Application of lactobacillus rhamnosus in improving treatment effect of hepatocellular carcinoma immune checkpoint inhibitor
By using a combination of mouse lactobacillus and immune checkpoint inhibitors in hepatocellular carcinoma (HCC) treatment, the problem of poor efficacy of HCC immunotherapy was solved, achieving significant inhibition of tumor growth and enhanced treatment effects.
Patent Information
- Application Number
- CN202511373370.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-24
- Publication Date
- 2025-12-12
AI Technical Summary
Existing immune checkpoint inhibitors have poor efficacy in treating hepatocellular carcinoma (HCC), and the overall response rate to immunotherapy is low. Therefore, personalized treatment plans are needed to improve their efficacy and patient prognosis.
The combined use of *Ligilactobacillus murinus* and immune checkpoint inhibitors such as PD-1 antibodies, administered orally or via intratumoral injection, enhances the efficacy of immunotherapy.
It significantly inhibits the growth of HCC, enhances the efficacy of immune checkpoint inhibitors, reduces the risk of side effects, provides a safe and effective administration method, and improves the treatment response rate and patient prognosis of HCC.
Smart Images
Figure CN121102291A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the application of *Ligilactobacillus murinus* in improving the efficacy of immune checkpoint inhibitor therapy for hepatocellular carcinoma, and belongs to the field of biomedical technology. Background Technology
[0002] Immune checkpoint inhibitors (ICIs), as an emerging treatment approach, have shown significant efficacy in some cancers, but their effectiveness in hepatocellular carcinoma (HCC) has been less than satisfactory. Nevertheless, combining different treatment strategies may improve the efficacy of immunotherapy. For example, studies have shown that the combination of PD-1 / PD-L1 antibodies with VEGF inhibitors has demonstrated better efficacy in HCC. This combination not only produces an additive effect by inhibiting tumor growth but also reprograms the immunosuppressive microenvironment into an immunostimulatory microenvironment. Furthermore, the combination of localized treatment with ICIs is also considered to enhance the potency of anti-tumor immunity, thereby improving treatment outcomes. However, despite these potential combination strategies, the overall response rate of immunotherapy in HCC remains low. Clinical trial results show that only a small percentage of HCC patients respond to immunotherapy, and the objective response rate is moderate. Therefore, further research needs to focus on how to improve the effectiveness of immunotherapy and patient prognosis through personalized treatment regimens.
[0003] In conclusion, although immunotherapy for HCC faces challenges, combining different treatment strategies and in-depth translational research may help improve its efficacy. Future research should continue to explore how to optimize immunotherapy regimens to achieve higher treatment response rates and better patient outcomes.
[0004] Microbiome-based immunotherapy is a field of extensive research, with *Ligilactobacillus murinus* being a recognized probiotic. As a gut microbiota, it has numerous effects, including inhibiting Helicobacter pylori-induced damage to the gastric mucosa; inhibiting the progression of Alzheimer's disease; suppressing intestinal inflammation and colon cancer; and regulating glucose and lipid metabolism. Furthermore, as a ectopic colonizing bacterium (e.g. in the lungs), it can inhibit lung inflammation caused by Mycobacterium tuberculosis; inhibit lung cancer; inhibit COPD; and inhibit COVID-19. However, there are currently no reports on combined interventions with *Ligilactobacillus murinus* to improve HCC immunotherapy. Summary of the Invention
[0005] The objective of this invention is to provide an application of *Lactobacillus murineis* in enhancing the efficacy of immune checkpoint inhibitor therapy for hepatocellular carcinoma (HCC). This invention validated in two constructed animal models of HCC that *Lactobacillus murineis* and its anti-PD-1 antibody can significantly inhibit HCC growth, and that *Lactobacillus murineis* can significantly enhance the efficacy of immune checkpoint inhibitor therapy for HCC.
[0006] To achieve the above objectives, the present invention provides the use of *Ligilactobacillus murinus* or its combination with immune checkpoint inhibitors in the preparation of medicaments for the prevention and / or treatment of hepatocellular carcinoma.
