Lactobacillus reuteri strain that enhances the therapeutic effect of pd-1 mab and uses thereof

Abstract

The application provides a Lactobacillus reuteri strain for enhancing the treatment effect of PD-1 monoclonal antibody and application thereof, the strain is named Lactobacillus reuteri shanghai 2021, Lactobacillus reuteri shanghai 2021 (hereinafter referred to as L.reuteri-sh2021), and the preservation number is CCTCC NO: M 2021619. The L.reuteri-sh2021 provided by the application can improve the tumor microenvironment of a tumor model mouse, recruit and increase the infiltration and activation of killing CD8 T lymphocytes in the tissue of the tumor mouse, efficiently promote the treatment effect of a PD-1 antibody, and significantly inhibit the growth of a tumor.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a Lactobacillus reuteri strain for enhancing the therapeutic effect of PD-1 monoclonal antibodies. Background Technology

[0002] Programmed death-1 (PD-1) monoclonal antibodies are immune checkpoint inhibitors that block the inhibitory effect of tumor cells on immune cells through immune checkpoints, enabling immune cells to kill tumor cells. Since their introduction, PD-1 monoclonal antibodies have been used to treat various cancers, achieving positive efficacy in 20%-30% of patients. Some patients with advanced cancer have even achieved complete remission and long-term survival after PD-1 monoclonal antibody treatment, marking a significant milestone in the history of cancer immunotherapy and bringing new hope to patients with malignant tumors. However, most cancer patients do not respond to initial PD-1 monoclonal antibody treatment or develop resistance after initial treatment. Discovering methods to overcome PD-1 monoclonal antibody resistance will help further improve the efficacy of PD-1 monoclonal antibodies.

[0003] The complex interactions between cancer cells and immune cells mediate the emergence of PD-1 monoclonal antibody resistance. Besides tumor cell-derived factors, the chemotaxis and infiltration of immune cells, immune recognition and antigen presentation, and cytokine-mediated immune regulation within the tumor microenvironment play crucial roles. Currently, research on overcoming PD-1 monoclonal antibody resistance is in its initial stages. While there have been attempts to combine different treatment methods to reduce tumor PD-1 monoclonal antibody resistance, effective clinical treatment strategies for overcoming this resistance are still lacking.

[0004] From an ecological perspective, the human body is a micro-ecosystem. The human gut contains the most microorganisms, approximately 1.5 kg, primarily bacterial flora; hence, gut microbiota is often referred to as the gut microbiota. The gut microbiota plays a crucial regulatory role in the host's immune system. The development and maturation of the body's immune system depend on the stimulation of abundant antigens. The gastrointestinal tract is where the body's immune cells are most concentrated; over 70% of the body's lymphocytes are distributed in intestinal tissues and lymph nodes. Gut microorganisms express a large number of antigens, making them the most important and abundant source of microbial antigens for the human immune system. Simultaneously, gut microorganisms produce important immunomodulatory substances such as lipoteichoic acid (LTA) and peptidoglycan (PG). The gut microbiota is a vital foundation for promoting the development and maturation of the immune system and maintaining immune homeostasis. The host strictly controls the gut microbiota through intestinal immune cells, reducing tissue inflammation and bacterial translocation; thus, the human immune system is stimulated and trained by the gut microbiota.

[0005] The therapeutic effect of PD-1 monoclonal antibodies depends on the body's own immune cells' ability to kill tumor cells. Recent studies have shown that the gut microbiota is one of the reasons for individual differences in the efficacy of PD-1 monoclonal antibody therapy. In 2018, Science published three articles focusing on the importance of gut microbiota in PD-1 monoclonal antibody therapy for tumors. Following this, in 2019, Nature reported that a team of Japanese scientists identified an "11-mix" of 11 bacterial strains in the gut microbiota that synergistically enhances the effects of immune checkpoint inhibitors. In August 2021, a research team at Rockefeller University discovered that Enterococcus faecalis can synergize with PD-1 monoclonal antibodies, significantly enhancing the latter's therapeutic effect.

[0006] Significant progress has also been made in clinical research recently. In December 2020, Science published online a study from Tel Aviv University Medical Center in Israel, which found that after transplanting gut microbiota from patients who responded to PD-1 antibody therapy into 10 patients who did not respond to PD-1 monoclonal antibody therapy, 3 of these patients responded to PD-1 monoclonal antibody therapy, with one advanced-stage patient achieving complete remission. These patients also showed increased CD8 levels in their tumor tissue. + A significant increase in T lymphocytes was observed. All three patients received gut microbiota from the same donor, while five patients who received gut microbiota from a different donor showed no effect. In February 2021, *Science* published research findings from UPMC Hillman Cancer Center and the National Cancer Institute: a single fecal microbiota transplantation (FMT) from a responder combined with PD-1 monoclonal antibody therapy can provide clinical benefit to some patients with PD-1-refractory melanoma. In this study, researchers performed FMT and PD-1 monoclonal antibody immunotherapy on melanoma patients who were PD-1 resistant. Among 15 patients with advanced melanoma who received the combination of FMT and PD-1 monoclonal antibody therapy, six patients experienced tumor reduction or disease stabilization for more than one year. Immune CD8+ in the tumor microenvironment of these responders... + Increased T cell activation and increased immunosuppressive cells in non-responders.

