WO2022222591A1

WO2022222591A1 - Lactobacillus paracasei strain for enhancing therapeutic effect of immune checkpoint inhibitor and use thereof

Info

Publication number: WO2022222591A1
Application number: PCT/CN2022/077453
Authority: WO
Inventors: 王立顺; 韩冰; 张世龙
Original assignee: 上海耀旦生物科技有限公司
Priority date: 2021-04-22
Filing date: 2022-02-23
Publication date: 2022-10-27
Also published as: CN114540229A; CN112877268A

Abstract

Provided are a Lactobacillus paracasei strain for enhancing the therapeutic effect of an immune checkpoint inhibitor and the use thereof, wherein the strain has a deposit number of CCTCC NO: M 2020474. In addition, the strain can recruit and increase the infiltration and activation of killer CD8 T lymphocytes in a tumor cell tissue, and promote the therapeutic effect of a PD-1 antibody.

Description

A strain of Lactobacillus paracasei that enhances the therapeutic effect of immune checkpoint inhibitors and its application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a Lactobacillus paracasei strain for increasing the therapeutic effect of an immune checkpoint inhibitor and its application.

Background technique

Immune checkpoint inhibitors (ICIs) are monoclonal antibodies targeting the corresponding immune checkpoints, blocking the inhibitory effect of tumor cells on immune cells through immune checkpoints, enabling immune cells to kill tumor cells. effect. There are currently 3 types of ICIs approved for marketing, including programmed death-1 (PD-1) monoclonal antibody, programmed death ligand-1 (PD-L1) monoclonal antibody, and Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) monoclonal antibody. ICI has been used to treat a variety of tumors since its inception, and has achieved positive results in 20%-30% of patients. Some patients with advanced tumors even have complete remission after ICI treatment and can survive for a long time, which has become an important part in the history of tumor immunotherapy. A milestone, bringing new hope to patients with malignant tumors. However, the initial treatment of ICI in most tumor patients is ineffective, or secondary drug resistance occurs after the initial treatment. Finding a method to overcome the resistance of ICI and further improve the efficacy of ICI has become a difficulty in the field of oncology treatment.

Studies in recent years have shown that the intestinal flora of patients is one of the reasons for the individual differences in the efficacy of ICIs such as PD-1 antibodies. In December 2020, Science published a study online from Tel Aviv University Medical Center in Israel. It found that 10 patients who did not respond to PD-1 antibody therapy were transplanted with the intestinal flora of patients who responded positively to PD-1 antibody therapy. Three patients responded positively to PD-1 antibody therapy, and one of the advanced patients achieved complete remission, and these patients had a significant increase in CD8 ⁺ T lymphocytes in the tumor tissue. All three patients received gut microbiota from the same donor, while five patients who received gut microbiota from another donor had no effect. In February 2021, "Science" published research results from UPMC Hillman Cancer Center and the US National Cancer Institute: Altering the gut microbiota can improve the efficacy of immunotherapy in patients with advanced melanoma. In this study, the researchers performed gut microbiota transplantation and PD-1 antibody immunotherapy in melanoma patients who were refractory to anti-PD-1 antibodies. Of the patients with tumor, 6 had tumor reduction or stable disease lasting more than one year. These responders had increased activation of immune CD8 T cells in the tumor microenvironment, whereas non-responders had increased immunosuppressive cells.

In recent years, multiple strains have been isolated from the intestinal flora, which can promote the anti-tumor effect of PD-1 antibodies. French immunologists studied 249 tumor patients with PD-1 antibodies and found that taking antibiotics before or during PD-1 antibody treatment significantly reduced the efficacy of PD-1 antibody treatment. Mice transplanted with gut microbiota that responded well to PD-1 antibodies responded better to PD-1 antibodies. Akkermansia muciniphila was significantly enriched in the gut microbiota of PD-1 treatment responders. Mice that responded poorly to PD-1 antibodies responded well by feeding Akkermansiac, with a significant increase in effector lymphocytes in the tumor microenvironment. The MD Anderson Cancer Center study found that those patients with high diversity of gut microbial species had a high response rate to PD-1 antibody therapy, a significant increase in CD8 ⁺ T lymphocytes, and Clostridiales bacteria and Faecalibacterium in their guts. Faecalibaterium); similarly, tumor-bearing mice transplanted with microbiota that responded to PD-1 antibody treatment had a better therapeutic effect on PD-1 antibody. The University of Chicago also found that in patients with metastatic melanoma, the intestinal commensal microbiome was associated with the efficacy of PD-1 antibodies, and Bifidobaterium was significantly enriched in the intestinal flora. Next, Nature reported that a team of Japanese scientists identified 11 bacterial strains "combinations" (11-mix) in the gut microbiota that synergistically enhance the effects of immune checkpoint inhibitors. In mouse experiments, they found that oral administration of 11-mix could increase IFN-γ ⁺ CD8 T cells in the tumor microenvironment and improve the anti-tumor effect of immune checkpoint inhibitors (PD-1 antibody, CTLA-4 antibody).

