Use of a novel Lactobacillus plantarum strain and a polysaccharide derived from the strain for combined administration for the prevention or treatment of tumors

Abstract

The present invention relates to a novel Lactobacillus plantarum IMB19 strain and / or polysaccharides derived from the strain for use in combination with an anticancer agent for the prevention or treatment of tumors. The novel Lactobacillus plantarum IMB19 strain and polysaccharides derived from the strain of the present invention exhibit excellent CD8+ T cell activity stimulating ability and Treg cell suppressive activity, and stimulate and enhance antitumor immune responses through various mechanisms such as increased macrophage infiltration in CPS tumors and differentiation / reprogramming of macrophages to an inflammatory (M1) phenotype. Furthermore, when administered in combination with an immune anticancer agent such as an anticancer agent, particularly a PD-1 or PD-L1 inhibitor (e.g., PD-1 antibody or PD-L1 antibody), the strain and polysaccharides can significantly increase antitumor immune responses and exhibit a significant tumor growth inhibitory effect compared to the use of the anticancer agent alone. Thus, when administered in combination with various anticancer agents, they can be usefully used for the prevention, improvement or treatment of tumors.

Description

Technical Field

[0001] The present invention relates to the use of the novel Lactobacillus plantarum IMB19 strain KCTC 14337BP and / or a polysaccharide derived from the strain in combination with an anti-cancer agent for the prevention or treatment of tumors. Specifically, the present invention relates to the combined use of the Lactobacillus plantarum IMB19 strain KCTC 14337BP having immunostimulatory and anti-tumor activities or the polysaccharide of Chemical Formula I derived from the strain in combination with an anti-cancer agent for the prevention or treatment of tumors.

[0002]

Background Art

[0003] Mammals harbour a series of microorganisms that constantly interact with the immune system. Symbiotic microorganisms establish a symbiotic relationship with the host and interact with the host in various processes such as digestion, behaviour, and maturation of the immune system (Cerf-Bensussan N, Gaboriau-Routhiau V. The immune system and the gut microbiota: friends or foes? Nat Rev Immunol 2010;10(10):735-44.). Similarly, fungi exist in the human body and affect the host's immune system (Wheeler ML, Limon JJ, Underhill DM. Immunity to Commensal Fungi: Detente and Disease. Annu Rev Pathol 2017;12:359-85.). Innate immune cells detect various pathogen-associated molecular patterns (PAMPs) on the surface of fungal cells, including polysaccharides, through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). Upon detecting a signal, innate immune cells alter their gene expression profile and produce immune signalling molecules such as cytokines to mediate adaptive immunity (Iliev ID, Leonardi I. Nat Rev Immunol 2017;17(10):635-46., Underhill DM, Iliev ID. Nat Rev Immunol 2014;14(6):405-16. and Brubaker SW, Bonham KS, Zanoni I, et al. Annu Rev Immunol 2015;33:257-90.).

[0004] The World Health Organization (WHO) defines probiotics as live microorganisms that, when administered in appropriate amounts, confer health benefits on the host (Nat Rev Gastroenterol Hepatol 11, 506 - 514 (2014)). Probiotics have been reported to act as supplements to the host's gut microflora, effectively improving gut barrier function and regulating the host's immune system (Microb Ecol Health Dis 26, 25877 (2015)). Most probiotics belong to the phylum Firmicutes, which is the largest single bacterial phylum, and most of them are Gram - positive bacteria with a low "G + C" content, mainly classified into the Bacilli and Clostridia groups. Lactobacillus species are lactic acid bacteria (LAB) belonging to the family Lactobacillaceae. Lactobacillus is a well - known microbial community with a distinct ecological niche. Various lactic acid bacteria are traditionally known to be associated with foods such as milk, dairy products, fermented foods, and sausages. Lactobacillus is a microbial group recognized as GRAS (Generally Regarded As Safe) by the FDA and is widely used in food and other industrial fields. Lactobacillus is classified as a facultative anaerobic, non - spore - forming, non - motile, rod - shaped, Gram - positive bacterium and is generally regarded as catalase - negative. Lactobacillus can exhibit homofermentative or heterofermentative characteristics and produce lactic acid as the end - product of primary fermentation (Front Cell Infect Microbiol 2, 86 (2012)). Lactobacillus shows smooth and bulging colonies.

[0005] Due to the similar biochemical and morphological characteristics among LABs, molecular identification methods are required to identify individual strains. Various lactic acid bacteria have been isolated and characterized in foods, especially fermented foods. Fermentation generally means biochemical changes caused by microorganisms. Kimchi, a traditional Korean food, is mainly a fermented food of Chinese cabbage and shows nutritionally and healthily beneficial effects (Crit Rev Food Sci Nutr 34, 175-203 (1994)). It is known that kimchi has a unique microbiota. According to various reports, it is mainly known that lactic acid bacteria are present in kimchi, and Weisellia, Lactobacillus, and Leuconostoc are dominant species. In particular, Lactobacillus plantarum strains are known as the most dominant species (Food Sci Biotechnol 19, 641-646 (2010)). Lactobacillus plantarum is one of the most studied strains due to its strain-specific probiotic profile and technical applications in the food industry. Most of the kimchi microbiota can be cultured (Int J Food Microbiol 102, 143-150 (2005)), and individual microorganism isolation is essential to study the health benefits they confer.

[0006] On the one hand, cancer growth and proliferation are strictly regulated by the host immune response. In order for tumor cells to grow and proliferate, it is necessary to avoid the surveillance of the immune system that induces the initial death of tumor cells. Tumor cells form an immunosuppressive tumor microenvironment through various pathways such as cytokine secretion and cell surface molecule expression, and grow and proliferate by evading the immune system. As a cancer treatment strategy targeting such immune evasion mechanisms, various efforts have been made to induce or enhance the immune system. Tumor immunotherapy can be said to be a treatment method that restores or enhances the tumor recognition ability or destruction ability of the immune system in order to overcome the immunosuppression or immune evasion mechanisms acquired by tumors. Since ipilimumab was developed as an immunotherapeutic agent that successfully treated patients with malignant melanoma in 2011, various immunotherapeutic agents such as nivolumab and pembrolizumab have been continuously developed.

[0007] As tools for such tumor immunotherapy, reports and cases regarding the importance of the gut microbiota and its applications have been increasing. The gut microbiota plays an essential role in shaping the host's local and systemic immune responses (Science 330, 1768 - 1773 (2010), Cell 148, 1258 - 1270 (2012)). The diversity and composition of the gut microbiota have also been found to affect the responsiveness to chemotherapy (Cancer Immunol Immunother 55, 1470 - 1479 (2006); Science 342, 971 - 976 (2013)). In particular, certain commensal microorganisms have been shown to be associated with the activation of spontaneous anti - tumor immunity and are known to exhibit a synergistic effect on the therapeutic efficacy of immunotherapeutic agents in experiments (Science 350, 1084 - 1089 (2015), Science 350, 1079 - 1084 (2015)) and human cancers (Science 359, 91 - 97 (2018), Nature 453, 620 - 625 (2008)). Therefore, it is becoming clear that specific strains such as those in the gut microbiota can also affect the progression of extra - mucosal and distant tumors. Based on such various reports, the modification of the gut microbiota is regarded as one of the effective and feasible clinical treatment options. Currently, most studies have demonstrated the comprehensive effects of specific strains or populations of strains on the host immune system or anti - tumor immunity from the perspective of probiotics, but little information has been revealed about the active ingredients derived from specific strains showing such effects and the mechanisms such as their signaling pathways.

[0008] Combination therapy is one of the treatment strategies for many infectious, autoimmune, nervous system, and cardiovascular diseases including cancer, and is a classical approach that combines one or more therapeutic agents or therapies to improve efficacy compared to monotherapy. Generally, drugs with orthogonal activities have been combined for cancer treatment, but currently, combinations of drugs or therapies with additional effects or synergistic effects to increase efficacy while reducing side effects are required.

[0009] Chemotherapy and targeted therapies, including small molecule inhibitors, have been the main clinical methods used for the treatment of tumors in the clinic for the past half century. However, recently, immune checkpoint inhibitors have emerged as a more effective treatment option. Immune checkpoint inhibitors (ICIs) are applicable to a variety of tumor types, show sustained reactivity, and exhibit minimal toxic effects and drug resistance, thus showing very good clinical results.

[0010] However, one of the biggest drawbacks of immune checkpoint inhibitors is the limited responsiveness of ICIs in the general population. Although various mechanisms can act due to factors related to non-responsive populations, patient genetics, or acquired resistance, recently, it has been reported that the gut commensal microbiota affects the responsiveness of patients to immunotherapy. FMT from donors responsive to ICIs has been shown to improve the objective response of non-responsive patients. The role of individual gut microbes in enhancing the anti-cancer effect was first recognized while the enhanced effects of Bacteroides fragilis and Bifidobacterium against anti-CTLA-4 and anti-PD-L1 were reported. Since then, various bacterial strains show similar effects but different efficiencies from each other, so combinations of immune checkpoint inhibitors and strains showing better efficiency are needed.

[0011] Under such a technical background, as a result of the inventors' intensive efforts to clarify the clear correlation and mechanism between the components, structure, molecular weight, etc. of polysaccharides derived from microorganisms and the immunomodulatory function, a strain belonging to the novel Lactobacillus plantarum with significantly high immune-enhancing activity was identified from kimchi, a traditional Korean food. It was confirmed that the capsular polysaccharide of the novel strain and a fraction (CPS-100) having a specific structure thereof are effective molecules showing immune stimulation and enhancement effects. Specifically, the novel strain and the novel polysaccharide with a specific structure of the inventors activate CD8+ T cell function, increase macrophage infiltration in the CPS tumor, and differentiate / reprogram macrophages into an inflammatory phenotype, etc. By stimulating the anti-tumor immune response through various mechanisms, it was confirmed that the growth of tumors can be significantly suppressed, and a patent application was filed (Republic of Korea Patent Application Nos. 2021-0003432 and 2021-0003433).

[0012] Furthermore, as a result of the inventors' intensive efforts to develop a cancer treatment strategy through the combined therapy of various therapeutic uses used in conventional cancer treatments and the strain or polysaccharide derived from the strain, based on the fact that the novel strain stimulates cytotoxic T cell activity through antigen-presenting cells, when the Lactobacillus plantarum IMB19 strain is administered in combination with various immunotherapeutic anti-cancer agents, such as anti-PD-L1 antibodies, it was confirmed that the anti-tumor immune effect is significantly improved, and thus the present invention was completed.

[0013]

[0014] The information described in this background art section is merely for improving the understanding of the background of the present invention. Therefore, it may not include information forming prior art already known to those having ordinary knowledge in the technical field to which the present invention pertains.

[0015]

[0016]

Summary of the Invention

Problems to be Solved by the Invention

[0017] An object of the present invention is to provide a novel strain having immunostimulatory activity and a use of combined administration with an anticancer agent.

[0018] Another object of the present invention is to provide a use of combined administration with a polysaccharide derived from the above strain having immunostimulatory activity and an anticancer agent.

[0019] Still another object of the present invention is to provide an immunomodulation method through combined administration of the above strain and / or a polysaccharide derived from the strain with an anticancer agent, and / or a method for preventing, improving or treating a tumor or an infectious disease.

[0020]

Means for Solving the Problems

[0021] To achieve the above object,

[0022] The present invention provides a composition for preventing or treating a tumor, which comprises Lactobacillus plantarum IMB19 strain KCTC 14337BP and is administered in combination with an anticancer agent.

[0023] Also, the present invention provides a composition for preventing or treating a tumor, which comprises Lactobacillus plantarum IMB19 strain KCTC14337 and an anticancer agent.

[0024] The present invention provides a composition for preventing or treating a tumor, which comprises a polysaccharide represented by the following Chemical Formula I as an active ingredient and is administered in combination with an anticancer agent:

[0025] [Chemical Formula I]

[0026] -[-D-B-I-F-G-C-E-H-A-] n -

[0027] In the formula I,

[0028] A and D are galactose,

[0029] B, C, E, G and H are rhamnose,

[0030] F is N-acetylglucosamine,

[0031] I is glucose,

[0032] n is an integer from 1 to 10.

[0033]

[0034] The present invention also provides a composition for preventing or treating tumors, comprising a polysaccharide represented by the following formula I and an anti-cancer agent:

[0035] [Chemical formula I]

[0036] -[-D-B-I-F-G-C-E-H-A-] n -

[0037] In the formula I,

[0038] A and D are galactose,

[0039] B, C, E, G and H are rhamnose,

[0040] F is N-acetylglucosamine,

[0041] I is glucose,

[0042] n is an integer from 1 to 10.

[0043] In addition, the present invention provides a use for co-administration with an anti-cancer agent for the prevention, improvement, or treatment of tumors of the Lactobacillus plantarum IMB19 strain.

[0044] In addition, the present invention provides a use for co-administration with an anti-cancer agent for the prevention, improvement, or treatment of tumors of the polysaccharide of Chemical Formula I.

[0045] In addition, the present invention provides a method for the prevention, improvement, or treatment of tumors, comprising the step of co-administering the Lactobacillus plantarum IMB19 strain and an anti-cancer agent.

[0046] In addition, the present invention provides a method for the prevention, improvement, or treatment of tumors, comprising the step of co-administering the polysaccharide of Chemical Formula I and an anti-cancer agent.

[0047] In addition, the present invention provides a use of the strain and / or polysaccharide for manufacturing a composition for co-administration for the prevention or treatment of tumors or infectious diseases.

[0048]

Brief Description of the Drawings

[0049]

Figure 1

[0050] A serial dilution of the kimchi suspension was made, streaked on an MRS agar plate, cultured at 37°C for 48 hours, and then strains were obtained from 14 colonies and further cultured in MRS broth for 24 hours. Spleen cells and each strain were mixed at 1:10 and cultured at 37°C under 5% CO2 conditions for 48 hours. Activation of spleen cells was evaluated by measuring the cytokine values in the culture supernatant by ELISA. The data are mean ± SEM values (n = 2). Statistical significance was calculated against Mock by one-way ANOVA. * p < 0.5, ** p < 0.01, *** p < 0.001

[0051]

Figure 2

[0052] 1710137507964_0 A of which shows a TEM photograph of Lactobacillus plantarum strain IMB19. The arrow indicates the capsular layer around the cell wall.

[0053] B of Figure 2: Hemolytic activity of Lactobacillus plantarum strain IMB19 after culturing on 5% sheep blood agar for 48 hours, positive control group: Bacillus cereus ATCC27348

[0054] C of Figure 2: Gelatin hydrolysis test for Lactobacillus plantarum strain IMB19. Positive control group: B. cereus ATCC11778

[0055]

Figure 3

[0056] The phylogenetic tree was constructed based on the differences among Lactobacillus plantarum strains. The average nucleotide index (ANI) was calculated using the OrthANI algorithm. The distance is directly proportional to the genomic difference.

[0057]

Figure 4

[0058] Flow cytometry data showing the effects of L. plantarum IMB19, L. plantarum 3105, L. plantarum 3106, and L. plantarum 3107 on naive CD4+ T cells. Dendritic cells primed with L. plantarum IMB19 were cultured with naive CD4+ T cells, anti-CD3 (10 ng / ml), and IL-2 (100 U / ml).

[0059] In panel A of Figure 4, i is a FACS plot depicting the generation of Th17 cells. Cells were gated as live CD4+ RORγ cells.

[0060] In panel A of Figure 4, ii is a bar graph showing the average cell number for Th17 cells gated as live CD4+ RORγ cells.

[0061] Panel B of Figure 4 shows Th1 cells gated as live CD4+ T-bet+ T cells.

[0062] Panel C of Figure 4 shows Th2 cells gated as live CD4+ GATA3+ T cells.

[0063] Panel D of Figure 4 shows Tregs gated as live CD4+ Foxp3+ regulatory T cells.

[0064] Data are shown as mean ± SD, and statistical significance was calculated against Mock by one-way ANOVA. ** p < 0.01

[0065]

Figure 5

[0066] i in panel A of Fig. 5 is a representative FACS plot and bar graph showing the effect of L. plantarum IMB19 on the generation of Th17 cells. Dendritic cells primed with L. plantarum IMB19 were co-cultured with naïve CD4+ T cells, anti-CD3 (0.1 μg / ml), TGF-β, IL-6 (2 ng / ml), IL1-β IL-2 (100 U / ml), anti-IL4 (10 μg / ml) and anti-IFNγ (μg / ml). Cells were gated as live CD4+RORγ cells.

[0067] ii in panel A of Fig. 5 is a representative FACS plot and bar graph showing the level of interleukin-17 in Th17 cells generated with L. plantarum IMB19. Cells were gated as live CD4+RORγ cells.