[0007] Although preclinical animal studies and small-sample clinical trials have found that gut microbiota influences the therapeutic effect of PD-1 monoclonal antibodies, the mechanisms by which this effect remains unclear. Different donor gut microbiota have varying effects on PD-1 antibody efficacy; the same donor's microbiota also has different effects on PD-1 antibody efficacy when transplanted to different recipients; furthermore, scientists from France, the United States, and Japan have isolated completely different gut bacteria that influence the therapeutic effect of PD-1 monoclonal antibodies. These differences suggest that discovering new gut microbiota species that influence the therapeutic effect of PD-1 monoclonal antibodies could help effectively overcome PD-1 antibody resistance. Summary of the Invention

[0008] The first objective of this invention is to provide a Lactobacillus reuteri strain that enhances the therapeutic effect of PD-1 monoclonal antibodies, said strain being able to effectively enhance the therapeutic effect of PD-1 monoclonal antibodies.

[0009] A second objective of this invention is to provide the application of Lactobacillus reuteri strains that enhance the therapeutic effect of PD-1 monoclonal antibodies.

[0010] To achieve the above objectives, the present invention provides a Lactobacillus reuteri strain that enhances the therapeutic effect of PD-1 monoclonal antibodies. The strain is named Lactobacillus reuteri shanghai 2021 (hereinafter referred to as L.reuteri-sh2021) and has the accession number CCTCC NO: M 2021619.

[0011] This invention provides the application of the Lactobacillus reuteri strain that enhances the therapeutic effect of PD-1 monoclonal antibodies in the preparation of food or pharmaceuticals that enhance the therapeutic effect of PD-1 monoclonal antibodies.

[0012] This invention provides the application of the Lactobacillus reuteri strain that enhances the therapeutic effect of PD-1 monoclonal antibodies in the preparation of food or pharmaceuticals that overcome PD-1 monoclonal antibody resistance.

[0013] This invention provides the application of the Lactobacillus reuteri strain that enhances the therapeutic effect of PD-1 monoclonal antibody in combination with PD-1 monoclonal antibody in the preparation of antitumor drugs.

[0014] The strain is deposited at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on May 27, 2021.

[0015] The viable count of the Lactobacillus reuteri-sh2021 bacterial powder of this invention is 2.0 × 10⁻⁶. 10 ~4.0×10 11 CFU / g.

[0016] The method for preparing the mycelial powder of the Lactobacillus reuteri-sh2021 includes the following steps:

[0017] The L. reuteri-sh2021 strain was inoculated into MRS culture medium and fermented at 36-38°C for 12-36 h to obtain the fermentation broth of L. reuteri-sh2021 strain;

[0018] Centrifuge the fermentation broth of L. reuteri-sh2021 above, collect the precipitate, and obtain L. reuteri-sh2021 bacterial sludge;

[0019] The above-mentioned Lactobacillus reuteri-sh2021 mycelial sludge was freeze-dried under vacuum to obtain Lactobacillus reuteri-sh2021 mycelial powder.

[0020] Studies have found that gut microbiota from healthy donors can enhance the efficacy of anti-PD-1 antibody therapy for tumors, with significantly higher levels of Lactobacillus in the gut microbiota of healthy donors compared to tumor patients. We further isolated and purified a single *Lactobacillus reuteri* strain from the gut microbiota of healthy donors and administered this bacterium to tumor-bearing mice treated with antibiotics. We found that *Lactobacillus reuteri* significantly improved the efficacy of PD-1 antibody therapy for tumors. Whole-genome sequencing analysis revealed that this *Lactobacillus reuteri* strain is a novel strain and has been deposited at the China Center for Type Culture Collection. The *Lactobacillus reuteri* strain that we discovered affecting the therapeutic effect of anti-PD-1 antibodies is completely different from other gut microbiota such as *Ackermannii*, *Femtobacterium*, and *Bifidobacterium* discovered by other international teams.