Although preclinical animal experiments and small clinical studies have found that the intestinal flora affects the therapeutic effect of ICI, the mechanism by which the intestinal flora affects the therapeutic effect of ICI is still unclear. Different donors have different effects on the effect of ICI; the same donor's flora has different effects on ICI treatment after transplantation to different recipients; at the same time, scientists from France, the United States and Japan isolated and obtained The gut bacteria that influence the effects of ICI treatment are also quite different. These differences mean in-depth study of how the gut microbiota affects the therapeutic effect of ICI preparations such as PD-1 antibodies, discovering new bacterial species in the gut microbiota that affect the therapeutic effect of ICI, and analyzing its molecular mechanism, which is essential for effectively overcoming ICI resistance. Medicine is important.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a Lactobacillus paracasei strain, which can effectively enhance the therapeutic effect of immune checkpoint inhibitors.

The second object of the present invention is to provide the application of the Lactobacillus paracasei strain in preparing food or medicine for enhancing the therapeutic effect of immune checkpoint inhibitor.

In order to achieve the above purpose, the present invention provides a Lactobacillus paracasei strain, which is named Lactobacillus paracasei Shanghai 2020 Lacticaseibacillus paracasei strain Shanghai 2020 (hereinafter referred to as L.paracasei-sh2020), and the deposit number is CCTCC NO:M 2020474.

In order to achieve the above second objective, the present invention provides the application of the Lactobacillus paracasei strain in preparing food or medicine for enhancing the therapeutic effect of immune checkpoint inhibitor.

As a preferred solution, the immune checkpoint inhibitor includes PD-1 antibody, PD-L1 antibody and CTLA-4 antibody.

The strain of the present invention is preserved in the China Collection Center for Type Cultures (CCTCC), and the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation date is September 9, 2020. The strain of the present invention was originally named "Lactobacillus casei strain Shanghai 2020" (application number 202110434508.2 application date: 2021-04-22), and after uploading the measured whole genome sequencing to the NCBI database, NCBI performed a comparison , in view of the higher homology between the strain and Lactobacillus paracasei, in this application, the name of the strain is modified to "Lactobacillus paracasei Shanghai 2020 Lacticaseibacillus paracasei strain Shanghai 2020", and issued by the China Collection of Type Cultures (CCTCC) Corresponding amendments have also been made to the deposit certificate of .

The viable count of the bacterial powder of the L. paracasei-sh2020 strain of the present invention is 1.0×10 ¹⁰ to 3.0×10 ¹¹ CFU/g.

The preparation method of the bacterial powder of described Lactobacillus paracasei strain L.paracasei-sh2020 comprises the following steps:

Inoculating the L. paracasei strain L.paracasei-sh2020 in an MRS culture solution and fermenting at 36-38° C. for 12-36 hours to obtain a L. paracasei-sh2020 fermentation solution;

The above-mentioned Lactobacillus paracasei strain L.paracasei-sh2020 fermented liquid is centrifuged, and the sediment is collected to obtain the Lactobacillus paracasei strain L.paracasei-sh2020 bacterial slurry;

The above-mentioned L. paracasei strain L. paracasei-sh2020 bacterial slurry was vacuum freeze-dried to obtain the bacterial powder of L. paracasei-sh2020 strain.

The study found that gut microbiota from healthy donors can enhance the effect of anti-PD-1 antibody in the treatment of tumors. ; We further isolated multiple strains of lactic acid bacteria from the intestinal flora of healthy donors, and administered the bacteria to tumor-bearing mice transplanted with the intestinal flora of tumor patients, and found that Lactobacillus paracasei can significantly increase PD-1 antibody The effect of treating tumors, through the whole genome sequencing analysis, found that the Lactobacillus paracasei is a new strain, which has been sent to the China Center for Type Culture Collection for preservation. The Lactobacillus paracasei we found that affects the therapeutic effect of anti-PD-1 antibodies is completely different from the intestinal bacteria such as Ackermann, Faecalibacterium, and Bifidobacterium discovered by other international teams.