[0068] Panel B of Fig. 5 is a representative FACS plot and bar graph showing the inhibitory effect of L. plantarum IMB19 on in vitro Treg cell generation. Dendritic cells primed with L. plantarum IMB19 were co-cultured with naïve CD4+ T cells with anti-CD3 (0.1 μg / ml), IL-2 (100 U / ml) and various concentrations of TGF-β. Data are presented as mean ± SEM. Statistical significance was analyzed by one-way ANOVA. ** p < 0.01, *** p < 0.001

[0069]

Figure 6

[0070] Panel A of Fig. 6 shows the results of cytokine analysis using ELISA of spleen cell-bacteria co-culture supernatants.

[0071] Panel B of FIG. 6 shows the quantification of IFNγ cells from co - cultures of spleen CD11c+ dendritic cells and naive CD8+ T cells primed with L. plantarum IMB19 or L. murinus.

[0072] Panel C of FIG. 6 shows the quantification of IFNγ cells from co - cultures of CD11b+F4 / 80+ peritoneal macrophages and naive CD8+ T cells primed with L. plantarum IMB19.

[0073] Panel D of FIG. 6 shows the quantification of Foxp3+CD4+ T cells from co - cultures of spleen CD11c+ dendritic cells, naive CD4+ T cells and 2 ng / ml of TGFβ primed with L. plantarum IMB19.

[0074] The data are mean ± SEM values and panel B was analyzed by one - way ANOVA with Tukey’s multiple comparison. ** p < 0.01, **** p < 0.0001

[0075] Panel E of FIG. 6 schematically shows an in vivo OVA - expressing Listeria monocytogenes (LM - OVA) cytotoxicity assay.

[0076] Panel F of FIG. 6 shows L. plantarum IMB19 - mediated CD8+ T cell - specific cytotoxicity against OVA+ spleen cells in mice infected with LM - OVA.

[0077] Panel G of FIG. 6 schematically shows an in vivo B16.F10 melanoma mouse model.

[0078] Panel H of FIG. 6 shows B16.F10 melanoma growth kinetics in C57 / BI6 germ - free mice treated or not treated with L. plantarum IMB19 or L. murinus.

[0079] Panel I in Fig. 6 shows the B16.F10 melanoma growth kinetics in C57 / BI6 SPF mice treated or not with L. plantarum IMB19 or L. murinus.

[0080] The data are mean ± SEM values, panel F in Fig. 6 is a non-parametric two tailed t-test, and panels H and I in Fig. 6 were analyzed by two-way ANOVA with Dunnet’s multiple comparisons. ** P < 0.01, *** P < 0.001, **** P < 0.0001

[0081]

Figure 7

[0082] Panels A and B in Fig. 7 quantify IFNγ cells in co-cultures of splenic (panel A in Fig. 7) or mLN (panel B in Fig. 7) CD11c+ dendritic cells primed with different ratios of L. plantarum IMB19 and naïve CD8+ T cells.

[0083] Panel C in Fig. 7 quantifies IFNγ cells in co-cultures of splenic CD11c+ dendritic cells primed with different ratios of L. plantarum IMB19 and naïve pmel TCR-transgenic CD8+ T cells in the presence of 100 ng / ml of gp-100. The data are mean ± SEM and were analyzed by one-way ANOVA with Tukey’s multiple comparisons. ** p < 0.01, *** p < 0.001, **** p < 0.0001

[0084]

Figure 8

[0085] Panel A of FIG. 8 quantifies Foxp3+ CD4+ T cells in co-cultures of spleen CD11c+ dendritic cells primed with L. plantarum IMB19 and naive CD4+ T cells at different TGFβ concentrations.

[0086] Panel B of FIG. 8 quantifies cytokines in co-cultures of spleen CD11c+ dendritic cells primed with L. plantarum IMB19 and naive CD4+ T cells at a TGFβ concentration of 2 ng / ml, gated on total CD4+ T cells.

[0087] Panel C of FIG. 8 shows IL-6 - / - Quantifies Foxp3+ CD4+ T cells in co-cultures of spleen DC and spleen CD11c+ dendritic cells primed with L. plantarum IMB19 and naive CD4+ T cells at a TGFβ concentration of 0.01 ng / ml.

[0088] Data are mean ± SEM and were analyzed by one-way ANOVA with Tukey's multiple comparisons. *** p < 0.001, ns: not significant

[0089]

Figure 9

[0090] Panel A of FIG. 9 shows the results of phylogenetic analysis of phyla (%) present in fecal samples from tumor-bearing mice administered L. plantarum IMB19 in H of FIG. 6.

[0091] B and C in Fig. 9 show the alpha (α) diversity analysis (B) and principal coordinates analysis of the bacterial β-diversity that appeared in the fecal samples of tumor-bearing mice administered with L. plantarum IMB19 in H of Fig. 6. The data show the values of two independent tests.

[0092]

Figure 10

[0093] a in Fig. 10 is the L. plantarum IMB19 bacterial community, and the arrow indicates individual bacteria.

[0094] b in Fig. 10 is L. plantarum IMB19, and the arrow indicates the thick capsular layer around the cell wall.

[0095]

Figure 11

[0096]

Figure 12

[0097]

Figure 13

[0098]

Figure 14

[0099]

Figure 15

[0100]

Figure 16

[0101]

Figure 17

[0102]

Figure 18

[0103]

Figure 19

[0104]

Figure 20

[0105]

Figure 21

[0106]

Figure 22

[0107]

Figure 23

[0108]

Figure 24

[0109]

Figure 25

[0110] Figure 25A shows the B16.F10 melanoma growth kinetics in C57 / Bi6 SPF mice treated or not treated with L. plantarum IMB19 or CPS.

[0111] B in Figure 25 is a photograph showing a tumor isolated from a mouse.

[0112] C and D in Figure 25 show the ratios of tumor-infiltrating CD8+ and CD4+ T cells determined by flow cytometry 16 - 18 days after the start of treatment with L.plantarum IMB19 (C) or CPS (D).

[0113] Data are mean ± SEM values. The data were analyzed by two-way ANOVA using Dunnett's multiple comparison (A) or non-parametric two-tailed t-test (C - D). * p < 0.05, ** p < 0.01, *** p < 0.001.

[0114] E and F in Figure 25 show the percentages of tumor-infiltrating IFNγ cells and the amounts of mean fluorescence intensity (MFI) of tumor-infiltrating CD8+ T cells at the time of treatment or non-treatment with L.plantarum IMB19 (E) or CPS (F). G and H in Figure 25 show the frequencies of IFNγ cells at the time of treatment or non-treatment with L.plantarum IMB19 (G) or CPS (H). Data are mean ± SEM values. The data were analyzed by non-parametric two-tailed t-test. * P < 0.05, ns: not significant.

[0115]

Figure 26

[0116] A and B in Fig. 26 show the percentage of tumor-infiltrating CD4+Foxp3+ regulatory T cells in tumor-infiltrated lymphocytes during the treatment with L. plantarum IMB19 (A) or CPS (B). The data were analyzed by non-parametric two tailed t-test. * P < 0.05, ns: not significant.

[0117]

Figure 27

[0118] A in Fig. 27 shows the EMT-6 breast cancer growth kinetics in Balb / c SPF mice treated or not treated with L. plantarum IMB19. The data are mean ± SEM and were analyzed by two-way ANOVA together with Dunnett's multiple comparisons. * P < 0.05, *** P < 0.001.

[0119]

Figure 28

[0120] A in Fig. 28 shows the number and percentage of CD45+CD11c+CD11b+ tumor-infiltrating macrophages at 40 hours after tumor inoculation during the treatment with CPS.

[0121] B and C in Fig. 28 show the activation markers, CD11b, MHC I, MHC II, CD86 and CD40, on tumor-infiltrating macrophages (B) and dendritic cells (C) during the treatment with CPS.

[0122] D in Fig. 28 shows the percentage of CD8+CD69+ T cells in tumor draining lymph nodes during CPS treatment. Data are mean ± SEM and were analyzed by two-way ANOVA (A) or non-parametric two tailed t-test (B) with Dunnett's multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001.

[0123] E and F in Fig. 28 show activation markers, MHC I, MHC II, iNOS2, CD68, CD40, CD80, and CD86 on mouse peritoneal macrophages treated with CPS (10 μg / ml) in vitro immediately after isolation (E) or after 24-hour IL-4 treatment (F).

[0124] G in Fig. 28 shows the result of analyzing the enriched M1 macrophage gene signature in CPS- or PBS-treated tumor-derived macrophages isolated 40 hours after tumor inoculation by DAVID pathway analysis.

[0125]

Figure 29

[0126] A in Fig. 29 shows biological pathways and functions within a subset of genes upregulated in each macrophage evaluated by DAVID pathway analysis, where macrophages were isolated from tumors of CPS- and PBS-administered mice initially infiltrated and subjected to mRNA sequencing for gene expression profiling (fold change ≥ 1.5).

[0127] B - D in Fig. 29 are heatmaps showing the relative abundance of genes showing significant changes among genes related to iron sequestration (B), TLR (C), and inflammatory macrophage markers (D).

[0128] E in Fig. 29 shows the results of quantifying IFN-γ T cells after co-culturing spleen CD11c+ dendritic cells and immature CD8+ T cells derived from wild-type mice primed with CPS (10 μg / ml) and LpIMB19. - / - This is the result of quantifying IFN-γ T cells after co-culturing spleen CD11c+ dendritic cells and immature CD8+ T cells derived from wild-type mice.

[0129] F in Fig. 29 shows the activity evaluated by SEAP secretion in the culture supernatant by QUANTI-BLUE SEAP reporter assay after culturing HEK-TLR2 cells with TLR2 ligand Pam3Csk4 or CPS for 24 hours. In Fig. 29E - F, the data are mean ± SD analyzed by two-way ANOVA and Dunnett's multiple comparison. * <0.05, ** P < 0.01, *** P < 0.001.

[0130] F in Fig. 29 shows the quantification and average of the fluorescence intensity of iron (Fe ++ ) in CD45+CD11c+CD11b+F4 / 80+ tumor-infiltrating macrophages 18 - 20 days after transplantation of B16.F10 melanoma.

[0131] G in Fig. 29 shows the result of quantifying iron uptake 30 minutes after exposure to iron in the medium with or without CPS treatment. The data are mean ± SD values analyzed by non-parametric two-tailed t-test, and ns indicates non-significance.

[0132]

Figure 30

[0133]

Figure 31

[0134]

Figure 32

[0135] A in Fig. 32 schematically shows a treatment therapy.

[0136] B in Fig. 32 shows a representative in vivo bioluminescence image (BLI) on the 20th day. BLI was performed to track the growth of renal cancer on the 10th, 13th, 18th, 20th, and 24th days.

[0137] C in Fig. 32 shows a longitudinal BLI indicating the progression of the tumor.

[0138] D in Fig. 32 schematically shows the combination administration for the B16.F10 mouse melanoma model.

[0139] E in Fig. 32 shows the progression of the tumor in melanoma. B16.F10 cells (0.2 milli / mouse) were subcutaneously injected, treated with various treatment therapies, and the progression of the tumor was tracked up to 20 days. Mean ± SEM values were displayed and analyzed by two-way analysis of variance (ANOVA). * P < 0.05 、** P < 0.001, *** P < 0.0001, **** P < 0.00001.

[0140]

[0141]

Mode for Carrying Out the Invention

[0142] Unless otherwise specified, all technical and scientific terms used in this specification have the same meaning as commonly understood by a skilled person in the technical field to which the present invention belongs. Generally, the nomenclature in this specification is well known and commonly used in this technical field.

[0143]

[0144] In one embodiment of the present invention, the inventors identified a novel Lactobacillus plantarum IMB19 strain showing high immune-stimulating activity from kimchi, a traditional fermented food in Korea, and deposited it with the Korean Collection for Type Cultures (deposit number: KCTC 14337BP).

[0145] In another embodiment of the present invention, in a co-culture system designed to include both the innate immune system and the adaptive immune system, the strain was shown to increase immune-stimulatory cytokines (e.g., IFN-γ) and suppress anti-inflammatory cytokines (e.g., IL-10), significantly increase effector T cells, and suppress the generation of Tregs, thereby stimulating the immune system in the host and suppressing tumor growth and proliferation.

[0146] In another embodiment of the present invention, the inventors confirmed that the antitumor effect is significantly improved when the strain and an immune checkpoint inhibitor are administered in combination, and that tumor growth can be effectively suppressed compared to the case where the strain or the immune checkpoint inhibitor is administered alone.

[0147]

[0148] Accordingly, in one aspect, the present invention relates to a composition for preventing or treating tumors, comprising the Lactobacillus plantarum IMB19 strain KCTC 14337BP and being administered in combination with an anticancer agent.

[0149]

[0150] In the present invention, the Lactobacillus plantarum IMB19 strain KCTC 14337BP may be characterized by showing excellent immune-enhancing activity and antitumor activity.

[0151] More specifically, the strain of the present invention can be characterized by exhibiting various immune-enhancing activities and / or anti-tumor activities such as i) inducing T cell differentiation in an inflammatory direction such as inducing helper T cells and suppressing the differentiation of Treg cells, ii) stimulating CD8+ T cells, enhancing their activity and improving tumor infiltration, iii) suppressing Treg cell activity, iv) increasing macrophage infiltration in tumors, v) activating macrophages into inflammatory cells, and reprogramming M2 macrophages into M1 macrophages.

[0152] In the present invention, the Lactobacillus plantarum IMB19 strain (hereinafter abbreviated as L. plantarum IMB19) was isolated from kimchi, identified as a novel strain through 16S rRNA analysis, recA amplification / band comparison, whole gene sequence analysis, and phylogenetic, morphological, and physiological analyses, and deposited.

[0153] In the present invention, the L. plantarum IMB19 strain may be characterized by being derived from fermented foods, such as kimchi.

[0154] In the present invention, the L. plantarum IMB19 strain may be characterized by showing small, smooth, circular, translucent colonies.

[0155] In the present invention, the L. plantarum IMB19 strain may be characterized by being non-flagellated.

[0156] In the present invention, the L. plantarum IMB19 strain may be characterized by showing rod-shaped colonies.

[0157] In the present invention, the L. plantarum IMB19 strain may be characterized by having a capsular layer.

[0158] In the present invention, the L. plantarum IMB19 strain may be characterized by being non-hemolytic or γ-hemolytic.

[0159] In the present invention, the L. plantarum IMB19 strain may be characterized by being negative for gelatinase activity.

[0160] In the present invention, the L. plantarum IMB19 strain may be characterized by not producing biogenic amines such as histamine, cadaverine, tyramine, and / or putrescine.

[0161] In the present invention, the L. plantarum IMB19 strain may be characterized by having resistance to kanamycin.

[0162] In the present invention, the L. plantarum IMB19 strain may be characterized by having immunopotentiating and / or antitumor activities. More specific examples include that the L. plantarum IMB19 strain may have various immunopotentiating and antitumor activities such as i) induction of T cell differentiation in an inflammatory direction such as induction of helper T cells and suppression of Treg cell differentiation, ii) stimulation, activation improvement, and intratumoral infiltration improvement of CD8+ T cells, iii) suppression of Treg cell activity, iv) increase in intratumoral macrophage infiltration, v) activation of macrophages into inflammatory cells and reprogramming from M2 to M1 macrophages, but is not limited thereto.

[0163] In the present invention, the L. plantarum IMB19 strain may be characterized by suppressing tumor growth.

[0164] In the present invention, the anticancer agent may be, for example, a chemical anticancer agent (cytotoxic anticancer agent), a targeted anticancer agent, an immunological anticancer agent, a hormonal anticancer agent, a cell therapy agent, etc., but is not limited thereto.

[0165] In the present invention, the chemical anti-cancer agent is also called a cytotoxic anti-cancer agent, which directly attacks cancer cells and exhibits an anti-cancer effect. Examples of such anti-cancer agents include alkylating agents such as cyclophosphamide, ifosfamide, bendamustine, melphalan, and cisplatin; antimetabolites such as capecitabine, cytarabine, doxifluridine, fluorouracil, clofarabine, fludarabine, and decitabine; DNA gyrase inhibitors such as doxorubicin, daunorubicin, idarubicin, etoposide, and topotecan; microtubule inhibitors such as cabazitaxel, docetaxel, vincristine; and other chemical anti-cancer agents such as mitomycin C, bleomycin, and hydroxyurea, but are not limited thereto.