[0021] The advantages of this invention are that the Lactobacillus reuteri-sh2021 provided by this invention can improve the tumor microenvironment in tumor model mice, recruit and increase the infiltration and activation of cytotoxic CD8 T lymphocytes in tumor mouse tissues, effectively promote the therapeutic effect of PD-1 antibody, and significantly inhibit tumor growth. Attached Figure Description

[0022] Figure 1 PD-1 antibody showed a better anti-tumor effect in tumor-bearing mice transplanted with gut microbiota from healthy donors. (A) Construction of humanized tumor-bearing mice with gut microbiota and PD-1 antibody treatment procedures: oral administration (PO), intraperitoneal injection (IP), and antibiotic mixture (ATB). (B) PD-1 antibody showed a better anti-tumor effect in mice transplanted with gut microbiota from healthy donors, including healthy donors (HD), colorectal cancer patient donors (CRC), with control mice (Control) and tumor-bearing mice treated with PD-1 antibody alone (PBS) as controls. (CE) Tumor growth curves in mice transplanted with gut microbiota from healthy donors. (FH) Tumor growth curves in mice transplanted with gut microbiota from colorectal cancer patient donors. *P<0.05,**P<0.01,***P<0.001,****P<0.0001.

[0023] Figure 2 A new strain of Lactobacillus reuteri was isolated and purified from the intestinal flora of a healthy donor.

[0024] Figure 3Supplementation with *Lactobacillus reuteri* significantly enhanced the therapeutic effect of PD-1 antibody. (A) After antibiotic elimination of intestinal flora, mice were inoculated with MC38 cells to construct a tumor-bearing mouse model. The mice were then treated with *Lactobacillus reuteri* L. reuteri-sh2021 and PD-1 antibody via oral (PO), intraperitoneal (IP), or antibiotic mixture (ATB). (B) Tumor growth curves were plotted for each group of mice. *Lactobacillus reuteri* significantly enhanced the therapeutic effect of PD-1 monoclonal antibody. (C) Tumor morphology in each group of mice. (D) Tumor size was statistically analyzed in each group of mice. *P<0.05,**P<0.01,****P<0.0001.

[0025] Figure 4 Lactobacillus reuteri combined with PD-1 antibody enhances CD8 + T cell killing ability. (A) Flow cytometry detection of CD8 in tumor tissues of mice treated with PD-1 antibody alone and in combination with Lactobacillus reuteri-sh2021 and PD-1 antibody. + T cells, IFN-γ + CD8 + T cell images. (B) Statistical analysis of CD8 in tumor tissues of mice in each group. + T cell proportion. (C) Statistical analysis of IFNγ in tumor tissues of mice in each group. + CD8 + T cell percentage. **P<0.01,***P<0.001. Detailed Implementation

[0026] The present invention will now be described in detail with reference to specific embodiments. It should be understood that the following specific embodiments are only for the purpose of helping those skilled in the art to understand the present invention, and are not intended to limit the present invention. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available.

[0027] Example 1. Gut microbiota from healthy donors enhances the antitumor effect of PD-1 antibodies.

[0028] In C57BL / 6 mice, the intestinal flora was first eliminated by treatment with an antibiotic combination (ATB) consisting of vancomycin, neomycin, metronidazole, and ampicillin for 3 days. Then, 200 μl of intestinal flora from healthy donors was administered by gavage for 3 days, with 200 μl of intestinal flora from tumor patients serving as a control. Next, the mice were inoculated with MC38 cells, and tumor volume was measured twice weekly after tumor implantation to plot tumor growth curves. After 7 days, the tumor size was 150-200 mm. 3 PD-1 antibody therapy was administered on days 7, 10, and 13 after tumor implantation. Twenty-eight days after tumor implantation, tumor size was assessed, mice were euthanized, and intestinal, blood, spleen, colon, and fecal samples were collected.

[0029] Figure 1 To demonstrate the superior antitumor effect of PD-1 antibodies in tumor-bearing mice transplanted with gut microbiota from healthy donors, we examined the following: (A) Construction of humanized tumor-bearing mice with gut microbiota and PD-1 antibody treatment protocol (FMT, microbiota transplantation; C, tumor patient; H, healthy donor; ATB, antibiotic mixture); (BH) PD-1 antibodies showed a superior antitumor effect in mice transplanted with gut microbiota from healthy donors. We found that in tumor-bearing mice with gut microbiota established by antibiotic depletion of mouse gut microbiota, the tumor proliferation rate and volume were significantly lower than in mice with gut microbiota from tumor patients after PD-1 antibody treatment, and the effect was better than in tumor-bearing mice treated with PD-1 antibody alone. The best antitumor effect was observed in healthy donor 2 (HD 2). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. These results suggest that gut microbiota from healthy donors can enhance the effect of anti-PD-1 antibody treatment for tumors.

[0030] Example 2. Isolation and purification of a new strain of Lactobacillus reuteri from the intestinal flora of a healthy donor.

