in lung cancer patients in lung cancer progression is unclear, we provide the first evidence that the respiratory commensal bacteria oral streptococcus has anti-tumor effects. When oral streptococcus is delivered back into the respiratory tract, it can stimulate an anti-tumor immune response, and its efficacy can be further enhanced by adenosylcobalamin. Attached Figure Description

[0014] Figure 1 This is a schematic diagram illustrating the growth-promoting effect of adenosylcobalamin on oral streptococci.

[0015] Figure 2 This is a schematic diagram illustrating the synergistic immune activation effect of adenosylcobalamin on oral streptococci.

[0016] Figure 3 This is a schematic diagram showing the size of lung tumors in different groups of mice in an in vivo experiment.

[0017] Figure 4 This is a schematic diagram showing the changes in body weight of mice in different groups during in vivo experiments.

[0018] Figure 5 This diagram illustrates the changes in immune cell infiltration in mice from different intervention groups.

[0019] Figure 6 CD4 in mice of different intervention groups + T cells and CD8 + Schematic diagram of changes in the stem cell characteristics of T cells. Detailed Implementation

[0020] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0021] I. Validation of the Supporting Effect of Adenosylcobalamin on Oral Streptococci by Detection at In Vitro Levels

[0022] (1) To verify the growth-promoting effect of adenosylcobalamin on oral streptococci, the bacterial concentration of different concentrations of adenosylcobalamin after incubation with oral streptococci for 24 hours was detected using an enzyme-linked immunosorbent assay (ELISA) reader. The results are as follows: Figure 1 As shown, adenosylcobalamin can promote the growth of oral streptococci within a certain concentration range;

[0023] (2) To verify the synergistic immune effect of adenosylcobalamin on oral streptococci, 10 μM adenosylcobalamin and 10 μM adenosylcobalamin were used at the cellular level. 5 CFU / mL of oral streptococci, or a combination of both, induces the maturation of bone marrow-derived dendritic cells and the killing effect of T cells. Flow cytometry analysis showed the following results: Figure 2 As shown, adenosylcobalamin was found to synergistically activate the immune response of oral streptococci.

[0024] II. Preparation of S. oralis and S. oralis_AdCbl solutions

[0025] (1) In a 15ml shake tube, culture oral streptococci (ATCC 35037) overnight in Brain Heart Infusion (BHI) medium until they amplify to the logarithmic growth phase;

[0026] (2) Collect the bacterial culture into a 50ml centrifuge tube, centrifuge at 1500rpm for 5min, remove the supernatant, and harvest the precipitate;

[0027] (3) Resuspend the precipitate in 1 ml PBS, collect it in a 50 ml centrifuge tube, add 19 ml PBS, centrifuge at 1500 rpm for 5 min, remove the supernatant, and collect the precipitate;

[0028] (4) Resuspend the precipitate in 1 ml PBS, collect it in a 50 ml centrifuge tube, add 19 ml PBS, centrifuge at 1500 rpm for 5 min, remove the supernatant, and collect the precipitate;

[0029] (5) Resuspend the precipitate and adjust the bacterial concentration to 10 by turbidimetric method. 9 CFU / ml;

[0030] (6) Take 10 μl of bacterial culture and add 5 ml of PBS to obtain S. oralis or add 5 ml of 0.8 mg / mL adenosylcobalamin to obtain S. oralis_AdCbl.

[0031] III. Animal-level validation of S. oralis_AdCbl inhibiting the growth of in situ lung tumors

[0032] At the animal level, we inoculated the left lung of C57BL / 6 mice with the lung cancer TC-1 cell line and randomly divided 40 mice into 4 groups (control group, S. oralis group, AdCbl group, and S. oralis_AdCbl group), with 10 mice in each group. The nebulized dose allocated to each mouse was: 500 μl of PBS, 10 6 CFU's S. oralis, 400ug of AdCbl, 10 6 CFU of *S. oralis* mixed with 400 μg of AdCbl (the latter three groups were dissolved in 500 μl PBS) was administered via nebulization every two days for a total of 5 interventions. Mouse weight was measured every other day, and the size of the in situ lung tumor was assessed using a small animal in vivo imaging system before the experiment ended. After 5 interventions, the *S. oralis*_AdCbl group showed significantly higher inhibition of in situ lung tumors than the single-drug treatment group, as shown in the results below. Figure 3 (left) and Figure 4 As shown, Figure 3 In this context, IGG represents the isotype control of the PD-L1 antibody.