[0166] In the present invention, the target anti-cancer agent is an anti-cancer agent mainly composed of a substance that targets and binds to or interacts with a specific antigen expressed by cancer or an antigen associated with cancer. Examples of such anti-cancer agents include antibody therapeutics such as cetuximab, trastuzumab, bevacizumab, rituximab, ibritumomab, alemtuzumab, brentuximab, and erlotinib; and signal transduction inhibitors such as erlotinib, gefitinib, vandetanib, afatinib, lapatinib, axitinib, pazopanib, sunitinib, sorafenib, but are not limited thereto.

[0167] In the present invention, the immune anti-cancer agent includes CTLA-4 antibodies such as ipilimumab, PD-1 antibodies such as pembrolizumab and nivolumab, PD-L1 antibodies such as atezolizumab, and immune checkpoint inhibitors such as IDO inhibitors, but is not limited thereto.

[0168] In the present invention, the hormonal anti-cancer agent includes male hormone suppressants such as bicalutamide and enzalutamide, and female hormone suppressants such as tamoxifen, anastrozole, and letrozole, but is not limited thereto.

[0169] In the present invention, the cell therapy agent refers to a therapeutic agent containing living immune cells as an active ingredient, including, but not limited to, cytotoxic T cell therapy agents, CAR-T cell therapy agents, CAR-NK cell therapy agents, etc.

[0170] The L. plantarum IMB19 strain of the present invention has various immunopotentiating and antitumor activities such as i) induction of T cell differentiation in an inflammatory direction such as induction of helper T cells and suppression of Treg cell differentiation, ii) stimulation of CD8+ T cells, improvement of activity and improvement of intratumoral infiltration, iii) suppression of Treg cell activity, iv) increase in intratumoral macrophage infiltration, v) activation of macrophages into inflammatory cells, and reprogramming from M2 to M1 macrophages. Therefore, in the present invention, preferably, the anticancer agent may be characterized in that it is an immunological anticancer agent.

[0171] In the present invention, more preferably, the immunological anticancer agent may be characterized in that it is an immune checkpoint inhibitor.

[0172] In the present invention, the immune checkpoint inhibitor is a preparation that blocks signal transduction by a receptor or its ligand involved in an immune checkpoint, and may be characterized by suppressing an immune checkpoint protein selected from the group consisting of PD-1 / PD-L1, CTLA-4, IDO, B7-1, and B7-2.

[0173] In the present invention, the immune checkpoint inhibitor may be an antibody that specifically binds to the immune checkpoint protein. In the present invention, the antibody that specifically binds to the immune checkpoint protein may be, for example, a PD-1 inhibitor such as pembrolizumab, nivolumab, and cemiplimab, a PD-L1 inhibitor such as atezolizumab, avelumab, and durvalumab, a CTLA-4 blocker such as atezolizumab, etc., but is not limited thereto.

[0174]

[0175] In one embodiment of the present invention, in an animal tumor model administered with the strain, it was confirmed that significant immune enhancement effects and antitumor activities were exhibited through various mechanisms such as stimulation of CD8+ T cells, differentiation in macrophages, phenotype induction and iron sequestration, suppression of Tregs, improvement of tumor infiltration of immune cells, and regulation of the TCR repertoire of CD8+ T cells.

[0176] In addition, in another embodiment of the present invention, it was confirmed that tumor growth was significantly suppressed during the treatment with CPS.

[0177]

[0178] The term "tumor" of the present invention includes any phenomenon in which cells grow autonomously and excessively, detached from the body's regulatory mechanism, or a neoplasm or hyperplastic lesion generated thereby. The tumor includes, for example, any of benign, pre-malignant, and malignant tumors. More specific examples include histiocytoma, glioma, astrocytoma, osteoma, various cancers, such as lung cancer, small cell lung cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, skin cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, brain cancer, sarcoma, osteosarcoma, melanoma, lymphoma (such as Hodgkin lymphoma, FL, MCL, MZBL, CLL, T-ALL, AML, ALL, etc.), blood cancer, leukemia, psoriasis, bone diseases, fibroproliferative disorders, atherosclerosis, etc., and preferably can be melanoma, breast cancer, kidney cancer, lung cancer, bladder cancer, rectal cancer, but is not limited thereto.

[0179] The term "prevention" used in the present invention means any act of suppressing a disease or delaying the onset of a disease by administering the composition according to the present invention.

[0180] The term "treatment" used in the present invention means any act in which the symptoms of a disease improve or are beneficially changed by administering the composition according to the present invention.

[0181] The composition of the present invention exhibits prevention or treatment and anti-inflammatory effects against various diseases due to the above-described immune enhancement effect and / or antitumor effect of its active ingredient.

[0182] The composition of the present invention can be characterized in that it is a composition for combined administration with an anticancer agent.

[0183] In the present invention, the L. plantarum IMB19 strain and the anticancer agent can be included in a composition as one dosage form for simultaneous administration after being mixed, but are not limited thereto, and as separate dosage forms, a composition containing the L. plantarum IMB19 strain and a composition containing the anticancer agent can be manufactured as their respective independent dosage forms.

[0184] In the present invention, the L. plantarum IMB19 strain and the anticancer agent can be included so as to be administered via the same or different administration routes. For example, the L. plantarum IMB19 strain is in a dosage form suitable for oral administration, and the anticancer agent can be manufactured and administered as an injection dosage form, but this is merely an example for explanation and is not limited thereto.

[0185] In the present invention, the composition containing the L. plantarum IMB19 strain is preferably manufactured as an oral dosage form, but is not limited thereto. In the present invention, the composition containing the L. plantarum IMB19 strain can be used in the form of a composition suitable for administration to humans or animals.

[0186] The L. plantarum IMB19 strain, which is an active ingredient of the composition of the present invention, can be provided as a composition in a freeze-dried or encapsulated form, a culture suspension, a fermentation broth, a dry powder, or a granular form when manufactured as an oral dosage form.

[0187] When prepared as a parenteral dosage form of a composition containing the L. plantarum IMB19 strain, which is the active ingredient of the composition of the present invention, it may include a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried preparation, and a suppository. As the non-aqueous solvent and the suspending solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As the base of the suppository, witepsol, macrogol, tween 61, cocoa butter, laurin fat, glycerogelatin, etc. may be used. Furthermore, it can be formulated by an appropriate method in the art or using the methods disclosed in Remington’s Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA.

[0188] In addition to containing the L. plantarum IMB19 strain or its culture solution of the present invention, the composition may further contain suitable carriers, excipients, and diluents commonly used in compositions containing microorganisms.

[0189] Examples of the carriers, excipients, and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating the composition, it is usually prepared by using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0190] The composition according to the present invention can be formulated and used in various forms by ordinary methods. Suitable dosage forms include oral dosage forms such as tablets, pills, powders, granules, dragees, hard or soft capsules, solutions, suspensions, emulsions, injections, aerosols, etc., external preparations, suppositories, and sterile injection solutions, but are not limited thereto.

[0191] The composition according to the present invention can be manufactured as an appropriate dosage form using an organic or inorganic carrier that is pharmaceutically inert. That is, when the dosage form is a tablet, a coated tablet, a dragee, or a hard capsule, it can contain lactose, sucrose, starch or its derivatives, talc, calcium carbonate, gelatin, stearic acid or its salts. Further, when the dosage form is a soft capsule, it can contain vegetable oils, waxes, fats, semi-solid and liquid polyols. Further, when the dosage form is in the form of a solution or syrup, it can contain water, polyols, glycerol, and vegetable oils, etc.

[0192] In addition to the above carriers, the composition according to the present invention can further contain preservatives, stabilizers, wetting agents, emulsifiers, solubilizers, sweeteners, coloring agents, osmotic pressure regulators, antioxidants, etc.

[0193] The composition of the present invention can be manufactured as an enteric-coated preparation using methods known in the art so as to reach the small intestine after passing through the gastrointestinal tract and rapidly release the active ingredient, the microorganism, Lactobacillus plantarum IMB19 strain, into the intestine.

[0194] Further, the composition of the present invention can be manufactured as a capsule-shaped composition using ordinary encapsulation methods. For example, after manufacturing pellets containing the microorganism of the present invention freeze-dried using a standard carrier, these can be filled into hard gelatin capsules. Or, after manufacturing suspensions and dispersions using the microorganism of the present invention and any appropriate pharmaceutical carrier, such as aqueous gums, celluloses, silicates or oils, such dispersions or suspensions can also be filled into soft gelatin capsules.

[0195] In the present invention, the composition can be provided, in particular, as an enteric-coated enteric preparation in an oral unit dosage form. "Enteric coating" as used herein includes all pharmaceutically acceptable known types of coatings that are not decomposed by gastric acid and maintain the coating, but are sufficiently decomposed in the small intestine to allow the active ingredient to be released into the small intestine. The material for the enteric coating can be appropriately selected from known polymer substances. Suitable polymer substances are listed in a number of known references (L. Lachman et al., The Theory and Practice of Industrial Pharmacy, 3rd edition, 1986, pp. 365-373; H. Sucker et al., Pharmazeutische Technologie, Thieme, 1991, pp. 355-359; Hagers Handbuch der pharmazeutischen Praxis, 4th edition, Vol. 7, pp. 739-742, and 766-778, (Springer-Verlag, 1971); and Remington’s Pharmaceutical Sciences, 13th edition, pp. 1689-1691 (Mack Publ., Co., 1970)), and may include, but are not limited to, cellulose ester derivatives, cellulose ethers, methyl acrylate copolymers of acrylic resins, and copolymers of maleic acid and phthalic acid derivatives.

[0196] The composition according to the present invention is administered in an effective amount. For example, when the composition is a food composition or a pharmaceutical composition, it may be characterized by being administered in a food scientifically effective dosage or a pharmaceutically effective amount, respectively. In the present invention, "effective dosage", "food scientifically effective dosage" and "pharmaceutically effective amount" mean an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to the prevention or treatment that is the object of the present invention, and the effective dosage level can be determined by factors including the type and severity of the disease of the subject, the activity of the drug, the sensitivity to the drug, the administration time, the administration route, the excretion ratio, the treatment period, drugs used simultaneously, and other factors well known in the medical field.

[0197] The composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered in single or multiple doses. Considering all of the above elements, it is important to administer an amount that provides the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

[0198] For example, in the present invention, the L. plantarum IMB19 strain is about 1x10 1 cfu to about 1x10 20 cfu, preferably about 1x10 5 cfu to about 1x10 15 cfu, and can be characterized by being administered at a dose of, and in one embodiment of the present invention, was administered at about 1x10 9 cfu, but is not limited thereto.

[0199] In the present invention, the anti-cancer agent administered in combination with the composition of the present invention can be administered at a pharmaceutically effective dose known in the art by the anti-cancer agent. In one embodiment of the present invention, the PD-L1 antibody administered in combination with the anti-cancer agent was administered at a dose of 100 μg, but is not limited thereto.

[0200]

[0201] The composition of the present invention can be administered to an individual by various routes. The mode of administration can be, for example, subcutaneous, intravenous, intramuscular or intrauterine epidural or intracerebrovascular injection. The composition of the present invention is determined by the type of drug that is the active ingredient, together with various relevant factors such as the disease to be treated, the route of administration, the age, sex, weight of the patient and the severity of the disease.

[0202] The administration method of the composition according to the present invention can be easily selected according to the dosage form and can be administered orally or parenterally. The dosage can vary depending on the patient's age, sex, weight, degree of disease condition, and administration route. The composition of the present invention can be administered in combination with other treatment therapies or therapeutic agents. When used for the prevention or treatment of tumors, preferably, the other treatment therapy or therapeutic agent can be, but is not limited to, immunotherapy or an immunocyte therapeutic agent, and can be used in various combinations according to the judgment of the clinician.

[0203] The term "combined administration" in the present invention means that when two or more active ingredients are administered simultaneously or sequentially, or when each active ingredient is independently administered by the action of two or more active ingredients, an effect improved from the effects expected when administered independently can be shown, and it means administering simultaneously, sequentially, or at a specific interval.

[0204] In the present invention, the combined administration can be characterized by administering the L. plantarum IMB19 strain of the present invention and an anticancer agent in combination, and in addition, it can be characterized by being carried out in parallel with a composition containing other active ingredients or other therapies.

[0205] In the present invention, each active ingredient to be administered in combination can be characterized by being administered via an independent route. Each active ingredient can be independently administered at appropriate administration methods and dosages by an ordinary technician. For example, as in one embodiment of the present invention, preferably, the L. plantarum IMB19 strain is administered orally, and the anticancer agent is administered through intravenous injection, but it is not limited thereto.

[0206] In the present invention, the combined administration of the L. plantarum IMB19 and the anticancer agent can be characterized by being carried out simultaneously.

[0207] In the present invention, the combined administration of the L. plantarum IMB19 strain and the anti-cancer agent may be characterized by being performed sequentially. For example, the anti-cancer agent may be administered after the administration of the L. plantarum IMB19 strain, or the strain may be administered after the administration of the anti-cancer agent.

[0208] In the present invention, when the L. plantarum IMB19 strain and the anti-cancer agent are administered sequentially, it can be characterized by being administered with a certain time interval. As a non-limiting example, they can be sequentially administered at intervals of 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, 1 day, several days, or 1 to several weeks, but it is not limited thereto, and can be sequentially administered at an appropriate interval by an ordinary technician.

[0209] In the present invention, the administration of the L. plantarum IMB19 strain and the anti-cancer agent can be independently repeated one or more times. In the present invention, when the L. plantarum IMB19 strain and the anti-cancer agent are repeatedly administered, the respective administration intervals can be easily adjusted by an ordinary technician independently according to the state of the subject and the level of the intended effect. For example, the strain and the anti-cancer agent can be independently administered at intervals of 1 hour, 6 hours, 8 hours, 12 hours, 1 day, 2 days, 1 week, 2 weeks, or 1 month, but it is not limited thereto.

[0210] The composition of the present invention can be manufactured as a pharmaceutical composition or a food composition.

[0211] In the present invention, the composition containing the L. plantarum IMB19 strain can be administered in a form independent of the dosage form of the anti-cancer agent to be administered in combination, for example, in the form of a pharmaceutical composition or a food composition. Preferably, the pharmaceutical composition containing the L. plantarum IMB19 strain and the pharmaceutical composition containing the anti-cancer agent may be administered in effective doses respectively, or the food composition containing the L. plantarum IMB19 strain and the pharmaceutical composition containing the anti-cancer agent may be administered in effective doses respectively, but it is not limited thereto.

[0212] The term "food composition" of the present invention is used in a broad sense to include substances containing nutritional components, including meats, sausages, bread, chocolate, candies, snacks, confectioneries, pizza, ramen, other noodles, gums, dairy products (milk products) including ice creams, various soups, drinking water, tea, drink agents, alcoholic beverages, vitamin complexes, prebiotics, probiotics, postbiotics, health supplements, health functional foods, and health foods, etc. It includes not only all foods in the ordinary sense but also "food additives" or "food additive compositions" added to foods.

[0213] In the present invention, when the food is provided as feed for animals excluding humans, it can be used interchangeably with "feed" in substantially the same meaning. Therefore, in the present invention, the food composition may mean a feed composition or a feed additive (feed additive composition).

[0214] The term "functional food" of the present invention is a term having the same meaning as "food for special health use" (FoSHU), and means a food with high medical and therapeutic effects that is processed so that a biological regulatory function can be efficiently exhibited in addition to nutrient supply. Here, "functionality" means obtaining effects useful for health purposes such as regulating nutrients with respect to the structure and function of the human body or physiological actions. The food of the present invention can be manufactured by methods commonly used in the industry, and during the manufacturing, raw materials and components commonly added in the industry can be added for manufacturing. Also, the dosage form of the food can be manufactured without limitation as long as it is a dosage form recognized as a food, and the health functional food according to the present invention can be in the form of powder, granule tablets, capsules, or beverages.

[0215] The above-mentioned health food refers to food that has a more proactive health maintenance and promotion effect compared to general food, and health supplement food refers to food for the purpose of health supplementation. In some cases, the terms functional food, health food, and health supplement food are used interchangeably.

[0216] The above-mentioned food composition may further contain a physiologically acceptable carrier, but the type of the carrier is not particularly limited, and any carrier commonly used in the technical field can be used.

[0217] Also, the above-mentioned composition can contain additional ingredients commonly used in food compositions that can improve odor, taste, visual appearance, etc. For example, the composition can contain vitamin A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc., and can also contain minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr), etc., and can also contain amino acids such as lysine, tryptophan, cysteine, valine, etc.

[0218] Also, the above-mentioned composition can contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (sun-dried powder and highly sun-dried powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc.), colorants (tar dyes, etc.), color developers (sodium nitrite, sodium acetate, etc.), bleaching agents (sodium sulfite), seasonings (MSG, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavors (vanillin, lactones, etc.), swelling agents (alum, potassium hydrogen D-tartrate, etc.), fortifiers, emulsifiers, thickeners (paste), film-forming agents, gum bases, bubble inhibitors, solvents, improvers, etc. The above-mentioned additives can be selected according to the type of food and used in appropriate amounts.

