Novel lactobacillus plantarum strains, polysaccharides derived from the strains and uses thereof

Abstract

The present invention relates to a novel Lactobacillus plantarum IMB19 strain, polysaccharides derived from the strain and uses thereof. The novel Lactobacillus plantarum IMB19 strain and polysaccharides derived from the strain of the present invention exhibit superior CD8+ T cell activity stimulating capacity and Treg cell inhibitory activity and stimulate and improve anti-tumor immune responses through various mechanisms such as increasing macrophage infiltration in CPS tumors as well as macrophage differentiation and reprogramming into inflammatory phenotype (M1). Thus, the strain and polysaccharides derived from the strain of the present invention can be effectively used for immunomodulation, especially immune boosting, in a subject and can inhibit tumor growth by inducing and enhancing anti-tumor immune responses. The novel strain and polysaccharides derived from the strain of the present invention are useful for preventing, ameliorating or treating various immunological diseases such as tumors, infectious diseases and various immunological diseases caused by or as symptoms of immune dysfunction.

Description

TECHNICAL FIELD

[0001] The present invention relates to a novel Lactobacillus plantarum IMB19 strain, polysaccharides derived from the strain and uses thereof, and more particularly to a Lactobacillus plantarum IMB19 strain having the accession number KCTC 14337BP and having immunostimulatory and anti-tumor activities, polysaccharides derived from the strain and uses thereof for immunomodulation, tumor suppression and treatment of infectious diseases. BACKGROUND

[0002] Mammals have a microbiota that constantly interacts with the immune system. Commensal microorganisms enter into a symbiotic relationship with the host and interact with the host in various processes such as digestion, behavior 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.). Likewise, fungi are present in the human body and influence the host's immune system (Wheeler M.L., Limon J.J., Underhill D.M. Immunity to Commensal Fungi: Detente and Disease. Annu. Rev. Pathol. 2017; 12: 359-85.). Innate immune cells detect a variety of pathogen-associated molecular patterns (PAMPs), including polysaccharides, on the cell surface of fungi through pattern recognition receptors (PRRs) such as toll-like receptors (TLRs). Upon detection of the signal, innate immune cells produce immune signaling molecules such as cytokines in order to change the gene expression profile and control adaptive immunity (Iliev I.D., Leonardi I. Nat. Rev. Immunol. 2017; 17(10): 635-46.; Underhill D.M., Iliev I.D., Nat. Rev. Immunol. 2014; 14(6): 405-16.; and Brubaker S.W., Bonham K.S., Zanoni I. et al. Annu. Rev. Immunol. 2015; 33: 257-90.).

[0003] WHO defines probiotics as live microorganisms that, when administered in adequate amounts, have health benefits for the host (Nat. Rev. Gastroenterol Hepatol 11, 506-514 (2014)). Probiotics act as supplements to the host's intestinal microbiota and have been reported to be effective in improving intestinal barrier function and modulating 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 low "G+C" content and are mainly classified as the genera Bacillus and Clostridium. Lactobacillus species are lactic acid bacteria (LAB) belonging to the family Lactobacillaceae. Lactobacillus is a well-known microorganism with different ecological states. Several types of LAB are known to be traditionally associated with foods such as milk, dairy products, fermented foods, and sausages. Lactobacillus is a group of microorganisms recognized by the FDA as GRAS (Generally Regarded as Safe) and is widely used in the food and other industries. Lactobacillus is classified as a conditional anaerobe, non-spore forming, non-motile, rod-shaped, and gram-positive bacterium and is generally considered to be 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 forms smooth and convex colonies.

[0004] Due to similar biochemical and morphological characteristics in LAB, a molecular identification method is required to identify individual strains. Several types of lactic acid bacteria have been isolated and characterized from foods, particularly fermented foods. Fermentation generally refers to a biochemical change caused by microorganisms. Kimchi, a traditional Korean food, is mainly fermented cabbage and has beneficial effects on nutrition and health (Crit. Rev. Food Sci. Nutr. 34, 175-203 (1994)). It is known that kimchi contains a unique microbial community. According to some reports, it is known that lactic acid bacteria are mainly present in kimchi, and Weissella, Lactobacillus, and Leuconostoc are dominant species, and in particular, Lactobacillus plantarum is considered the most dominant strain (Food Sci. Biotechnol. 19, 641-646 (2010)). Lactobacillus plantarum is one of the most studied strains due to strain-specific probiotic profiles and technical applications in the food industry. Most of the kimchi microorganisms can be cultured (Int. J. Food Microbiol. 102, 143-150 (2005)), and isolation of individual microorganisms is essential to study their health benefits.