[0007] Preferably, the drug comprises an active ingredient and a pharmaceutically acceptable carrier or excipient, wherein the active ingredient is a combination of *Ligilactobacillus murinus* or *Ligilactobacillus murinus* and an immune checkpoint inhibitor.
[0008] Preferably, the immune checkpoint inhibitor includes PD-1 (anti-PD-1 antibody) and / or PD-L1 inhibitor (anti-PD-L1 antibody).
[0009] Preferably, the dosage form of the drug is tablets, granules, pills, powders, or capsules.
[0010] Preferably, the dosage form of the drug is an injection, enema, oral administration, or tube feeding preparation.
[0011] Compared with the prior art, the present invention has the following beneficial effects:
[0012] The *Ligilactobacillus murinus* proposed in this invention is a recognized probiotic with a low risk of side effects. In two hepatocellular carcinoma (HCC) models, *Ligilactobacillus murinus* and its synergistic anti-PD-1 antibody significantly inhibited HCC growth. *Ligilactobacillus murinus* significantly enhanced the efficacy of HCC immunotherapy, and the combined drug regimen showed a synergistic effect. Furthermore, *Ligilactobacillus murinus* can be administered orally or via intratumoral injection (surgery, percutaneous liver biopsy, or transhepatic artery administration), both of which are common and safe clinical methods with good safety profiles. Therefore, the *Ligilactobacillus murinus* and its combined drug regimen provided by this invention have promising clinical application prospects. Attached Figure Description
[0013] Figure 1 This is a schematic diagram of the animal experiment process in Example 1.
[0014] Figure 2To evaluate the therapeutic effect of *Lactobacillus simulans* combined with *Lactobacillus* in the Hep-53.4 hepatocellular carcinoma orthotopic model: A. Tumor mapping and quantitative analysis of number and size; B. Immunohistochemical Ki67 and TUNEL staining and quantification; **P<0.01, ***P<0.001, ****P<0.0001 indicate statistically significant differences between the two groups.
[0015] Figure 3 This is a schematic diagram of the animal experiment process in Example 2.
[0016] Figure 4 The therapeutic effect of mouse combined with Lactobacillus in a spontaneous model of liver cancer induced by (DEN) and carbon tetrachloride (CCl4): A. Tumor mapping and quantitative analysis of number and size; B. Immunohistochemical Ki67 and TUNEL staining and quantification; **P<0.01, ***P<0.001, ****P<0.0001 indicate statistically significant differences between the two groups.
[0017] Figure 5 This is a schematic diagram of the animal experiment process in Example 3.
[0018] Figure 6 The therapeutic effect of combined Lactobacillus and anti-PD-1 antibody in murine hepatocellular carcinoma in an orthotopic Hep-53.4 model was evaluated as follows: A. Tumor mapping and quantitative analysis of number and size; B. Immunohistochemical Ki67 and TUNEL staining and quantification; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 indicate statistically significant differences between the two groups.
[0019] Figure 7 This is a schematic diagram of the animal experiment process in Example 4.
[0020] Figure 8 The therapeutic effect of mouse combined with Lactobacillus and anti-PD-1 antibody in a spontaneous model of hepatocellular carcinoma induced by (DEN) and carbon tetrachloride (CCl4): A. Tumor mapping and quantitative analysis of number and size; B. Immunohistochemical Ki67 and TUNEL staining and quantification; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 indicate statistically significant differences between the two groups. Detailed Implementation
[0021] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.