[0219] The composition of the present invention may further include a food additive acceptable in food science together with the L. plantarum IMB19 strain, can be used together with other foods or food ingredients, and can be appropriately used by ordinary methods. The mixing amount of the active ingredient can be appropriately determined according to its purpose of use (prevention, health or therapeutic treatment).

[0220] In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating tumors, which comprises Lactobacillus plantarum IMB19 strain KCTC 14337BP and an anticancer agent.

[0221] In yet another aspect, the present invention relates to a method for preventing or treating tumors, which comprises a step of co-administering the Lactobacillus plantarum IMB19 strain KCTC 14337BP and an anticancer agent.

[0222] In still another aspect, the present invention relates to the use of co-administration of the Lactobacillus plantarum IMB19 strain KCTC 14337BP and an anticancer agent for preventing, improving or treating tumors.

[0223] In yet another aspect, the present invention relates to the use for manufacturing a composition for co-administering with an anticancer agent of the Lactobacillus plantarum IMB19 strain KCTC 14337BP.

[0224]

[0225] In one embodiment of the present invention, it was confirmed that capsular polysaccharides are the active ingredient showing the immune-enhancing activity of L. plantarum IMB19 of the present invention.

[0226] In another embodiment of the present invention, it was confirmed that the separated capsular polysaccharide exhibits excellent immunopotentiating activity and antitumor activity by various mechanisms such as i) induction of T cell differentiation in an inflammatory direction such as induction of helper T cells and suppression of Treg cell differentiation, ii) stimulation of CD8+ T cells, improvement of activity and improvement of intratumoral infiltration, iii) suppression of Treg cell activity, iv) increase in intratumoral macrophage infiltration, v) activation of macrophages into inflammatory cells, and reprogramming from M2 to M1 macrophages, vi) alteration of gene profiles in macrophages and iron sequestration, and / or vii) regulation of the TCR repertoire of CD8+ T cells, similar to the L. plantarum IMB19 strain.

[0227] In another embodiment of the present invention, the capsular polysaccharide was structurally analyzed by NMR.

[0228]

[0229] Therefore, from another aspect, the present invention relates to a composition for preventing or treating tumors, which contains capsular polysaccharides (CPS) derived from Lactobacillus plantarum IMB19 strain KCTC 14337BP having immunopotentiating activity and is administered in combination with an anticancer agent.

[0230] In the present invention, the capsular polysaccharide may be characterized by containing a polysaccharide of the following Chemical Formula I:

[0231] [Chemical Formula I]

[0232] -[-D-B-I-F-G-C-E-H-A-] n -

[0233] A and D are galactose,

[0234] B, C, E, G, and H are rhamnose,

[0235] F is N-acetylglucosamine,

[0236] I is glucose,

[0237] n is an integer of 1 or more.

[0238]

[0239] In the present invention, the polysaccharide of Chemical Formula I may have a polymer (n = 1 or more) structure of a repeating unit represented by [-D-B-I-F-G-C-E-H-A-] in Chemical Formula I.

[0240] In the present invention, when n of Chemical Formula I is 2 or more, the repeating units may be linked without limitation in various ways in addition to direct covalent bonds. For example, the repeating units may be linked by chemical bonds such as glycosidic bonds and phosphodiester bonds, or may be linked through a linker.

[0241] In the present invention, when n of Chemical Formula I is 2 or more, the repeating unit ([-D-B-I-F-G-C-E-H-A-]) may be characterized in that it is linked by a glycosidic bond (-O-) between A and D.

[0242] In the present invention, when n of Chemical Formula I is 2 or more, the repeating unit ([-D-B-I-F-G-C-E-H-A-]) may be characterized in that it is linked by a phosphodiester linkage between A and D.

[0243] In the present invention, when n of Chemical Formula I is 2 or more, it may be characterized in that the carbon at the 1-position of A of the repeating unit and the carbon at the 6-position of D of another repeating unit are linked by a phosphodiester linkage.

[0244] In the present invention, one or more of A and D in the chemical formula I may be phosphorylated. Preferably, in the case of A, it can be characterized in that the hydroxyl group at the carbon atom at the 1-position is phosphorylated, and in the case of D, it can be characterized in that the hydroxyl group at the carbon atom at the 6-position is phosphorylated.

[0245] In the present invention, the galactose is used in the sense of including galactose generally present in nature or its derivatives. For example, in the present invention, the galactose may be an isomer and may be in the α-configuration, β-configuration, D-configuration or L-configuration, preferably α-galactose, more preferably α-D-galactose, but is not limited thereto.

[0246] In the present invention, the rhamnose is used in the sense of including any of rhamnose generally present in nature or its derivatives. For example, in the present invention, the rhamnose may be an isomer and may be in the α-configuration, β-configuration, D-configuration or L-configuration, preferably α-rhamnose, more preferably α-L-rhamnose, but is not limited thereto.

[0247] In the present invention, the N-acetylglucosamine is used in the sense of including both N-acetylglucosamine and its derivatives.

[0248] In the present invention, the glucose is used in the sense of including any of general glucose or its derivatives. For example, in the present invention, the glucose may be an isomer, and may be of the α-form, β-form, D-form or L-form. Preferably, the glucose may have a β-form sequence, and more preferably D-glucose, but is not limited thereto.

[0249] In the present invention, D and B (D-B), and B and I (B-I) may be characterized by being linked by an α-1,3-glycosidic bond.

[0250] In the present invention, I and F (I-F) may be characterized by being linked by a β-1,6-glycosidic bond.

[0251] In the present invention, F and G (F-G), G and C (G-C), C and E (C-E), and E and H (E-H) may be characterized by being linked by an α-1,2-glycosidic bond.

[0252] In the present invention, H and A (H-A) may be characterized by being linked by an α-1,6-glycosidic bond.

[0253] In this specification, the glycosidic bond is indicated by α or β according to the bonding orientation, and the subsequent numbers respectively mean the carbon numbers linked by the glycosidic bond (-O-) in two monosaccharides. For example, the statement that "I and F (I-F) are linked by a β-1,6-glycosidic bond" means that the carbon at the 1-position of I and the carbon at the 6-position of F are linked by a β-sequence glycosidic bond with I as the reference.

[0254] In the present invention, the capsular polysaccharide can include a polysaccharide of Chemical Formula I where n is an integer of 1 or more, and can include a number of polysaccharides of Chemical Formula I polymerized with various n. Preferably, the polysaccharide of Chemical Formula I may be characterized in that n is 1 to 10.

[0255] In one embodiment of the present invention, the average degree of polymerization of the polysaccharide of Chemical Formula I contained in the capsular polysaccharide was about 4, and the average molecular weight was confirmed to be about 6.0 kDa. Each repeating unit ([-D-B-I-F-G-C-E-H-A-]) was confirmed to have a molecular weight (MW) of about 1.5 kDa.

[0256] In the present invention, the "average of n" of the polysaccharide of Chemical Formula I contained in the capsular polysaccharide may preferably be 1 to 10, and most preferably about 4.

[0257] In the present invention, the average molecular weight of the polysaccharide of Chemical Formula I contained in the capsular polysaccharide may be characterized as 1.5 to 15 kDa, preferably about 6.0 kDa.

[0258] In one embodiment of the present invention, the polysaccharide of Chemical Formula I contained in the capsular polysaccharide was separated by an ion exchange chromatography method using capsular polysaccharides (CPS) derived from the L. plantarum IMB19 strain with about 100 mM NaCl as an eluent. Although the detailed separation method is described in detail in the examples, it is not limited thereto, and based on the polysaccharide structure and characteristics of Chemical Formula I described in the present invention, it can be obtained by various conventional purification methods.

[0259] In the present invention, the capsular polysaccharide may further contain teichoic acid.

[0260] As confirmed from one embodiment of the present invention, in the present invention, the capsular polysaccharide may further contain two or more teichoic acids.

[0261] In the present invention, the teichoic acid may be of the Gro type or the Rbo type. When the capsular polysaccharide further contains two or more teichoic acids, it may be contained in a single type or a mixture of two types.

[0262] In the present invention, the capsular polysaccharide may be characterized in that, in addition to the polysaccharide of Chemical Formula I and / or teichoic acid, it further contains other biomolecules such as other polysaccharides or lipids.

[0263] In still another aspect, the present invention relates to a pharmaceutical composition for preventing or treating tumors, which contains capsular polysaccharides (CPS) derived from Lactobacillus plantarum IMB19 strain KCTC 14337BP having immunopotentiating activity and an anticancer agent.

[0264] In still another aspect, the present invention relates to a method for preventing or treating tumors, which includes the step of administering to a subject capsular polysaccharides (CPS) derived from Lactobacillus plantarum IMB19 strain KCTC 14337BP having immunopotentiating activity and an anticancer agent.

[0265] In still another aspect, the present invention relates to the use of the capsular polysaccharides (CPS) derived from Lactobacillus plantarum IMB19 strain KCTC 14337BP in combination with an anticancer agent for preventing, ameliorating or treating tumors.

[0266] In still another aspect, the present invention relates to the use for producing a composition for combined administration with an anticancer agent of the capsular polysaccharides (CPS) derived from Lactobacillus plantarum IMB19 strain KCTC 14337BP.

[0267]

[0268] The method for producing the capsular polysaccharide of the present invention and the step of obtaining an effective polysaccharide fraction having immunopotentiating activity are described in detail in the examples of the present invention as an example, but are not limited thereto.

[0269]

[0270] In an embodiment of the present invention, capsular polysaccharides (CPS) derived from the L. plantarum IMB19 strain were fractionated using ion exchange chromatography with various concentrations of NaCl eluent, and the structure of the polysaccharides contained in each fraction was analyzed by NMR.

[0271] In another embodiment of the present invention, among various fractions of capsular polysaccharides (CPS) derived from the L. plantarum IMB19 strain, only in CPS-100 purified using 100 mM NaCl, significant levels of IFN-γ, TNF-α, IL-6 and IL-12 were produced, and negligible levels of IL-10, IL-17, IL-1β production were shown. In contrast, in other fractions (such as CPS-400 (containing teichoic acid)), no significant cytokine production was detected. It was confirmed that the active molecule showing the immune enhancing activity of the L. plantarum IMB19 strain is a polysaccharide (chemical formula I) having a polymer structure of a specific repeating unit contained in CPS-100.

[0272]

[0273] Therefore, the present invention further relates to a composition for preventing or treating tumors, which contains a polysaccharide represented by the following chemical formula I and is administered in combination with an anticancer agent.

[0274] [Chemical formula I]

[0275] -[-D-B-I-F-G-C-E-H-A-] n -

[0276] In the chemical formula I,

[0277] A and D are galactose,

[0278] B, C, E, G, and H are rhamnose,

[0279] F is N-acetylglucosamine,

[0280] I is glucose,

[0281] n is an integer of 1 or more.

[0282] In the present invention, the polysaccharide of Chemical Formula I may have a polymer (n = 1 or more) structure of a repeating unit represented by [-D-B-I-F-G-C-E-H-A-] in Chemical Formula I.

[0283] In the present invention, when n of Chemical Formula I is 2 or more, the repeating units may be linked without limitation in various ways in addition to direct covalent bonds. For example, the repeating units may be linked by chemical bonds such as glycosidic bonds and phosphodiester bonds, or may be linked via a linker.

[0284] In the present invention, when n of Chemical Formula I is 2 or more, the repeating unit ([-D-B-I-F-G-C-E-H-A-]) may be characterized by being linked by a glycosidic bond (-O-) between A and D.

[0285] In the present invention, when n of Chemical Formula I is 2 or more, the repeating unit ([-D-B-I-F-G-C-E-H-A-]) may be characterized by being linked by a phosphodiester linkage between A and D.

[0286] In the present invention, when n of Chemical Formula I is 2 or more, it may be characterized in that the carbon at the 1-position of A of the repeating unit and the carbon at the 6-position of D of another repeating unit are linked by a phosphodiester linkage.

[0287] In the present invention, it may be characterized in that one or more of A and D in the chemical formula I are phosphorylated. Preferably, in the case of A, it can be characterized in that the hydroxyl group of the carbon at the 1-position is phosphorylated, and in the case of D, it may be characterized in that the hydroxyl group of the carbon at the 6-position is phosphorylated.

[0288] In the present invention, the galactose is used in the sense of including galactose generally present in the natural system or its derivatives. For example, in the present invention, the galactose may be an isomer and may be in the α configuration, β configuration, D configuration or L configuration, preferably α-galactose, more preferably α-D-galactose, but is not limited thereto.

[0289] In the present invention, the rhamnose is used in the sense of including any of rhamnose generally present in the natural system or its derivatives. For example, in the present invention, the rhamnose may be an isomer and may be in the α configuration, β configuration, D configuration or L configuration, preferably α-rhamnose, more preferably α-L-rhamnose, but is not limited thereto.

[0290] In the present invention, the N-acetylglucosamine is used in the sense of including any of N-acetylglucosamine generally present in the natural system or its derivatives.

[0291] In the present invention, the term "glucose" is used to mean either ordinary glucose or its derivatives. For example, in the present invention, the glucose may be an isomer, which may be of the α-form, β-form, D-form or L-form, preferably β-glucose having a β-form sequence, and more preferably D-glucose, but is not limited thereto.

[0292] The term "derivative" in the present invention means a compound whose structure is sufficiently similar to the structure of the compounds disclosed in the present application, and which exhibits the same or similar activities and uses as the claimed compound based on such similarity, or a compound having a structure derived from the structure of a parent compound (for example, a polysaccharide of Chemical Formula I described in the present application) that is expected to induce the same or similar activities and uses as the claimed compound as a precursor. For example, the derivatives include, but are not limited to, salts of the parent compound, isomers, esters, amides, salts of esters or amides, N-oxides, etc.

[0293] In the present invention, D and B (D-B), and B and I (B-I) of Chemical Formula I may be characterized by being linked by an α-1,3-glycosidic bond.

[0294] In the present invention, I and F (I-F) of Chemical Formula I may be characterized by being linked by a β-glycosidic bond.

[0295] In the present invention, F and G (F-G), G and C (G-C), C and E (C-E), and E and H (E-H) of Chemical Formula I may be characterized by being linked by an α-1,2-glycosidic bond.

[0296] In the present invention, H and A (H-A) of Chemical Formula I may be characterized by being linked by an α-1,6-glycosidic bond.

[0297] In the present specification, the linkage is indicated by α or β according to the bonding orientation, and the following numbers mean the carbon numbers of the (-O-) bonds of two monosaccharides.

[0298] In the present invention, n in the chemical formula I may be an integer of 1 or more, preferably an integer of 1 to 10, and more preferably 4.

[0299] In the present invention, the polysaccharide may be characterized by having a structure of the following chemical formula II:

[0300] [Chemical formula II]

[0301] JPEG0007710106000001.jpg87170

[0302] n is an integer of 1 or more.

[0303]

[0304] In the present invention, n in the chemical formula II may be preferably an integer of 1 to 10, and more preferably 4.

[0305] In one embodiment of the present invention, the average degree of polymerization of the polysaccharide of the chemical formula I contained in CPS-100, which is an effective fraction of the capsular polysaccharide, was about 4, and the average molecular weight was confirmed to be about 6.0 kDa. Each repeating unit ([-D-B-I-F-G-C-E-H-A-]) was confirmed to have a molecular weight (MW) of about 1.5 kDa.

[0306] The polysaccharide of the chemical formula I contained as an active ingredient in the composition of the present invention may be characterized by containing two or more polysaccharides having other degrees of polymerization (n).

[0307] In the present invention, the "average of n" of the polysaccharide of the chemical formula I contained in the composition may be preferably 1 to 10, and most preferably about 4.

[0308] In the present invention, the polysaccharide of the chemical formula I may be characterized by having a molecular weight of at least about 1.5 kDa, preferably about 1.5 kDa to about 15 kDa, and more preferably about 4 kDa, depending on the degree of polymerization, the degree of phosphorylation, etc.

[0309] In the present invention, the polysaccharide may be characterized in that it is derived from Lactobacillus plantarum IMB19 strain KCTC 14337BP.