The advantage of the present invention is that the L. paracasei-sh2020 strain provided by the present invention can improve the intestinal microecology of tumor model mice, promote the expression of CXCL10 chemokine by tumor cells, and recruit and increase the lethality of tumor cells in tissue. The infiltration and activation of CD8 T lymphocytes can effectively promote the therapeutic effect of PD-1 antibody and significantly inhibit tumor growth.

Description of drawings

Figure 1. PD-1 antibody has better antitumor effect in tumor-bearing mice transplanted with healthy donor gut flora.

Figure 2. Significantly increased infiltration of effector immune cells in tumor tissues of mice transplanted with healthy donor gut microbiota.

Figure 3. Lactic acid bacteria were elevated in the gut of healthy donors and positively correlated with immune killer cells.

Figure 4. Supplementation with Lactobacillus paracasei significantly enhances the therapeutic effect of PD-1 antibodies.

Figure 5. Lactobacillus paracasei enhances the efficacy of PD-1 antibody dependent on CD8 ⁺ T cells.

Figure 6. Genome sequencing analysis found this strain to be a new strain of Lactobacillus paracasei.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention.

Unless otherwise specified, the equipment and reagents used in each example and test example can be obtained from commercial sources.

Example 1. Gut flora from healthy donors enhances the antitumor effect of PD-1 antibody

In C57BL/6 mice, an antibiotic combination (ATB) consisting of vancomycin, neomycin, metronidazole, and ampicillin was first treated for 7 days to clear the mouse gut microbiota; then the guts of healthy donors were administered 20 μl of microflora was administered by gavage for 10 days, and 20 μl of intestinal microflora of tumor patients was used as a control; then, mice were inoculated with MC38 cells, and tumor volume was measured twice a week after tumor inoculation, and tumor growth curves were drawn; after 7 days, tumors were 150-200 mm ³ in size, treated with PD-1 antibody for 21 days; 28 days after tumor seeding, tumor size was assessed, mice were sacrificed, and intestinal, blood, spleen, colon and stool samples were taken from the mice.

Figure 1 shows that PD-1 antibody has better anti-tumor effect in tumor-bearing mice transplanted with healthy donor intestinal flora. (A-C) After antibiotics cleared the intestinal flora of mice, PD-1 antibody lost tumor cells MC38 inhibitory effect; (D) Construction of humanized tumor-bearing mice with gut microbiota and treatment procedure with PD-1 antibody; (E-G) PD-1 antibody is better in mice transplanted with healthy donor gut microbiota antitumor effect. FMT, flora transplantation; C, tumor patient; H, healthy donor. ATB, antibiotic cocktail. We found that PD-1 antibody can significantly inhibit tumor growth in normal mice (Fig. 1A), and after the application of antibiotics to clear the intestinal flora of mice, PD-1 antibody lost its inhibitory effect on tumor cells MC38 (Fig. 1B-C). ); (D) In the humanized tumor-bearing mice with intestinal flora established by transplantation of the intestinal flora of tumor patients, the tumor proliferation rate and volume were significantly higher than those of healthy donors with PD-1 antibody. Colony-constructed mice (Fig. 1D-F) had significantly lower survival than healthy donor gut microbiota-constructed mice (Fig. 1G).

We further found that lymphocytes CD4 ⁺ T (Fig. 2A), CD8 ⁺ T (Fig. 2B), ICOS expression in CD4 ⁺ T (Fig. 2C) and CD8 ⁺ T (Fig. 2D), and INF-γ that promote antitumor effects The expression of CD8 ⁺ T ( FIG. 2E ) was significantly increased in the tumor tissues of mice transplanted with healthy donor gut flora, while Treg (CD4 ⁺ CD25 ⁺ FoxP3 ⁺ ) cells, which inhibited the antitumor effect, were not significantly changed. *P<0.05, **P<0.01, ***P<0.001. These results suggest that gut microbiota from healthy donors can enhance the effect of anti-PD-1 antibodies on tumor therapy.

Example 2. Correlation between intestinal flora and the therapeutic effect of PD-1 antibody

Lactobacillus in healthy donors was significantly higher than that in tumor patients, and was positively correlated with the therapeutic effect of anti-PD-1 antibodies.

The gut, blood, spleen, colon and fecal samples of mice with gut microbiota constructed from the gut microbiota of the above tumor patients and healthy donors were collected. 16S was used to sequence the intestinal flora in stool samples, and analyze the expression of cytokines and the number and classification of immune cells in mice treated with PD-1 antibody, to explore the relationship between the intestinal flora and the therapeutic effect of PD-1 antibody. Correlation. We found that the taxonomic evolutionary tree (Fig. 3A), LDA score (Fig. 3B), genus-level analysis (Fig. 3C), and relative abundance comparison (Fig. 3D) of gut microbiota all indicated that Lactobacillus was significantly increased in the gut of healthy donors; And the correlation analysis between immune cells and intestinal flora (Figure 3E-I) suggested that the number of lactic acid bacteria in the intestine was positively correlated with immune killer cells.