[0022] Example 1
[0023] Implementation materials:
[0024] Hep-53.4 mouse hepatocellular carcinoma cells were purchased from Wuhan Pronosei Life Sciences Co., Ltd. Cells were cultured at 37℃ in a 5% CO2 incubator on DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. *Lactobacillus aspergillus* (catalog number: BMZ144994) was purchased from Ningbo Mingzhou Biotechnology Co., Ltd., and cultured at 37℃ in an aerobic environment on MRS agar medium (10.0g peptone, 10.0g beef extract, 5.0g yeast extract, 20.0g glucose, 1.0g Tween 80, 2.0g ammonium citrate, 5.0g sodium acetate, 0.1g magnesium sulfate, 0.05g manganese sulfate, 2.0g dipotassium hydrogen phosphate, 15.0g agar, 1L water, pH 6.5±0.2, sterilized at 121℃ for 15min). C57BL / 6 mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
[0025] Implementation method:
[0026] Hep-53.4 mouse liver cancer cells were cultured to approximately 80% confluence, then digested with trypsin and resuspended in 1×PBS. (The solution was then added at 1×10⁻⁶ ppm.) 6 Cells / mouse were inoculated into the left lobe of the liver of 6-week-old male C57BL / 6 mice. On day 5 post-inoculation, mice underwent two surgical injections of 2 × 10⁻⁶ cells / mouse. 7 CFU-lactobacter BMZ144994 in mice, or gavage of CFU-lactobacter BMZ144994 starting on day 5 post-inoculation (2×10⁻⁶). 8 CFU / time, once every other day, for a total of 8 times). Mice were sacrificed 21 days after cell inoculation, and samples were collected for observation. Figure 1 Ki67 (Proteintech, 27309-1-AP, 1:15000) and Tunel (Beyond the Clouds, C1091) staining were performed by immunohistochemistry.
[0027] Implementation results:
[0028] Oral / intratumoral injection of mouse lactobacillus combined with other methods can significantly reduce tumor volume. Figure 2 A) inhibits tumor proliferation and promotes tumor apoptosis, manifested by a decrease in the number of Ki67-positive cells and an increase in the number of Tunel-positive cells within the tumor. Figure 2 B).
[0029] Example 2
[0030] Implementation materials:
[0031] DEN (73861) and CCl4 (319961) were purchased from Sigma-Aldrich. C57BL / 6 mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.
[0032] Implementation method:
[0033] DEN was dissolved in physiological saline and administered intraperitoneally at a dose of 25 mg / kg to male C57BL / 6 mice at 2 weeks of age. At 6 weeks of age, mice were given intraperitoneal injections of CCl4 (2 mL / kg, twice weekly for 14 weeks). At 14 weeks of age, mice were given combined oral administration of Lactobacillus (2 × 10⁻⁶) via gavage. 8 CFU / time, every other day for 6 weeks), sample taken at 20 weeks of age for observation. Figure 3 Ki67 (Proteintech, 27309-1-AP, 1:15000) and Tunel (Beyond the Clouds, C1091) staining were performed by immunohistochemistry.
[0034] Implementation results:
[0035] Oral administration of mouse lactobacillus significantly reduced tumor volume. Figure 4 A) inhibits tumor proliferation and promotes tumor apoptosis, manifested by a decrease in the number of Ki67-positive cells and an increase in the number of Tunel-positive cells within the tumor. Figure 4 B).
[0036] Example 3:
[0037] Implementation materials:
[0038] Hep-53.4 mouse hepatocellular carcinoma cells were purchased from Wuhan Pronosei Life Sciences Co., Ltd. Cells were cultured at 37℃ in a 5% CO2 incubator on DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. *Lactobacillus aspergillus* (catalog number: BMZ144994) was purchased from Ningbo Mingzhou Biotechnology Co., Ltd., and cultured at 37℃ in an aerobic environment on MRS agar medium (10.0g peptone, 10.0g beef extract, 5.0g yeast extract, 20.0g glucose, 1.0g Tween 80, 2.0g ammonium citrate, 5.0g sodium acetate, 0.1g magnesium sulfate, 0.05g manganese sulfate, 2.0g dipotassium hydrogen phosphate, 15.0g agar, 1L water, pH 6.5±0.2, sterilized at 121℃ for 15min). C57BL / 6 mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The anti-PD-1 mouse monoclonal antibody (BE0146) and the isotype control monoclonal antibody (BE0089) were purchased from BioXCell.