[0310] In the present invention, the polysaccharide of Chemical Formula I may be characterized in that it has immunopotentiating and / or antitumor activities. As a more specific example, the L. plantarum IMB19 strain may have various excellent immunopotentiating and antitumor activities such as i) induction of T cell differentiation in an inflammatory direction such as induction of helper T cells and suppression of differentiation of Treg cells, ii) stimulation of CD8+ T cells, improvement of activity and improvement of intratumoral infiltration, iii) suppression of Treg cell activity, iv) increase in intratumoral macrophage infiltration, v) activation of macrophages into inflammatory cells and reprogramming from M2 to M1 macrophages, vi) alteration of gene profiles in macrophages and iron sequestration, and / or vii) regulation of the TCR repertoire of CD8+ T cells, but is not limited to such mechanisms.

[0311] In the present invention, the polysaccharide of Chemical Formula I may be characterized in that it suppresses tumor growth.

[0312] In the present invention, an ordinary technician or the like can produce / separate the polysaccharide using the strain of the present invention, and can also induce or synthesize the polysaccharide of the present invention by other chemical or biological methods.

[0313] Furthermore, it is obvious that the structure of the CPS can be modified for improving the pharmacokinetics and / or pharmacodynamics characteristics of the polysaccharide of the present invention or for clinical formulation (e.g., solubility). For example, i) addition of one or more functional groups, ii) modification of the carbon chain, iii) addition of one or more hydrogen or hydroxyl groups, iv) modification of the end group (e.g., addition of a signal molecule such as a dye), v) binding with other known sugar molecules (e.g., for oral or systemic delivery formulations), etc. can be carried out, but not limited thereto.

[0314] Therefore, the polysaccharide of the present invention should not be limited to the polysaccharide of the above Chemical Formula I or Chemical Formula II, but should be interpreted as a concept including any of variants, derivatives, analogs, etc. of the above Chemical Formula I or Chemical Formula II as long as it has the immunostimulatory and immunopotentiating effects of the present invention.

[0315] Therefore, in another aspect, the present invention relates to an immunomodulatory composition containing the polysaccharide of the above Chemical Formula I as an active ingredient.

[0316] In yet another aspect, the present invention relates to an immunomodulatory method including the step of administering the polysaccharide to a subject.

[0317] In yet another aspect, the present invention relates to the immunomodulatory use of the polysaccharide.

[0318] In yet another aspect, the present invention relates to the use of the polysaccharide for manufacturing an immunomodulatory composition.

[0319] Hereinafter, unless otherwise specified regarding the terms, it will be understood to have the same meaning as understood by a person of ordinary skill in the art or as defined in other aspects of the present invention.

[0320] In one embodiment of the present invention, the 50% effective concentration (half maximal effective concentration, EC50) of the polysaccharide was confirmed to be 3.16 μM. Therefore, preferably, the pharmaceutical composition of the present invention may be characterized by containing at least 3.16 μM or more of the polysaccharide.

[0321] In the present invention, the polysaccharide and the anticancer agent may be included in a composition as one dosage form for combined administration after being mixed, but are not limited thereto, and as separate dosage forms, a composition containing the polysaccharide and a composition containing the anticancer agent may be manufactured as respective independent dosage forms.

[0322]

[0323] In the present invention, unless otherwise specified, the descriptions regarding the dosage form, administration method, administration dose, combined administration, etc. of the composition containing the polysaccharide have substantially the same characteristics as those described from the perspective of a composition for preventing or treating tumors, which contains Lactobacillus plantarum IMB19 strain KCTC 14337BP and is characterized by being administered in combination with an anticancer agent.

[0324] In the present invention, the combined administration of the polysaccharide and the anticancer agent may be characterized by being administered simultaneously or sequentially. For example, the anticancer agent may be administered after the administration of the polysaccharide, or the polysaccharide may be administered after the administration of the anticancer agent.

[0325] In the present invention, when the polysaccharide and the anticancer agent are administered sequentially, it may be characterized by being administered at a certain time interval. As a non-limiting example, they may be sequentially administered at intervals of 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, 1 day, several days, or 1 week to several weeks, but are not limited thereto, and may be sequentially administered at an appropriate interval by an ordinary technician.

[0326] In the present invention, the administration of the polysaccharide and the anticancer agent can each be independently repeated one or more times. In the present invention, when the polysaccharide and the anticancer agent are repeatedly administered, the respective administration intervals can be independently easily adjusted by an ordinary technician according to the condition of the subject and the level of the intended effect. For example, the polysaccharide and the anticancer agent can be independently administered one or more times at intervals of 1 hour, 6 hours, 8 hours, 12 hours, 1 day, 2 days, 1 week, 2 weeks, or 1 month, but are not limited thereto.

[0327]

[0328] In another aspect of the present invention, it relates to a pharmaceutical composition for preventing or treating tumors, comprising the polysaccharide represented by the chemical formula I and an anticancer agent.

[0329] In another aspect of the present invention, it relates to a method for preventing or treating tumors, comprising the step of co-administering the polysaccharide and the anticancer agent to a subject.

[0330] In another aspect of the present invention, it relates to the use of the polysaccharide in combination with an anticancer agent for preventing, ameliorating, or treating tumors.

[0331] In another aspect of the present invention, it relates to the use for producing a composition for co-administering the polysaccharide of the chemical formula I with an anticancer agent for preventing, ameliorating, or treating tumors.

[0332] The term "tumor" in the present invention includes any of the phenomena in which cells grow autonomously and excessively detached from the body's regulatory mechanisms, or the neoplasms or hyperplastic lesions generated thereby. The tumors include, for example, any of benign, pre-malignant, and malignant tumors. More specific examples include histiocytoma, glioma, astrocytoma, osteoma, various cancers, such as lung cancer, small cell lung cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, skin cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, brain cancer, sarcoma, osteosarcoma, melanoma, lymphoma (Hodgkin lymphoma, FL, MCL, MZBL, CLL, T-ALL, AML, ALL, etc.), blood cancer, leukemia, psoriasis, bone diseases, fibroproliferative disorders, atherosclerosis, etc., but are not limited thereto.

[0333]

[0334]

Example

[0335] Hereinafter, the present invention will be described in more detail using examples. It will be apparent to those having ordinary knowledge in the art that these examples are merely for illustrating the present invention and should not be construed as limiting the scope of the present invention.

[0336] Example 1: Materials and Methods

[0337] 1. Bacterial culture and identification

[0338] Kimchi was homogenized and a suspension was collected. Then, it was serially diluted, streaked on MRS broth and agar, and colonies were isolated by culturing at 37 °C for 48 hours and further cultured for various analyses.

[0339] Lactobacillus plantarum IMB19 strain was generally cultured in MRS broth at 37 °C for 24 - 36 hours. For TEM (transmission electron microscope), after culturing the bacteria, they were streaked on MRS - 1.5% agar (Neogen Corp., USA) and cultured for 24 - 30 hours. Bacterial colonies were drop - cast on a 2000 - mesh graphene - coated copper grid. TEM imaging was performed at 80 kV using JEOL1220 and Hitachi HT7700.

[0340] For identification, the cell morphology of the selected isolated strains was examined under a microscope, and genetic characteristic analysis was performed using genomic DNA. 16S rRNA sequence analysis was carried out at Macrogen (Korea).

[0341] The 16S rRNA gene was amplified by direct PCR using universal primers of forward (27F primer) 5’ - AGAGTTTGATCMTGGCTCAG - 3’ and reverse (1492R primer) 5’ - TACGGYTACCTTGTTACGACTT - 3’.

[0342]

[0343] 2. Primary cell - based tests

[0344] 2 - 1. In vitro spleen cell stimulation

[0345] All animal experiments and procedures were conducted in accordance with the ethical regulations and approvals of the Pohang University of Science and Technology Animal Care and Use Committee. C57BL / 6 mice were bred and maintained in a pathogen-free animal barrier facility and used at 6 - 8 weeks of age. Spleens were harvested and splenocytes were released by gently grinding. The cell suspension was lysed with ammonium chloride buffer to lyse RBCs and then resuspended in complete RPMI medium (Welgene, S. Korea) containing 10% FBS (Hy-Clone, Australia). Cells were plated in 96-well plates at a density of 200k / well in 200 μL of medium / well containing anti-CD3 (Bio-Xcell, USA) at 10 ng / mL and GM-CSF (Peprotech, USA) at 2.5 ng / mL. Fractionated CPS-100, unfractionated whole CPS (tCPS), LPS (lipopolysaccharide from E. coli 0111:B4, Invivogen, USA), and medium were added as needed and cultured at 37 °C in 5% CO2 for 48 h. Following the manufacturer's instructions, supernatants were collected after centrifugation and frozen for cytokine estimation using Enzyme linked immunosorbent Assay (ELISA); e-Bioscience, Ready set go ELISA kits.

[0346] 2-2. Immune cell co-culture system

[0347] Spleen cell separation was performed as described above. CD11c+ APCs (Miltenyi biotec) and immature CD4+ T cells (naive CD4+ T-cells) (Stem Cell Technologies) were separated according to the manufacturer's protocol. APCs were cultured with probiotic strains at 37 °C in 5% CO2 for 18 - 20 h. Subsequently, the probiotic strains were washed and the primed APCs and CD4+ T cells were co-cultured together under specific conditions.

[0348]

[0349] 3. Safety evaluation

[0350] 3-1. Hemolysis Test

[0351] After growing L. plantarum IMB19 under optimal growth conditions, it was streaked on 5% sheep blood agar (Hanil, Komed) and cultured for 48 hours. Alpha (α) 2 hemolysis is regarded as partial degradation of hemoglobin in red blood cells (actually not showing hemolysis), beta (β) hemolysis observed as a clear zone on the agar plate is regarded as complete degradation of hemoglobin in red blood cells, and gamma (γ) hemolysis was regarded as insufficient hemolysis. Bacillus cereus ATCC 27348 was used as the positive control group.

[0352] 3-2. Gelatin degradation test

[0353] The basic protocol was carried out according to ASM Science Recommendation (Dela Cru et al., 2012). L. plantarum IMB19 grown under optimal growth conditions was inoculated into the gelatin medium with an inoculation loop and cultured at 30 °C for up to 5 days, and gelatin liquefaction and bacterial growth were confirmed daily. Gelatin is generally liquefied at temperatures above 28 °C. To confirm whether the liquefaction is due to gelatin-degrading enzyme activity, the tube was stored in the refrigerator for 30 minutes. Then, the tube was tilted and it was observed whether the gelatin was degraded. If the gelatin was degraded, it would appear as a liquefied medium even after exposure to low temperature. Bacillus cereus ATCC 11778 was used as the positive control group.

[0354] 3-3. Biologenic amine analysis

[0355] L. plantarum IMB19 was grown under optimal growth conditions and streaked onto a special medium containing precursors of histamine, cadaverine, tyramine, and putrescine by Bover-Cid and Holzapfel (Bover-Cid et al, 1999), and cultured at 37 °C, 30 °C, and 23 °C for 4 days. Subsequently, the change in the medium color phase was confirmed, and positive and negative were determined. E. coli ATCC 25922 was used as a positive control group. The culture was performed on agar using a basic culture medium of any one of ornithine, lysine, tyrosine, and histidine together with a bromo cresol purple indicator.

[0356] 3 - 4. Antibiotic Resistance Test

[0357] The basic protocol was carried out based on ISO recommendations (ISO - 10932, 2010). To evaluate the minimum inhibitory concentration (MIC) of the strain against antibiotics, the broth dilution method was used.

[0358] In broth microdilution, the test organisms were cultured purely in broth medium, and all organisms were washed with 1X PBS. The bacterial solution washed with PBS was adjusted to an optical density (OD) unit of 0.01 - 0.02 at 600 nm. 10 μL of the strain (1 - 2×10 5 CFU) was inoculated into a 96 - well plate containing 200 μL of LSM broth medium together with the antibiotic.

[0359] The strain was considered sensitive when it was inhibited at a concentration for a specific antibiotic that was the same as or lower than the cut - off value set by the parameters set by the European Food Safety Authority (EFSA, 2018), and was considered resistant when it was not inhibited at a concentration of a specific antibiotic higher than the cut - off value set by the regulation.

[0360]

[0361] 4. Isolation of crude capsular polysaccharide (CPS)

[0362] The isolation of crude capsular polysaccharide (CPS) was performed by modifying the previously described method (Verma et al., 2018). The bacterial culture broth was centrifuged and sonicated with a Branson digital sonicator for 15 min at 10% amplitude and 10 s pulses. The supernatant was treated with trichloroacetic acid (0.5% w / v) at 4 °C overnight. The samples were centrifuged at 6000 rpm for 20 min, and 100% ethanol was added to the supernatant at a ratio of 3:1 to precipitate the crude polysaccharide at -20 °C. The precipitate was resuspended in Tris buffer (100 mM, Sigma Aldrich, USA) containing magnesium chloride (20 mM, Sigma Aldrich) and calcium chloride (20 mM, Sigma Aldrich) prepared with endotoxin-free distilled water, and treated with DNAse (0.1 mg / mL, Roche, Germany) and RNAse (0.4 mg / mL, Sigma Aldrich, USA) at 37 °C for 4 - 6 h. To degrade protein contaminants, Pronase (0.3 mg / mL, Sigma-Aldrich, USA) was added and incubated overnight at 4 °C. The samples were treated with trichloroacetic acid (1 - 2% w / v) at 37 °C for 30 min to remove total proteins including the added enzymes. The total polysaccharide of the protein-free samples was precipitated again by ethanol precipitation. The pellet was resuspended in endotoxin-free water and dialyzed at 4 °C for 48 h with water changes twice a day (MW cut-off 12,000 Da). The total CPS fraction was obtained by lyophilization with a final yield of 20 mg per liter of culture.

[0363]

[0364] 5. GC-MS analysis conditions

[0365] All chemical derivatives were analyzed using a gas-liquid chromatography (GLC-MS) Agilent 7820A (Santa Clara, CA, USA) equipped with a mass selective detector 5973N and a Zebron ZB-5 capillary column (Phenomenex, 30 m × 0.25 mm i.d., film thickness 0.25 μm, flow rate 1 mL / min, He as carrier gas). Electron impact mass spectra were recorded at an ionization energy of 70 eV and an ionization current of 0.2 mA. The temperature program used was as follows: 5 min at 150 °C, from 150 °C to 300 °C at 10 °C / min, 12 min at 300 °C.

[0366]

[0367] 6. NMR Acquisition Medium Variables

[0368] For the structural analysis of the isolated polysaccharides, NMR spectra were recorded in D2O using a Bruker 600 MHz spectrometer with a reverse cryo-probe with a gradient formed along the Z-axis. The spectra were measured at 298 K or 310 K and corrected with acetone ( 1 H 2.225 ppm; 13 C 31.45 ppm) as an internal standard, acquired with Topspin 2.0 software (Bruker), and processed and studied with Topspin 3.6. 1 H- 1 H DQ-COSY (double quantum COZY spectrum; hereinafter, COSY), TOCSY, and NOESY spectra were acquired from a 2048 × 512 point data set (t1 × t2), and for the TOCSY and NOESY spectra, were collected with 24 scans with mixing times of 100 ms and 200 ms set, respectively. Heteronuclear 1 H- 13 C HSQC, HMBC, and HSQC-TOCSY spectra were acquired using a 2048 × 512 point data set 1Performed in the H-detection mode. HSQC and HSQC-TOSCY were performed by multiple editing in the selection stage to distinguish the "2" density from other densities. HMBC was optimized for long-range coupling constants using a low-pass J-filter to suppress one-bond correlation relationships, and a 60 ms delay was used for the evolution of long-range correlation relationships. In the case of HSQC-TOCSY, the mixing time was set to 100 ms. In all two-dimensional experiments, the data matrix was expanded to 4092×2048 points and transformed by applying a qsine or sine window function.

[0369]

[0370] 7. Animals and mouse tumor models

[0371] C57BL / 6 & Balb / c mice were secured and maintained at the POSTECH University animal facility. Pmel-1 TCR transgenic, TLR2 - / - , TLR4 - / - , TLR6 - / - , MyD88 - / - and IL-6 - / - mice were obtained from Jackson Lab and maintained at the POSTECH animal facility. The C57BL / 6-derived melanoma cell line B16.F10 and the Balb / c-derived breast cancer cell line EMT-6 were maintained according to the protocol procured from ATCC. Syngeneic tumor models were generated by subcutaneous injection of 200,000 B16.F10 tumor cells or 500,000 EMT-6 cells. Tumor size was measured every other day until the endpoint, and tumor volume was calculated as length × width 2 × 0.5. For the analysis of the initial infiltration of innate immune cells, tumor cells were subcutaneously injected at 5 mill / mouse, and the tumor cells were analyzed 40 hours later. All experimental animal procedures were performed with the approval of the POSTECH University Institutional Animal Care and Use Committee (IACUC).