[0033] IV. Verification that S. oralis_AdCbl can enhance the efficacy of immunotherapy

[0034] In animal studies, to verify that *S. oralis*_AdCbl could enhance the efficacy of immunotherapy, we inoculated the left lung of C57BL / 6 mice with the lung cancer TC-1 cell line and randomly divided the mice into four groups (control group, *S. oralis*_AdCbl group, antiPD-L1 group, and *S. oralis*_AdCbl+antiPD-L1 group). Dosage and administration: 100 μl PBS intraperitoneal injection, 10 6 CFU of S. oralis + 400ug of AdCbl nebulized inhalation, 100μg of antiPD-L1 intraperitoneal injection, 10 6 Mice were administered CFU (carbohydrated s. oralis) via nebulization with 400 μg of AdCbl and 100 μg of antiPD-L1 via intraperitoneal injection (antiPD-L1 dissolved in 100 μl PBS). Nebulization was repeated every two days for a total of 5 interventions, while intraperitoneal injection of antiPD-L1 was administered every three days. Mouse weight was measured every other day, and lung tumor size was assessed using a small animal in vivo imaging system before the experiment ended. After 5 interventions of inhalation and 3 interventions of intraperitoneal injection of antiPD-L1, the combined treatment group showed significantly higher lung tumor inhibition than the antiPD-L1-only group. The results are as follows: Figure 3As shown on the right, in the S. oralis_AdCbl combined with antiPD-L1 group, 50% of the mice showed no detectable tumor burden by in vivo imaging, indicating that S. oralis_AdCbl can enhance the efficacy of immunotherapy and achieve complete tumor regression in some tumor-bearing mice.

[0035] V. Animal-level validation: S. oralis_AdCbl inhibits the progression of in situ lung tumors in mice by remodeling the immune microenvironment.

[0036] To explore the pathways and mechanisms by which *S. oralis*_AdCbl inhibits the progression of orthotopic lung tumors in mice, and in conjunction with current research on the relationship between microorganisms and metabolites and tumor progression, we completed the treatment of orthotopic lung tumors in mice after treatment (control group, *S. oralis* group, AdCbl group, and *S. oralis*_AdCbl group) as described in Part III of the specific implementation plan. CD45+ immune cells were then sorted and sent for single-cell transcriptome sequencing to compare the distribution and functional differences of immune cells in different intervention groups. We found that *S. oralis*_AdCbl significantly reduced the infiltration of immunosuppressive tumor-associated macrophages and tumor-associated neutrophils in tumor tissue, while increasing T cell infiltration. Functionally, *S. oralis*_AdCbl regulates the stemness program of CD4+ T cells and CD8+ T cells. See the results below. Figure 5 , Figure 6 As shown.

[0037] in conclusion

[0038] 1. In response to the current clinical problem of the unclear role of respiratory flora in lung cancer patients in lung cancer progression, we provide the first evidence that the respiratory commensal bacteria oral streptococcus has anti-tumor effects. When oral streptococcus is delivered back into the respiratory tract, it can stimulate an anti-tumor immune response, and its efficacy can be further enhanced by adenosylcobalamin.

[0039] 2. Currently, there is a lack of non-invasive, non-invasive inhaled treatment methods for lung cancer in clinical practice. The anti-tumor preparation of this invention can be combined with a novel non-invasive inhaled bacterial-metabolite immunotherapy to achieve a long-term chronic disease management model, which is a safe and convertible treatment method.

[0040] 3. In addition to achieving effective lung tumor control when used alone, this therapy can synergistically enhance the efficacy of immunotherapy, inducing complete tumor regression in 50% of tumor-bearing mice.

[0041] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of antitumor agents based on respiratory symbiotic bacteria in the preparation of lung cancer therapeutic products, characterized in that, antitumor agent comprising a concentration of (1.5-2.5)*10 6 CFU / mL of Streptococcus oralis having the accession number ATCC 35037 and 0.6-1.0 mg / mL cobalamin, administered by nebulization.

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