[0039] Implementation method:
[0040] Hep-53.4 mouse liver cancer cells were cultured to approximately 80% confluence, then digested with trypsin and resuspended in 1×PBS. (The solution was then added at 1×10⁻⁶ ppm.) 6 Cells / mouse were inoculated into the left lobe of the liver of 6-week-old male C57BL / 6 mice. On day 5 post-inoculation, mice underwent two surgical injections of 2 × 10⁻⁶ cells / mouse. 7CFU-lactobacter BMZ144994 in mice, or gavage of CFU-lactobacter BMZ144994 starting on day 5 post-inoculation (2×10⁻⁶). 8 CFU / dose, every other day, for a total of 8 doses). Anti-PD-1 mouse monoclonal antibody (10 mg / kg, every three days, for a total of 5 doses) was injected intraperitoneally on day 7 post-inoculation. Mice were sacrificed 21 days later for observation. Figure 5 Ki67 (Proteintech, 27309-1-AP, 1:15000) and Tunel (Beyond the Clouds, C1091) staining were performed by immunohistochemistry.
[0041] Implementation results:
[0042] Oral / intratumoral injection of mouse lactobacillus combined with anti-PD-1 therapy significantly reduced tumor volume, and its ability to inhibit tumor growth was significantly enhanced compared to the anti-PD-1 antibody monotherapy group. Figure 6 A) It inhibits tumor proliferation and promotes tumor apoptosis, manifested by a decrease in the number of Ki67-positive cells and an increase in the number of Tunel-positive cells within the tumor. Compared with the anti-PD-1 antibody monotherapy group, its ability to inhibit tumor proliferation and promote tumor apoptosis is significantly enhanced. Figure 6 B). Oral / intratumoral injection of mouse lactobacillus combined with other drugs can enhance the efficacy of anti-PD-1 therapy.
[0043] Example 4:
[0044] Implementation materials:
[0045] DEN (73861) and CCl4 (319961) were purchased from Sigma-Aldrich. C57BL / 6 mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. Anti-PD-1 mouse monoclonal antibody (BE0146) and isotype control monoclonal antibody (BE0089) were purchased from BioXCell.
[0046] Implementation method:
[0047] DEN was dissolved in physiological saline and administered intraperitoneally at a dose of 25 mg / kg to male C57BL / 6 mice at 2 weeks of age. At 6 weeks of age, mice were given intraperitoneal injections of CCl4 (2 mL / kg, twice weekly for 14 weeks). At 14 weeks of age, mice were given a combination of mouse lactobacillus BMZ144994 via gavage (2 × 10⁻⁶). 8 CFU / dose, every other day for 6 weeks), and simultaneously, at 14 weeks of age, mice were intraperitoneally injected with anti-PD-1 mouse monoclonal antibody (10 mg / kg, every three days for 6 weeks). Samples were collected at 20 weeks of age for observation. Figure 7 Ki67 (Proteintech, 27309-1-AP, 1:15000) and Tunel (Beyond the Clouds, C1091) staining were performed by immunohistochemistry.
[0048] Implementation results:
[0049] Oral administration of mouse immunotherapy combined with Lactobacillus and anti-PD-1 immunotherapy significantly reduced tumor volume and, compared to the anti-PD-1 antibody monotherapy group, significantly enhanced the ability to inhibit tumor growth. Figure 8 A) It inhibits tumor proliferation and promotes tumor apoptosis, manifested by a decrease in the number of Ki67-positive cells and an increase in the number of Tunel-positive cells within the tumor. Compared with the anti-PD-1 antibody monotherapy group, its ability to inhibit tumor proliferation and promote tumor apoptosis is significantly enhanced. Figure 8 B). Oral administration of *Lactobacillus murineis* can enhance the efficacy of anti-PD-1 therapy.
[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.
Claims
1. The use of Ligilactobacillus murinus or its combination with immune checkpoint inhibitors in the preparation of drugs for the prevention and / or treatment of hepatocellular carcinoma.
2. The application according to claim 1, characterized in that, The drug comprises an active ingredient and a pharmaceutically acceptable carrier or excipient, wherein the active ingredient is *Ligilactobacillus murinus* or a combination of *Ligilactobacillus murinus* and an immune checkpoint inhibitor.
3. The application according to claim 1 or 2, characterized in that, The immune checkpoint inhibitors include PD-1 and / or PD-L1 inhibitors.
4. The application according to claim 1, characterized in that, The dosage forms of the drug are tablets, granules, pills, powders, and capsules.
5. The application according to claim 1, characterized in that, The dosage form of the drug is injection, enema, oral administration, or tube feeding preparation.