[0372]

[0373] 8. Cell-based in vitro analysis methods

[0374] Total cells harvested from the spleen and / or lymph nodes were used for whole spleen cell culture or applied to magnetic bead separation (Miltenyi Biotec) to enrich CD11c+ dendritic cells, immature CD8+ T cells (naive CD8+ T-cell) or immature CD4+ T cells. Total spleen cells (200,000 cells / well) were cultured in 96-well plates at a 1:1 ratio with bacteria, and the supernatant was harvested at 48 hours and used for ELISA (eBioscience Ready set Go kits). CD11c+ dendritic cells (200,000 cells / well) were primed with the appropriate ratio of bacteria for 18 - 20 hours, washed, and then T cells (200,000 cells / well) were added and cultured for 72 hours. As indicated, anti-CD3@0.01 μg / ml (BioXCell), GM-CSF@2.5 ng / ml (Peprotech) were added for the stimulation of CD8+ T cells, and anti-CD3@0.1 μg / ml, GM-CSF@10 ng / ml, IL2@100 U / ml and TGFβ were added for the stimulation of CD4+ T cells.

[0375] In the case of peritoneal macrophages, 2% BIogel (Bio-Rad) was injected intraperitoneally, and the cells were harvested 5 days later and cultured in vitro with recombinant murine MCSF 10 ng / ml (Peprotech). For the polarization of selectively activated macrophages, IL-4 (Peprotech) was added for 24 hours, and then treated with CPS, LPS (lipopolysaccharide derived from E-coli 0111:B4, Invivogen) or Pam3CSk4 (Sigma).

[0376]

[0377] 9. Metagenomics analysis and Whole genome sequencing

[0378] Stool pellets from SPF mice bearing tumors were sent for metagenomic analysis (Macrogen, S. Korea). Whole-genome sequencing of bacteria was performed on the Illumina platform (Macrogen, S. Korea), and bacterial culture samples were sent directly for analysis. Bioinformatics analysis was also outsourced to Macrogen.

[0379]

[0380] 10. Classification and gene expression profile analysis of tumor-infiltrating macrophages

[0381] After injecting 500 μg of CPS into mice twice at 24-hour intervals, 5×10 6 B16.F10 tumor cells were subcutaneously inoculated. Forty hours after tumor transplantation, whole tumors containing infiltrating immune cells were digested into single-cell suspensions with Liberase. Five to ten mouse samples were recruited from the same treatment group and stained with Fixable Viability-ef506 (eBioscience), CD45-AF488 (Bioloegend, 30-F11), CD3-ef450 (Ebioscience, 145-2C11), CD19-PB (Ebioscience, 1D3), I-A / I-E-PECy7 (Biolegend, M5 / 114.15.2), CD11c-PE (Ebioscience, N418), and CD11b-PerCpCy5.5 (BD, M1 / 70). Live CD45+CD3-CD19-MHCII hi CD11c + CD11b +Macrophages were classified in FBS-supplemented medium and centrifuged and stored in Trizol (Trizol, Sigma). Samples were analyzed for gene expression profiles at Macrogen, and the average fold change of gene transcripts over several weeks between treatment groups was calculated. Genes with a fold-change of 1.5 or more in both comparisons were input into DAVID v6.7 (The Database for Annotation, Visualization and Integrated Discovery v6.7) for pathway analysis. Genes determined to have significantly enhanced immune function were displayed in a heat map (p<0.05).

[0382]

[0383] 11. In vivo cytotoxicity analysis of Listeria monocytogenes

[0384] Analysis was performed by a conventionally known method. Specifically, Listeria monocytogenes expressing OVA peptide (LM-OVA) was injected into C57 / Bl6 mice at 5000 CFU / mice. The L. plantarum IMB19 strain was administered to the mice every other day. On day 6, spleen cells of naive C57 / bl6 mice were pulsed with OVA peptide, and peptide-pulsed or non-peptide-pulsed cells were stained with CFSE or CTV, mixed at a 1:1 ratio, and administered intravenously to the infected mice. Mice infected with LM-OVA were sacrificed 2 hours later, spleen cells / lymph nodes were harvested, and the cell death rate of peptide-pulsed spleen cells was detected using flow cytometry.

[0385]

[0386] 12. Purification using chromatography

[0387] The isolated total capsular polysaccharide (tCPS 28 mg) was purified by the anion-exchange Q-Sepharose high flow method (GE Healthcare; V = 4.4 mL, flow 16 mL / h). The resin was packed, washed with 1 M NaCl, and equilibrated with 10 volumes of 10 mM NaCl. Subsequently, the total capsular polysaccharide (tCPS) was dissolved in 10 mM NaCl (5 mL) and adsorbed onto the resin. Elution was performed stepwise by sequentially adding 16 mL of NaCl (10, 100, 200, 400, 700, and 1000 mM respectively). The eluates eluted with each concentration of NaCl were collected, desalted by dialysis (Cut-off 1 kDa), and lyophilized. The six fractions eluted from each concentration of NaCl were labeled as CPS-X (X is the NaCl concentration used for elution, mM).

[0388] Molecular weight determination was inferred for CPS-100 using an HPLC system Agilent 1100 with a TSK G-5000PWXL size exclusion column (30 cm × 7.8 mm) equilibrated with 50 mM NH4HCO3 as the eluent (flow = 0.8 mL / min), and the eluate was monitored with a refractive index detector. The column was calibrated by injecting dextran standards (50 μL of a 1 mg / mL solution) of known molecular weights (12, 50, 150, and 670 kDa respectively). The log of the molecular weight was plotted against the elution volume, and the MW of the polysaccharide was calculated using the established linear relationship (linear relationship, LogPM = -0.811 mL + 11.7; R2 = 0.98).

[0389]

[0390] 13. QUANTI-Blue SEAP assay

[0391] HEK293 cells transfected with the inducible SEAP reporter gene and human TLR 2 (InvivoGen) were cultured in 200 ml of DMEM (complete growth medium) supplemented with 10% FBS (fetal bovine serum), 1% penicillin / streptomycin, and 1% L-glutamine (L-glutamine). Specifically, the cells were seeded in a 96-well plate at 70,000 cells per well and cultured in the complete growth medium at 37 °C for 24 hours. The medium was removed from the 96-well plate and replaced with DMEM supplemented with 10% FBS (without 1% penicillin / streptomycin and 1% L-glutamine), and then treated with the appropriate concentration of the compound. Thereafter, the cells were cultured overnight at 37 °C for 18 - 24 hours. To detect SEAP activity, 20 mL of the medium was removed from each well and transferred to a transparent 96-well plate containing 180 ml of QUANTI-Blue reagent (InvivoGen), and cultured at 37 °C in the dark for 30 minutes to 2 hours.

[0392]

[0393] 14. TCR repertoire analysis

[0394] Tumor-infiltrating CD8+ T cells were classified by a MoFlow sorter (BD biosciences) as CD45 + Dump - CD3 + CD8a + (Dump CD19 / CD11c / Ly6c / Ly6G / CD11b) on days 18 - 20 of the experiment. The cells were collected in HEPES / PBS buffer and centrifuged, and the cell pellet was stored at -80 °C until DNA isolation (AbCam). DNA from 4 - 5 mice was pooled into a single sample, and TCR-β repertoire analysis was commissioned (Adaptive Bio). The data was analyzed on the Immunoseq platform provided by Adaptive Bio.

[0395]

[0396] 15. Statistical analysis

[0397] Tumor growth curves were analyzed by two-way ANOVA using Sidak's multiple comparison post-tests for comparisons between two groups, Dunnett's multiple comparison post-tests for comparisons between various groups and the control group, or Tukey's multiple comparison post-tests for comparisons of each of two or more groups. In other comparisons, an unpaired Student's t-test was used when comparing two groups, and Bonferroni correction for multiple testing and one-way ANOVA were used when comparing more than two groups. P < 0.05 was considered statistically significant ( * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). Statistical analysis was performed using GraphPad PRISM v8.0. Flow cytometry analysis data were analyzed using Flow-jo software v10.1.

[0398]

[0399] Example 2: Isolation and identification of L. plantarum IMB19

[0400] Lactobacillus plantarum IMB19 strain KCTC 14337BP was isolated from a homemade kimchi fermented mainly by raw material-derived microorganisms. To induce colonies of Lactobacillus sp., a serially diluted kimchi suspension was streaked on MRS broth (De Man, Rogosa and Sharpe broth, Becton-Dickinson, USA) plates. Single colonies obtained from the culture were isolated and further cultured in MRS broth. Since the colony morphology was not sufficient to distinguish each individual strain, 14 isolated bacteria were analyzed by PCR and 16s rRNA sequence analysis, and the sequence similarity was confirmed using BLAST of NCBI. All of the isolated bacteria were found to belong to lactic acid bacteria (LAB). Among the isolated bacteria, a strain with more than 99% similarity to L. plantarum was identified, and in addition, it was confirmed that many of the isolated bacteria were 99% similar to Weissellia koreensis on agar. Such results are consistent with existing reports that the dominant species of kimchi are L. plantarum and Weissellia koreensis.

[0401] The 16S rRNA sequence information of L. plantarum IMB19 analyzed is as follows.

[0402] -L.plantarum IMB19 16S rRNA(785 Forward)(SEQ ID NO: 3)

[0403]

[0404]

[0405] - L.plantarum IMB19 16S rRNA(907 Forward)(SEQ ID NO: 4)

[0406]

[0407]

[0408] Example 3: Screening of L. plantarum IMB19

[0409] To confirm the immunostimulatory effect of the isolated bacteria on immune cells, the effect of bacterial culture isolates on murine spleen cells was tested, and bacteria with a stimulatory effect on immune cells were identified. After culturing whole spleen cells with the isolated bacteria for 48 hours, the levels of cytokines in the culture supernatant were measured. Using IFN-γ, a well-known inflammatory marker, and IL-10, an anti-inflammatory cytokine, the effect of the isolated bacteria on immune cells was evaluated. Among all the isolated bacteria, a novel Lactobacillus plantarum strain that induced negligible levels of IL-10 and very high amounts of IFN-γ was selected and named L. plantarum IMB19 (Figure 1, colony 1).

[0410]

[0411] Example 4: Microbiological and biochemical characterization of L. plantarum IMB19

[0412] The colony morphology of L. plantarum is small and smooth circular, showing a translucent appearance. Non-flagellated, rod-shaped microbial colonies of L. plantarum were confirmed using cryosection TEM (A in Figure 2). A thick capsule layer of individual bacteria was generally clearly confirmed. When cultured on sheep blood agar, since it is transparent or there is no green light region, the strain was confirmed to be non-hemolytic or γ-hemolytic in a form similar to other Lactobacillus (B in Figure 2). Bacillus cereus ATCC 11778 used as a positive control group showed negative for gelatinase activity up to 5 days (C in Figure 2). Also, the production of four biogenic amines of the strain was tested in a specialized medium (histamine, cadaverine, tyramine, and putrescine). In the case of L. plantarum IMB19, unlike E-coli ATCC 25922, it was confirmed not to produce biogenic amines (Table 1).

[0413]

Table 1

[0414] The antibiotic susceptibility test results, as shown in Table 2 below, were confirmed to be sensitive to most clinically relevant antibiotics except kanamycin, similar to most Lactobacillus species (Appl Environ Microbiol 85, (2019)).

[0415]

Table 2

[0416] *EFSA = European Food Safety Authority, Amp = Ampicillin, Ery = Erythromycin, Gen = Gentamicin, Tet = Tetracycline, Str = Streptomycin, Chl = Chloramphenicol, Cli = Clindamycin, Kan = Kanamycin, Van = Vancomycin, n.r = not required, Q.C = Quality Control.

[0417]

[0418] Example 5: Genetic Characterization of L. plantarum IMB19

[0419] Sequence similarity comparison based on 16S rRNA sequence analysis confirmed the identity among L. plantarum species. In addition, analysis of the recA gene by PCR distinguished it from other genetically closely related species such as L. pentosus and L. paraplantarum (Appl Environ Microbiol 67, 3450 - 3454 (2001)).

[0420] DNA isolation, Pac - Bio & Illumina Hi - seq sequence analysis, and analysis based on bioinformatics were performed (Macrogen, Korea).

[0421]

[0422] L. plantarum IMB19 was differentiated from other genetically closely related species by amplifying the recA gene using primers derived from the recA gene and comparing the bands. Since several L. plantarum strains are known to have similarities in the overall genome, phylogenetic analysis was performed based on the average nucleotide index (OrthoANI) to identify L. plantarum IMB19 as a unique strain (Figure 3). Based on the virulent gene sequence homology to four known strains, the presence of pathogenic genes was confirmed using Virulence Finder 2.0. No significant hits to pathogenic genes were found using a 90% nucleotide cut-off. As confirmed by ResFinder (J Clin Microbiol 52, 1501 - 1510 (2014)), it was confirmed that there were no antibiotic resistance genes in the entire genome of L. plantarum IMB19.

[0423] As disclosed in each of the above examples, L. plantarum IMB19 is a novel strain with characteristics different from previously reported L. plantarum strains and was deposited at the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on October 21, 2020, under the accession number KCTC 14337BP.

[0424]

[0425] Example 6: Immunostimulatory effect of L. plantarum IMB19 on murine T cells

[0426] The beneficial effects of various strains belonging to L. plantarum on host immunity and health have been reported previously (Biomed Res Int 2018, 9361614 (2018)), and the direct effects of L. plantarum IMB19 of the present invention on murine immune cells were confirmed. The present inventors predicted that the uptake of bacterial antigens by antigen-presenting cells (APCs) could alter the activation state, thereby priming other immune cells and consequently enabling the regulation of the immune system. Therefore, a co-culture system was designed that included both APCs and CD4+ T cells, which represent the innate and adaptive immune systems, respectively. CD11c+ APCs were exposed to bacteria for 20 hours (APC:bacteria = 1:100). Such APCs were co-cultured with naive CD4+ T cells under sub-optimal external stimuli without distorting the immune phenotype. To confirm the differentiation of T cells into Th1, Th2, Th17, or regulatory T cells (Tregs), other transcription factors, Tbet, GATA3, RORγ, and Foxp3 were analyzed. Without special external influence, L. plantarum IMB19 significantly induced the generation of CD4+RORγ Th17 cells compared to the other three strains (A in Figure 4). The presence or absence of significant generation of other Th cell subtypes was not clearly defined and appeared in a similar manner to other L. plantarum strains (B in Figure 4).

[0427] To verify the above results, the generation of Th17 cells by L. plantarum IMB19 was tested under the minimal generation conditions of Th17 cells. L. plantarum IMB19 significantly increased the generation of CD4+RORγ Th17 cells that produce a substantial amount of IL-17 (A in Figure 5).

[0428] On the other hand, L. plantarum IMB19 did not induce a significant amount of Foxp3 under neutral conditions (B in Figure 5). Therefore, as a result of confirming whether L. plantarum IMB19 could generate Tregs from immature CD4+ T cells with a sufficient concentration of TGF-β, it was confirmed that the generation of Tregs was significantly suppressed rather under increasing concentrations of TGF-β.

[0429]

[0430] Example 7: Confirmation of the effect of promoting the anti-tumor immune response of L. plantarum IMB19 in vivo

[0431] The L. plantarum IMB19 strain and various Lactobacillus strains (14 species) as a control group were cultured together with spleen cells to compare the change in cytokine production by immune cells. As a result, L. plantarum IMB19 showed a significantly higher IFN-γ level and a significantly lower IL-10 level compared to other Lactobacillus strains (A in Figure 6). For reference, another strain, Lactobacillus murinus, showed the highest IL-10 inducing ability and low IFN-γ production (A in Figure 6).

[0432] Since CD8+ T cells are the main anti-tumor effector cells, the CD8+ T cell stimulating ability of the two isolated strains was confirmed. In the co-culture of dendritic cells (DC) and CD8+ T cells primed with bacteria, an increase in the IFN-γ level was confirmed when treated with L. plantarum IMB19, and in particular, it increased significantly compared to the case when treated with Lactobacillus murinus (B in Figure 1). During co-culture with spleen and mesenteric lymph node antigen-presenting cells, the activation of CD8+ T cells changed depending on the concentration of bacteria (A and B in Figure 7).

[0433] L. plantarum IMB19 did not directly stimulate CD8+ T cells in the absence of dendritic cells. The activation of TCR-bearing CD8+ T cells against the murine melanoma-specific antigen Pmel-1 was activated in a similar manner in the presence of the antigen gp-100 (C in Figure 7). In addition to DCs, macrophages are known as APCs that play an important role in tumor growth and suppression. Therefore, the effect of L. plantarum IMB19 on CD8+ T cell stimulation using CD11b+F4 / 80+ peritoneal macrophages was confirmed. Similar to dendritic cells, L. plantarum IMB19 greatly increased the ratio of IFN-γ cells during co-culture with macrophages-CD8+ T cells (C in Figure 6).