Example 3. A new strain of L. paracasei L. paracasei-sh2020 from healthy donors enhances the anti-tumor effect of PD-1 antibody

Using MRS lactic acid bacteria selective medium, we isolated and cultured fecal samples from healthy donors with good PD-1 antibody promoting effect, and we obtained multiple strains of lactic acid bacteria that can be efficiently amplified. Next, we treated C57BL/6 mice with an antibiotic combination (ATB) consisting of vancomycin, neomycin, metronidazole and ampicillin for 7 days to clear the intestinal flora of the mice; 20 μl of .paracasei-sh2020 was administered by gavage for 10 days, and 20 μl of other lactic acid bacteria species and the intestinal flora of healthy donors were administered as controls; then, mice were inoculated with MC38 cells, and tumor volumes were measured twice a week after tumor inoculation, and plotted Tumor growth curve; 7 days after tumor size 150-200mm ³ , PD-1 antibody treatment was given for 21 days; 28 days after tumor seeding, tumor size was assessed, mice were sacrificed and mouse intestinal, blood, spleen, colon and stool samples were taken . We found that after the gut microbiota was cleared with antibiotics, humanized tumor-bearing mice with intestinal microflora were made, and the lactic acid bacteria mixture was administered to significantly enhance the therapeutic effect of PD-1 antibody (Fig. 4A-D); Humanized tumor-bearing mice supplemented with L. paracasei strain L. paracasei-sh2020 had the most significant enhanced therapeutic effect of PD-1 antibody (Fig. 4E); in mice with antibiotic clearance of intestinal flora, administration of paracasei Lactobacillus strain L. paracasei-sh2020 also significantly enhanced the therapeutic effect of PD-1 antibody (Fig. 4F). Next, the control and L. paracasei strain L. paracasei-sh2020-treated tumors were examined for immune cell infiltration using flow cytometry, and the results showed that CD8 ⁺ T cells expanded significantly in L. paracasei-sh2020-treated tumors, while other No significant changes were found in immune cells (Fig. 5A). Furthermore, IFNγ ⁺ CD8 ⁺ T cells were significantly increased after L. paracasei-sh2020 treatment (Fig. 5B-C). To determine which T cell subsets were required for L. paracasei-sh2020 to enhance PD-1 antibody efficacy, CD4 ⁺ or CD8 ⁺ T cells were depleted using neutralizing antibodies against CD8, CD4 in vivo (Fig. 5D). Depletion of CD8 ⁺ T cells completely abolished the effect of L. paracasei-sh2020, indicating that CD8 ⁺ T cells are essential for L. paracasei-sh2020 to enhance anti-tumor immunity of PD-1 antibodies. In contrast, depletion of CD4 ⁺ T cells did not affect the antitumor effect of L. paracasei strain L. paracasei-sh2020 combined with PD-1 antibody (Fig. 5E). Overall, L.paracasei-sh2020 significantly enhanced the therapeutic effect of PD-1 antibody, promoted CD8 ⁺ T cell infiltration and induced CD8 ⁺ T cell-dependent antitumor immunity.

Further, the whole gene sequence of L. paracasei-sh2020 was sequenced, and the assembled genome was compared with L. paracasei in the NCBI data, and it was found that its sequence similarity with known strains was about 77.56%. The Lactobacillus strain is a new strain, named L. paracasei-sh2020 (Fig. 6), and has been sent to the China Center for Type Culture Collection for preservation under the number CCTCC NO: M 2020474.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can also be made, and these improvements and modifications should also be regarded as It is the protection scope of the present invention.

Claims

A Lactobacillus paracasei strain, characterized in that the strain is named Lactobacillus paracasei Shanghai 2020 Lacticaseibacillus paracasei strain Shanghai 2020, and the preservation number is CCTCC NO: M 2020474.
The application of the Lactobacillus paracasei strain of claim 1 in preparing food or medicine for enhancing the therapeutic effect of immune checkpoint inhibitor.
The application of the Lactobacillus paracasei strain according to claim 2 in the preparation of food or medicine for enhancing the therapeutic effect of an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor comprises PD-1 antibody, PD-L1 Antibodies and CTLA-4 Antibodies.