[0031] Intestinal and fecal samples were collected from humanized mice with gut microbiota constructed from the gut microbiota of cancer patients and healthy donors. 16S sequencing analysis of the fecal samples revealed that *Lactobacillus* abundance was highest in the feces of humanized mice with gut microbiota constructed from healthy donors, and significantly higher than in the cancer patient group. Figure 2 A). Given that the antitumor effect of healthy donor No. 2 was the best, the fecal microbiota from healthy donor No. 2 was further serially diluted, and strains were isolated and cultured using MRS medium preferred by Lactobacillus. The process involved repeated plating, single-clone picking, amplification, and re-plating, followed by isolation and purification, finally yielding a single strain capable of efficient amplification. Figure 2 B). Further, mass spectrometry and whole-genome sequencing were performed on the strain, identifying it as *Lactobacillus reuteri*. The assembled genome was compared with *Lactobacillus reuteri* data from NCBI, revealing a sequence similarity of approximately 46.07% with known strains. This *Lactobacillus reuteri* strain is a novel strain, named *Lactobacillus reuteri* shanghai 2021 (abbreviated as L. reuteri-sh2021). Figure 2 C), has been deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 2021619.

[0032] Example 3. A novel strain of Lactobacillus reuteri-sh2021 from a healthy donor enhances the antitumor effect of PD-1 antibody.

[0033] In C57BL / 6 mice, we first treated them with an antibiotic combination of vancomycin, neomycin, metronidazole, and ampicillin (ATB) for 5 days to eliminate the intestinal flora. Then, we inoculated the mice with MC38 cells and simultaneously administered 200 μl (1 × 10⁻⁶) of cultured L. reuteri-sh2021. 9 Mice were administered CFU via gavage for 14 consecutive days, with PD-1 antibody combined with treatment on days 7, 10, and 13 post-tumor implantation. Mice with tumors treated with PD-1 antibody alone served as controls. Tumor volume was measured twice weekly after implantation, and tumor growth curves were plotted. After 7 days, the tumor size was 150-200 mm. 3 This indicates successful establishment of the tumor-bearing mouse model; 28 days after tumor implantation, tumor size was assessed, mice were sacrificed, and intestinal, blood, spleen, colon, and fecal samples were collected. Figure 3 A). We found that compared with tumor mice treated with PD-1 antibodies alone, treatment with L. reuteri-sh2021 in mice with antibiotic-mediated gut microbiota clearance significantly enhanced the therapeutic effect of PD-1 antibodies, such as... Figure 3 As shown in BD, tumor volume was measured on day 28 after tumor implantation, revealing that the average tumor volume in untreated mice reached 1400 mmHg. 3 The average tumor volume in mice treated with PD-1 antibody alone was 500 mmHg. 3 The inhibition rate was approximately 64%, while the average tumor volume in mice treated with L. reuteri-sh2021 combined with PD-1 antibody was only 180 mm. 3 The inhibition rate was close to 90%, and the tumor growth rate was greatly reduced, indicating that L. reuteri-sh2021 combined with PD-1 antibody therapy significantly enhanced the anti-tumor effect of PD-1 monoclonal antibody. Figure 3 Furthermore, flow cytometry analysis revealed that CD8+ was present in tumor tissues of mice treated with PD-1 antibody alone. + The proportion of T cells was 38%, and CD8+ cells were present in tumor tissues of mice treated with L. reuteri-sh2021 in combination with PD-1 antibody. + The proportion of T cells increased to 50%. Figure 4 Further analysis revealed that, compared with mice treated with PD-1 antibody alone, mice treated with L. reuteri-sh2021 in combination with PD-1 antibody had significantly higher levels of IFNγ in tumor tissue. + CD8 + The significantly increased infiltration of T cells indicates that the combination of Lactobacillus reuteri and PD-1 antibody can enhance CD8 cell infiltration. +The killing ability of T cells is enhanced, thereby increasing the therapeutic effect of PD-1 monoclonal antibodies and resulting in better treatment of tumors. Figure 4 A, 4C)( Figure 4 ).

[0034] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A *Lactobacillus reuteri* strain that enhances the therapeutic effect of PD-1 monoclonal antibodies, characterized in that, The strain was named *Lactobacillus reuteri* (… Lactobacillus reuteri Shanghai 2021, accession number CCTCC NO: M 2021619.

2. The use of the Lactobacillus reuteri strain that enhances the therapeutic effect of PD-1 monoclonal antibody as described in claim 1 in the preparation of a drug that enhances the therapeutic effect of PD-1 monoclonal antibody.

3. The use of the Lactobacillus reuteri strain that enhances the therapeutic effect of PD-1 monoclonal antibody as described in claim 1 in combination with PD-1 monoclonal antibody in the preparation of anti-colorectal cancer drugs.

Images