[0434] CD4+Foxp3+ regulatory T cells (Tregs) accumulate in tumors, suppress the effector functions of CD8+ T cells and other immune cells, and increase tumor progression. In addition, various bacteria that promote Treg generation have been reported (Nature 453, 620-625 (2008); Sci Immunol 3, (2018)). Therefore, the present inventors confirmed whether L. plantarum IMB19 affects Treg induction during co-culture of CD11c+ DCs and CD4+ T cells. Under highly Treg skewed culture conditions, L. plantarum IMB19 showed significant suppression of Treg generation in the presence of TGF-β (D in Figure 6). This effect was sustained even at lower levels of TGF-β, and no Treg generation was observed under any of the tested conditions (A in Figure 8). On the other hand, interleukin-17A (IL-17A) increased (B in Figure 8), which means the generation of T-helper-17 cells (Th17). Interleukin-6 (IL-6) is essential for Th17 generation and suppresses Treg generation in the presence of TGF-β (Eur J Immunol 40, 1830-1835 (2010)). Thus, IL-6-deficient DCs generated Tregs in CD4+ T cell co-culture. Therefore, IL-6 production by L. plantarum IMB19-primed DCs suppresses Treg generation under Treg skewed culture conditions.

[0435] To test whether CD8+ T cells activated by L. plantarum IMB19 are functionally cytotoxic, cytotoxicity was tested using an acute Listeria monocytogenes infection model expressing the OVA antigen (LM-OVA) (E in Figure 6). LM-OVA-infected mice administered L. plantarum IMB19 showed a significant increase in the lysis of target cells loaded with OVA pulsed CFSE in vivo (F in Figure 6). Also, the effect of L. plantarum IMB19 on B16.F10 melanoma subcutaneously transplanted in both specific pathogen-free (SPF) and germ-free (GF) mice was evaluated (G in Figure 6). L. plantarum IMB19 showed a significant suppression of melanoma growth in both SPF and GF mice compared to L. murinus (H and I in Figure 6). To evaluate whether L. plantarum IMB19 induces any dysbiosis that leads to its anti-tumor effect, 16s ribosomal RNA sequencing was performed on fecal samples from tumor-bearing animals administered L. plantarum IMB19. However, no significant changes in microbial diversity were observed in the feces of L. plantarum IMB19-fed mice compared to the PBS control group (A-C in Figure 9). This means that the L. plantarum IMB19-mediated regulation of anti-tumor immunity corresponds to the L. plantarum IMB19-specific effect and is not the result of dysbiosis. Collectively, the data in this example indicate that L. plantarum IMB19 is a positive regulator of cytotoxic T cell-mediated anti-tumor immune responses in vivo.

[0436]

[0437] Example 8: Chemical Characterization of the Unpurified Polysaccharide Fraction from L. plantarum IMB19

[0438] As a result of the analysis by transmission electron microscopy (TEM), it was confirmed that the cells of L. plantarum IMB19 were surrounded by a layer of capsular material containing a carbohydrate composition composed of rhamnose, galactose, glucose, and glucosamine, and a small amount of glycerol and ribitol (A in FIGS. 10 and 11). The two polyols are generally associated with the presence of teichoic acids interconnected by phosphodiester bonds (Tomita, Tanaka, & Okada, 2017). Generally, methanolysis cannot completely cleave phosphodiester bonds, so it is very difficult to detect such polyols. Therefore, the sample was dephosphorylated with aqueous HF before methanolysis and acetylation, and GC-MS analysis was repeated, and the presence of teichoic acids was confirmed based on the increase in the amounts of the two polyols. In the case of monosaccharides, rhamnose has an L absolute configuration, and glucose and galactose have a D configuration (FIG. 12). On the other hand, in the case of glucosamine, the D configuration was assumed based on the exclusive presence of stereoisomers.

[0439]

[0440] Example 9: Purification of Unpurified Polysaccharide

[0441] The chemical analysis results of the carbohydrate component suggest that CPS is a mixture of polymers. Therefore, six fractions obtained by purifying CPS using ion exchange chromatography were designated as CPS-X according to the concentration (X) of the eluent used. The yields obtained by purification were as follows: CPS-10 11%, CPS-100 13%, CPS-200 9.0%, CPS-400 51%, CPS-700 7.0%, and CPS-1000 7.9%. Each fraction was 1 compared with the spectral profile of the original mixture (CPS) using 1H NMR analysis (FIG. 13).

[0442] Specifically, each fraction was as follows (FIG. 13).

[0443] CPS-100 (b in Figure 13): CPS-10 showed a somewhat heterogeneous spectrum. The anomeric region of CPS-10 contained two major signals with strong signals at 4.2 and 1.3 ppm associated with substances different from carbohydrates.

[0444] CPS-100 (c in Figure 13): The anomeric region (5.6 - 4.5 ppm) of CPS-100 contained nine major signals with a doublet of doublets at 5.5 ppm (Figure 14) indicating α - array ( 3 J H1,H2 = 3.4 Hz) and phosphorylated ( 3 J H1 ,P = 7.0 Hz) residues. Also, strong signals containing various methyl groups of acetyl groups (methyl group at 2.06 ppm) and dioxy residues (1.3 ppm) consistent with the presence of N - acetylglucosamine and rhamnose units respectively were confirmed.

[0445] CPS-200 (d in Figure 13): CPS-200 was shown as a mixture of CPS-100 and CPS-400, showing four somewhat broad signals in the anomeric region and a strong methyl signal in the congested carbinol region (4.4 - 3.2 ppm) at 1.6 ppm (a value not consistent with the methyl of rhamnose).

[0446] By integration, the ratio between anomeric protons and carbinol protons was shown to be 1.0:12, and the generally expected ratio is 1:6 or less, which means that CPS-400 does not have a typical structure of polysaccharide. The increase in the ratio is due to the presence of ribitol and glycerol units as confirmed by chemical analysis (Figure 15). Also, glucose is the most abundant monosaccharide, and then, as characteristics of peptidoglycan, two monosaccharides of a small amount of glucosamine (GlcN) and muramic acid (MurA) were abundantly found.

[0447] Such observations mean that CPS-400 is teichoic acid (TA).

[0448] CPS-700 and CPS-1000 (f and g in Figure 13): The anomeric region contained only some of the signals found in CPS-400, and the ratio between anomeric positive and carbinol positive was amorphous in both fractions. Signals from non-carbohydrate substances were also further confirmed.

[0449] In CPS, due to the low abundance of CPS-10, CPS-700, and CPS-1000, only small amounts were obtained.

[0450]

[0451] Example 10: NMR Analysis of CPS-100

[0452] The structure of the capsular polysaccharide was determined by analyzing a complete set of 2D NMR spectra of 1H-1H homonuclear (COSY, TOCSY, NOESY) and 1H-13C heteronuclear (HSQC, HMBC, HSQC-TOCSY) recorded at 310 K (Table 3). 1 H- 1 H homonuclear (COSY, TOCSY, NOESY) and 1 H- 13 13C heteronuclear (HSQC, HMBC, HSQC-TOCSY) (Table 3).

[0453] [Table 3]

[0454] * C-3 of C and E overlapped, and their exact chemical shifts were not determined with certainty.

[0455] ** C-5 of C, E, G, and H overlapped, and their exact chemical shifts were not determined with certainty.

[0456] The temperature increase from 297 K (c in Figure 13) to 310 K (Figure 14) decreased the overlap between the three anomeric signals at approximately 5.15 ppm and the simplified NMR attributes of the associated residues.

[0457] The HSQC spectrum (Figure 14) shows nine major anomeric densities at H 5.6 - 4.5, and all corresponding positive peaks have similar ratios. NMR analysis started with H-1 (5.51 ppm) of A, which shows three correlations in the TOCSY spectrum, common with 3.85 ppm in the COSY spectrum (Figure 16). Thus, this density was assigned to H-2, and H-3 (3.91 ppm) and H-4 (4.04 ppm) were also assigned by a similar approach. Since there is no additional correlation with H-1, A was identified as a galactose unit. H-5 was identified in the NOESY spectrum by a strong H-4 / H-5 correlation (Figure 17), and two H-6s were confirmed by the corresponding COZY correlation (Figures 16 and 17). 1 The HSQC and HSQC-TOCSY spectra (Figure 18a) defined all carbon chemical shifts of A (Table 3), an α-galactose unit linked to O-6 based on the high chemical shift of C-6 (67.1 ppm). The TOCSY spectrum correlation occurring at H-1 (5.12 ppm) of D has the same pattern as A, so D is galactose, and α is

[0458] configured based on the J 3 (3.9 Hz) value, and the chemical shifts of H-6s / C-6 (4.04 - 4.02 / 65.6 ppm) are consistent with previous literature data at this position, meaning it is phosphorylated (Sechenkova et al., 2004). Thus, D is 6P-α-Gal and was not further branched based on the high field values of all other carbon chemical shifts. H1,H2 (3.9Hz) value, and the chemical shifts of H-6s / C-6(4.04 - 4.02 / 65.6ppm) are consistent with previous literature data at this position, meaning it is phosphorylated (Sechenkova et al.,2004). Thus, D is 6P-α-Gal and was not further branched based on the high field values of all other carbon chemical shifts.

[0459] In B, H-1 (5.24 ppm) shows the two strongest TOCSY correlation relationships with H-2 (4.25 ppm), which is consistent with the COZY density (Figure 16). As a result of interpreting TOCSY in this second proton (Figure 16), all other protons of the unit containing methyl were confirmed at 1.28 ppm, which is a diagnosis of the entire pattern of rhamnose residues. Therefore, based on the similarity of its C-5 value (about 70.5 ppm) to the reference glycoside (69.4 or 73.6 ppm for α- or β-methylglycoside, respectively, Bock & Pedersen, 1983), B is rhamnose α-configured at the anomeric center and 3-substituted as defined from the glycosylation shift experienced at that carbon. The TOCSY patterns of residues C, E, G, and H (H-1 at 5.14, 5.10, 4.97, 4.88 ppm, respectively) are similar to that of B and are α-rhamnose units. Comparing with the reference value (71.0 ppm, Bock & Pedersen, 1983), the low field value of C-2 (77.8 - 79.6 ppm) means it is substituted at O-2.

[0460] In F, H-1 (5.01 ppm) has four TOCSY correlation relationships (Figure 16), which are due to H-2 - H-5 in the COSY spectrum and are the pattern of a gluco configurated residue. HSQC-TOCSY analysis of H-5 confirmed the correlation with the density at 69.5 ppm, which is due to C-6 and is gradually related to H-6s (4.14 and 3.96 ppm). Therefore, F is N-acetylglucosamine based on the C-2 (54.9 ppm) and H-2 (3.93 ppm) values and is substituted at C-6 (69.4 ppm). The α-configuration is inferred from the anomeric signal shape (broad singlet) and the C-3 value (71.8 ppm), which is very similar to the reference α-glycoside (72.0 ppm, Bock & Pedersen, 1983). Finally, H-1 (4.50 ppm) of I has a TOCSY pattern showing all the protons of the unit. Therefore, 13Based on the shift of C chemical shift, I is substituted at C-3 (82.8 ppm), 3 J H1,H2 and is β-configured glucose based on the coupling constant (7.9 Hz).

[0461]

[0462] The sequence between residues was deduced by analyzing the HMBC (B in Figure 5) and NOESY (Figure 4) spectra. First, the HMBC correlation and intensity of H-1 of B and C-3 of I were linked and denoted as B1I3 (b in Figure 18). Other correlations such as C1E2, D1B3, F1G2, H1A6, and I1F6 were discovered in the same format. Also, H-1 of E and H-1 of G showed long-range correlations with carbons at approximately 79 ppm, which are similar to C-2 of C and H. The exact assignment of such intensities to E1H2 and G1C2 was deduced by analyzing the NOESY spectrum (Figure 17) where H-1 of E and H-2 of H, and H-1 of G and H-2 of C are related. Such attributes were confirmed by observing the inverse correlation shown in detail for the H-2 protons of different rhamnose units (Figure 19).

[0463] Finally, the information that A and D are phosphorylated by phosphodiester bonds at O-1 and O-6, respectively, explains why no HMBC linkage appears between H-1 of A and C-6 of D, or H-6 of D and C-1 of A, or why these protons have no NOE correlation between residues. The HMBC spectrum could not actually detect any correlation between H-1 (or C-1) of A and C-6 (or H-6) of D because the number of couplings between them exceeds the limit (three couplings) of this sequence (five couplings). Similarly, the density of the phosphate moiety between A and D separates the protons of these units far enough to provide a detectable NOE effect. Therefore, the repeating unit structure of CPS-100 is non-saccharide as reported in Figure 20.

[0464] In addition, to understand its characteristics, minor NMR signals were investigated. In the HSQC spectrum (Figure 14), the carbon chemical shifts indicated at Galα and Galβ (H / 1 H / 13 C 5.26 / 93.5 and 4.57 / 97.8 ppm) represent free reducing form α or β residues. The TOCSY spectrum at H-1 (Figure 16) showed a typical pattern of the galacto constituent sugars (i.e., in the case of Galα, the third density almost overlaps with the COZY density). Such findings include ultrasonic treatment and trichloroacetic acid treatment for the separation of CPS. In the above two treatment processes, especially in the anomeric phosphate of galactose A, it can be explained from the point that the extreme flexibility of the phosphodiester bond can induce the cleavage of some of those bonds. Therefore, minor signals (indicated by " * " in Figure 14) near the strong signal were predicted to belong to the first repeating residue and be linked to galactose in the free reducing form. However, their low intensity and the density in the carbinolic region hindered the accurate determination of the attributes. Finally, in the HSQC spectrum (a in Figure 14), when integrating the anomeric density, the average degree of polymerization of the sample is 4, the average MW is about 6 kDa (the MW of the repeating unit is 1500 Da), and the 11 kDa calculated by HPSEC is a value similar to an overestimated value (Figure 21).

[0465]

[0466] Example 11: NMR Analysis of CPS-400

[0467] The structural characteristics of CPS-400 were analyzed in a manner similar to that described in the above example (Table 4).

[0468]

Table 4

[0469] First, the anomeric region of the HSQC spectrum (a and b in Figure 22) shows several residues, 1 The signal at H 5.3 - 5.1 ppm originated from monosaccharide residues, whereas 1 H / 13 C 5.39 / 75.5 ppm is not an anomeric signal but C-2 of the glycerol unit (Gro) denoted as A, which has shifted to a lower field due to acylation. The substituent at O-2 is alanine (Ala), 1 H / 13 confirmed by the methyl confirmed at H / C 1.64 / 16.5 ppm and Hα / Cα at 4.30 / 50.2 ppm. Also, H-1 / C-1 and H-3 / C-3 of A are equivalent and were confirmed at 4.11 / 65.0 ppm based on the correlation relationships of the HSQC-TOCSY (a in Figure 22) and TOCSY (g in Figure 22).

[0470] Finally, the C-1 (or, C-3) value indicates that A is phosphorylated at both ends, as in the case of Gro-type teichoic acids. In the HSQC analysis referring to Gerlach et al., 2018, the second 1,3-diphosphorylated Gro unit (B), and the 1,5-diphosphorylated ribitol unit (Rbo, C) were identified, and such final residues imply the presence of further other teichoic acids based on the ribitol-phosphate backbone. In the monosaccharide unit, the analysis focused on the strongest signals (D, E, F’, and F) identified as α-glucose units based on the efficient propagation of magnetization up to the maximum H-6 in the anomeric signals of the TOCSY spectrum. Such Glc units 13 were not further substituted based on the similarity of C chemical shifts (Bock & Pedersen, 1983).

[0471] The position of such units was inferred from HMBC spectral analysis and comparison with the data in the reference literature. In fact, E was linked to O-2 of the Gro unit (H), whereas (Shashkov, Potekina, Senchenkova, & Kudryashova, 2009), F’ was linked to O-4 of the Rbo unit (I) (Streshinskaya et al., 2011). In the case of D, H-1 has a long-range correlation with the carbon at 78.3 ppm (b in Figure 18) to which the proton (4.30 ppm) is linked to “CH2” at 69.9 ppm in the HSQC-TOCSY spectrum (Figure 23). Such a new unit was designated as G, 1 H / 13 The densities at C 4.30 / 78.3 and 4.14 / 66.9 ppm were assigned to G4 and G5 (c in Figure 18 and Figure 23). G was identified as ribitol, and other signals were searched for by HSQC-TOSCY spectral analysis. In fact, 1 H / 13 The “CH2” density at C 4.16 / 67.7 ppm has three correlations with the signal indicating G4, and this density was designated as G1. Also, C-1 (67.7 ppm) of G represents a phosphorylated carbon that is not glycosylated at the adjacent position as reported for I. Such information allowed the remaining two HSQC-TOSCY correlations to be assigned to C-2 (70.5 ppm) and C-3 (80.6 ppm), and the corresponding H-2 (4.15 ppm) and H-3 (3.97 ppm) were identified in the HSQC spectrum in turn (Figure 18, Figure 23, Table 4). Therefore, G is Rbo glycosylated at O-3 and O-4 together with the two attached units, F and D (HMBC in b of Figure 22).

[0472] Such types of substitutions have been confirmed from other Lactobacillus plantarum strains, but their NMR data reported Rbo units without phosphate, and such chemical shifts cannot be compared with the data of the present invention (Tomita et al., 2017). However, our results were similar to those of teichoic acids from Bifidobacterium strains that proposed an inverted substitution pattern of ribitol with glucose units linked to C-2 and C-3 (Valueva et al., 2013). Interestingly, the NMR data of the dephosphorylated form reported by Valueva et al. (2013) were consistent with the NMR data of 3,4-diglucosylated ribitol reported by Tomita et al. (2009). Therefore, such substitution patterns (2,3 or 3,4) of ribitol units have not yet been clearly defined. Therefore, the NMR data of the present invention indicated that CPS-400 is a mixture of two teichoic acids of the Gro- and rbo-types, each showing the presence of some substituents in a non-stoichiometric manner. In the case of Gro-type TA, the non-stoichiometric substituents were alanine and α-glucose. In the case of Rbo-type TA, α-glucose occurred at both O3 and O4 of ribitol, at O4 alone, or did not occur at either position. Attempts were made to separate the two TAs by size-exclusion chromatography, but CPS-400 appeared as a symmetric peak of about 45 kDa (Figure 21) and was not further separated.

[0473]

[0474] Example 12: Immunostimulatory activity of CPS-100 and CPS-400

[0475] The activities of the said CPS-100 and CPS-400 against immune responses were confirmed.

[0476] Spleen cells with all immune cells mixed at physiological ratios were used to confirm the effect of CPS on the immune system. Endpoint analysis was performed by ELISA to test individual cytokines. Cytokines refer to a group of secreted peptide / glycoprotein that are involved in cell signaling that mediates and regulates inflammatory or tolerogenic immune responses in vivo. Therefore, when exposed to CPS, it means that cytokine distortion in the immune cell pool plays a similar role in vivo. To confirm the immune responses generated by CPS-100 and CPS-400, interferon gamma (IFN-γ) was analyzed as an inflammatory marker and interleukin-10 (IL-10) as a regulatory cytokine. According to the research results, CPS-100 appears to be immunostimulatory, as shown by high IFN-γ and negligible levels of IL-10 production (a and b in Figure 24). On the other hand, in the TA fraction CPS-400, no IFN-γ levels were detected (a in Figure 24). Under similar conditions, other cytokines including TNF-α (tumor necrosis factor-α), IL-6 (interleukin 6), IL-12 (interleukin 12), IL-17 (interleukin 17) and IL1-β (interleukin 1β) were evaluated.

[0477] CPS-100 stimulated the cells and showed a significantly high level of production of TNF-α, IL-6 and IL-12 (c-e in Figure 24), while IL-17 and IL1-β were not detected. On the other hand, for CPS-400, no clear increase was seen in any of the cytokines measured (c and e in Figure 24). Since IFN-γ is a primary immune stimulation marker produced by various types of immune cells, to confirm the specificity of the immune stimulation response of CPS, it was confirmed whether the production of IFN-γ was concentration-dependent on CPS-100. The EC50 (half maximal effective concentration) of CPS-100 in the 48-hour IFN-γ induction was 3.16 μM (f in Figure 24), meaning that CPS-100 can be used as an efficient immunostimulant.

[0478] Consequently, CPS-100 exhibited immune-stimulatory properties similar to those of the L. plantarum IMB19 strain, which implies that the active molecule responsible for the immunopotentiating activity of the L. plantarum IMB19 strain is CPS-100.

[0479]

[0480] Example 13: Enhancement of CD8+ T cell function and anti-tumor immune effect of L. plantarum IMB19 and CPS

[0481] It was confirmed whether the CPS activity as an immunostimulant in vitro could lead to tumor-suppressive activity in vivo. In the orally administered L. plantarum IMB19 and intraperitoneally administered CPS treatment groups, it was confirmed that the growth of subcutaneous melanoma was significantly reduced (A and B in Figure 25). In both groups, the delay in tumor growth was associated with the infiltration of CD8+ T cells (C in Figure 25). The increased production and frequency of IFN-γ by tumor-infiltrating CD8+ T cells imply that the cytotoxic activity of CD8+ T cells was significantly enhanced by the administration (E and F in Figure 25). Also, tumor-infiltrating CD4+ T cells showed upregulation of IFN-γ production in both treatment groups (G and H in Figure 25). On the other hand, no difference in the intratumoral Treg population was observed compared to the PBS administration group (A and B in Figure 26). Oral administration of L. plantarum IMB19 regulated tumor growth in EMT-6 breast cancer (Figure 27). Consequently, the data imply that CPS and L. plantarum IMB19 improve the anti-tumor immune activity that suppresses cancer growth.

[0482]

[0483] Example 14: Increased intratumoral macrophage infiltration by CPS

[0484] CPS increases macrophage infiltration in tumors. To identify specific APC types that regulate the CD8+ T cell response, B16.F10 melanoma cells were transplanted along with intraperitoneal administration of CPS, and APC infiltration in the primary tumors was evaluated. CPS mainly increased the frequency of CD11c+CD11b+ macrophages in the tumors compared to CD11c+ DCs (A in Figure 28). As confirmed by flow cytometry, the number of macrophages appeared significantly higher in CPS-administered mice (A in Figure 28). Under the same conditions, activation markers of CD11c+CD11b+ macrophages and CD11c+ dendritic cells were identified. Surprisingly, the activation state of CPS-treated dendritic cells showed no significant difference from the control group (C in Figure 28). However, the macrophages were further activated, showing higher expression of CD11b, MHC I, MHC II, CD86, and CD40 (B in Figure 28). To confirm the activation of the systemic adaptive immune system in the same mice, CD69, an early activation marker of CD8+ T cells, was identified in the draining lymph nodes. Compared to the control group, CD69 was clearly and significantly upregulated by CPS administration (D in Figure 28). Such data means that CPS plays an important role in macrophage activation and can limit tumor growth.

[0485]

[0486] Example 15: Differentiation of Macrophages into Inflammatory Macrophages and Reprogramming of M2 Macrophages to M1 Phenotype by CPS

[0487] To characterize the effect of CPS on macrophages, phenotypic changes were examined when macrophages were exposed to CPS. Peritoneal CD11b+F4 / 80+ macrophages showed an activated phenotype when treated with CPS, with significant upregulation of MHC I, MHC II, CD68, iNOS2, and CD40 in CPS-treated macrophages compared to those treated with LPS or Pam3CSK4 (E in Figure 28), indicating an M1 phenotype or an inflammatory phenotype of macrophages. In particular, TLR2, similar to Pam3CSK4, was upregulated upon CPS treatment, suggesting that CPS may be a TLR2 ligand. However, in contrast to in vivo experiments, the expression of CD80 and CD86 was not altered (E in Figure 28).

[0488] As an alternative, activated macrophages or M2 phenotype macrophages greatly contribute to immunosuppression and improve tumor growth (Ann Oncol 28, xii18 - xii32 (2017)) (Front Oncol 9, 421 (2019)). Therefore, the above results indicate that CPS may reprogram M2 macrophages into the M1 phenotype in tumors. Indeed, IL-4-induced M2 phenotype peritoneal macrophages were significantly increased when treated with CPS compared to LPS and Pam3CSK4 (F in Figure 28). Also, the inflammatory macrophage marker iNOS2 was significantly upregulated separately from MHC I, MHC II, CD40, and CD68 (F in Figure 28). Such data imply that treatment with CPS generates inflammatory macrophages and reprograms immunosuppressive macrophages into an immunostimulatory phenotype.

[0489]

[0490] Example 16: Confirmation of the gene expression profile of CPS-modulated tumor-infiltrating macrophages

[0491] Macrophages that were initially infiltrated in the tumors of CPS- and PBS-administered mice were isolated, and mRNA sequencing was performed for gene expression profiling. Various genes related to cellular chemotaxis, inflammatory response, and iron ion homeostasis (Figure 29A) were upregulated in CPS-group macrophages. Iron regulation in the body is one of the main physiological roles mainly performed by macrophages to maintain iron circulation and solubility. A significant upregulation of genes involved in intracellular iron ion sequestration from macrophages in the tumor microenvironment was confirmed (Figure 29B). Also, Tlr2 and Tlr6 were significantly upregulated, but the gene expression level of Tlr6 was lower than that of Tlr2 (Figure 29C). To support the effect of CPS on macrophages, as a result of confirming the upregulation of genes showing M1 or M2 phenotypes, a significant upregulation of M1 inflammatory genes and a significant downregulation of M2 genes were confirmed (Figure 29D). Therefore, it means that Tlr2 and iron sequestration play important roles in the regulation of the inflammatory macrophage phenotype by CPS.

[0492]

[0493] Example 17: TLR2-Mediated Stimulation of CD8+ T-Cells by CPS

[0494] To confirm the role of TLRs in CPS-mediated CD8+ T-cell stimulation, first, the DC-specific MyD88 regulatory effect on CD8+ T-cell activation was confirmed. In a co-culture experiment using MyD88-deficient dendritic cells and CD8+ T cells derived from wild-type mice, a significant decrease in the proportion of IFN-γ cells was observed (A in Figure 30). Next, the roles of TLR2, TLR4, and TLR6 were confirmed under similar conditions, and it was confirmed that dendritic cells with TLR2 knockdown in CPS- or L. plantarum IMB19-treated cells significantly suppressed the activation of CD8+ T cells (E in Figure 29 and B in Figure 30). This is consistent with the RNA sequencing data showing upregulation of TLR2 (C in Figure 29), indicating that TLR2 plays an important role in CPS-mediated priming of CD8+ T cells.

[0495]

[0496] Example 18: Iron sequestration by CPS in tumor-associated macrophages

[0497] To confirm the role of iron sequestration by macrophages in relation to anti-tumor immunity, tumor-associated macrophages were isolated from melanoma tumor-bearing mice, and the total intracellular soluble iron (Fe++) and iron uptake by such macrophages were evaluated. Total immune cells isolated from tumors were stored in iron-supplemented medium for 30 minutes, and Fe++ ions were directly stained and analyzed through flow cytometry. Macrophages isolated from CPS-administered mice showed high intracellular labile iron levels (F in Figure 29). Also, iron uptake by macrophages isolated from CPS-administered mice appeared significantly higher (G in Figure 29). The intracellular iron level per cell appeared even higher in the CPS-treated group as reflected by MFI (F and G in Figure 29). Therefore, iron sequestration in CPS-primed macrophages can be an important mechanism for suppressing tumor growth.

[0498]

[0499] Example 19: Regulation of the CD8+ T cell TCR repertoire in tumors of CPS or L. plantarum IMB19

[0500] It was confirmed whether CPS or L. plantarum IMB19 caused the TCR repertoire of tumor-infiltrating CD8+ T-cells associated with an increase in cytotoxic response. Compared with the PBS group (D in Figure 31), it was confirmed that specific TCRs appeared abundantly during CPS or L. plantarum IMB19 treatment (Figure S14A). The Simpson’s diversity index decreased during L. plantarum IMB19 and CPS treatment, showing clonal expansion of a few selected clones compared with PBS, which showed more polyclonal responses (B in Figure 31).

[0501] Pielou evenness or Shannon equitability indicates an increase in the abundance of specific TCRs during CPS treatment compared with PBS, which shows the uniform abundance of all TCRs (C in Figure 31). Therefore, both CPS and L. plantarum IMB19 affect the TCR repertoire in the tumor microenvironment, including the expression of some specific TCRs, clearly showing a phenotypic increase in specific TCR sequences.

[0502]

[0503] Example 20: Combination administration therapy of CPS or L. plantarum IMB19 with anticancer agents

[0504] As confirmed in each of the above examples, L. plantarum IMB19 and the polysaccharide demonstrated significant suppression of tumor progression in the B16F10 mouse melanoma model, and experiments were conducted through combination administration in the same method to confirm the effect of the combination of L. plantarum IMB19 and the immune anticancer agent on tumors.

[0505] The syngeneic mouse melanoma model was generated by subcutaneous administration of 0.2 million B16F10 cells / mouse. L. plantarum IMB19 (1x10 9 CFU / mouse) was orally administered from day 0, and the anti-PD-L1 (100 μg i.p.) antibody was treated on days 7, 10, 13, and 16 (A in Figure 32). The tumor growth rate was significantly lower in the group co-administered with the L. plantarum IMB19 strain compared to the anti-PD-L1 antibody alone (B in Figure 32).

[0506] The synergistic effect was evaluated in a mouse renal cancer model using a treatment therapy against L. plantarum IMB19 (C in Figure 32). Renal cell carcinoma (RCC) is a cancer with an increasing incidence of 400,000 worldwide, and 40,000 Renca-luciferase cells were surgically transplanted into the renal capsule of the right kidney of the mouse. Mice were randomly selected and treatment was started on day 7 (C in Figure 32).

[0507] Bioluminescence imaging (BLI) was acquired using an IVIS Spectrum (Ami-HTX, Spectral Instruments Imaging, USA) at other time points to confirm tumor progression.

[0508] To normalize the bioluminescence intensity at all time points, the scale was manually set based on the analysis on the last day. A significant synergistic effect between L. plantarum IMB19 and anti-PD-L1 was observed in the longitudinal evaluation of tumor progression (D - E in Figure 32). The above results mean that L. plantarum IMB19 can significantly enhance the anti-tumor effect of anti-PD-L1 in both treatment and semi-treatment therapies.

[0509]

[0510]

Deposit Number

[0511] Name of depository institution: Korea Research Institute of Bioscience and Biotechnology

[0512] Accession No.: KCTC 14337BP

[0513] Date of deposit: 20201021

[0514]

Industrial Applicability

[0515] The novel Lactobacillus plantarum IMB19 strain KCTC 14337BP of the present invention and the polysaccharide derived from said strain exhibit excellent ability to stimulate CD8+ T cell activity and inhibitory activity of Treg cells, and stimulate and enhance the anti-tumor immune response through various mechanisms such as increased macrophage infiltration in CPS tumors and differentiation / reprogramming of macrophages into an inflammatory (M1) phenotype.

[0516] In particular, when the said strain and polysaccharide are co-administered with an anti-cancer agent, especially an immune anti-cancer agent such as a PD-1 or PD-L1 inhibitor (for example, a PD-1 antibody or a PD-L1 antibody), the anti-tumor immune response can be significantly increased. Therefore, by co-administering the composition containing the strain and / or polysaccharide of the present invention with various anti-cancer agents, it can be usefully used for the prevention, improvement or treatment of tumors.

[0517]

[0518] As described above, specific parts of the content of the present invention have been described in detail. However, it will be apparent to those of ordinary skill in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, it can be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0519]

Sequence Listing Free-Text

[0520] An electronic file is attached.

Claims

1. A composition for preventing or treating a tumor, comprising Lactobacillus plantarum IMB19 strain KCTC 14337BP and administered in combination with an anti-cancer agent, wherein the anti-cancer agent is an immune checkpoint inhibitor that inhibits PD-1 or PD-L1.

2. The composition according to claim 1, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.

3. The tumor according to claim 1, wherein the tumor is selected from the group consisting of histiocytoma, glioma, astrocytoma, osteoma, lung cancer, small cell lung cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, skin cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, brain cancer, sarcoma, osteosarcoma, melanoma, lymphoma, blood cancer, and leukemia.

4. The composition according to claim 1, wherein the composition is a pharmaceutical composition or a food composition.

5. A composition for preventing or treating a tumor, comprising a polysaccharide represented by the following Chemical Formula II and administered in combination with an anti-cancer agent, wherein the anti-cancer agent is an immune checkpoint inhibitor that inhibits PD-1 or PD-L1: [Chemical Formula II] n is an integer from 1 to 10.

6. The composition according to claim 5, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.

7. The tumor according to claim 5, wherein the tumor is selected from the group consisting of histiocytoma, glioma, astrocytoma, osteoma, lung cancer, small cell lung cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, skin cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, brain cancer, sarcoma, osteosarcoma, melanoma, lymphoma, blood cancer, and leukemia.

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