[0005] Meanwhile, the growth and proliferation of cancer are strictly regulated by the host immune response. For the growth and proliferation of tumor cells, it must be possible to escape the surveillance of the immune system, which induces the early death of tumor cells. Tumor cells grow and proliferate by forming an immunosuppressive tumor microenvironment through various pathways such as cytokine secretion, expression of molecules on the cell surface, etc. As a cancer treatment strategy targeting this immune escape mechanism, various efforts have been made to induce or enhance the immune system. Tumor immunotherapy is known as a treatment method for restoring or enhancing the ability of the immune system to recognize or destroy tumors to overcome the immunosuppression or escape mechanisms acquired by tumors. In 2011, the ipilimumab immunotherapy drug was used to successfully treat patients with malignant melanoma. Since then, various immunotherapeutic agents such as nivolumab and pembrolizumab have been continuously developed.

[0006] As a tool for such tumor immunotherapy, there are increasing reports and cases about the importance of gut microbiota and its application. Gut microbiota plays an important role in forming local and systemic immune responses of the host (Science 330, 1768-1773 (2010), Cell 148, 1258-1270 (2012)). The diversity and composition of gut microbiota have also been shown to affect the responsiveness to chemotherapy (Cancer Immunol. Immunother. 55, 1470-1479 (2006); Science 342, 971-976 (2013)). In particular, it has been found that certain symbiotic microorganisms are associated with the activation of spontaneous anti-tumor immunity, and have been found to exhibit synergistic effects on the therapeutic efficacy of immunotherapeutic agents in experiments (Science 350, 1084-1089 (2015); Science 350, 1079-1084 (2015)) as well as human cancers (Science 359, 91-97 (2018); Nature 453, 620-625 (2008)). Therefore, it is increasingly clear that certain strains (such as gut microbiota) can also affect the progression of tumors outside the mucosa and distant tumors. Based on these various reports, changes in gut microbiota are considered to be an effective and feasible clinical treatment option. Recent studies have confirmed the comprehensive effects of specific strains or strain communities on the host immune system or anti-tumor immunity from the perspective of probiotics, but little is known about the mechanisms, such as active ingredients derived from specific strains that show these effects and their signaling pathways.

[0007] In particular, as a consideration for treating patients using such microorganisms or their metabolites, some patients must suppress an excessively active immune response (i.e., allergy or autoimmune disease), and some patients must enhance the immune system (i.e., cancer or viral infection). For example, when Bifidobacterium, which is a Th17-inducing probiotic, was administered to an animal model of rheumatoid arthritis, the symptoms of arthritis were aggravated (Tze Guan Tan, 113(50): E8141-E8150, 2016). Therefore, it is very important in therapy to identify beneficial microorganisms and elucidate the mechanisms of their action factors.

[0008] In light of this technical background, the present inventors made great efforts to elucidate the clear correlation and mechanism between the components, structure, molecular weight, etc. of the polysaccharide of microbial origin and its immunomodulatory activity, and identified a novel Lactobacillus plantarum strain having a significantly high immune-enhancing activity from kimchi (a Korean traditional food) and deposited with the accession number KCTC 14337BP at the Korean Biological Resource Center. In addition, it has been confirmed that when a host is fed with the novel strain Lactobacillus plantarum KCTC 14337BP, the number of effector T cells is significantly increased and Treg production is inhibited, thereby stimulating the immune system in the host and inhibiting tumor growth and proliferation.

[0009] In addition, the present inventors not only identified and characterized the novel strain, but also determined that the capsular polysaccharide of the novel strain and a fraction thereof having a specific structure (CPS-100) are effective molecules exhibiting immune stimulation and enhancement effects, and also determined that by stimulating an anti-tumor immune response through various mechanisms such as activation of CD8+ T cell function, increase in macrophage infiltration in tumors by CPS, and differentiation / reprogramming of macrophages into an inflammatory phenotype, tumor growth can be greatly inhibited, thereby completing the present invention.

[0010] The information described in the background art section is merely for improving the understanding of the background of the present invention, and it should not be construed as including information forming the related art known to those skilled in the art to which the present invention pertains. SUMMARY

[0011] An object of the present invention is to provide a novel strain having an immune stimulation activity.

[0012] Another object of the present invention is to provide a polysaccharide derived from the strain having an immune stimulation activity.

[0013] Still another object of the present invention is to provide the use of the strain and the polysaccharide derived from the strain for immunomodulation and / or prevention, amelioration or treatment of a tumor or an infectious disease.

[0014] To achieve the above object, the present invention provides a Lactobacillus plantarum IMB19 strain having the accession number KCTC 14337BP.

[0015] In addition, the present invention provides a capsular polysaccharide derived from the strain having an immune-enhancing activity.

[0016] In addition, the present invention provides a method of producing a capsular polysaccharide having an immune-enhancing activity, which comprises culturing the strain and obtaining a capsular polysaccharide from the cultured strain.

[0017] In addition, the present invention provides a polysaccharide represented by the following formula I:

[0018] [Formula I]

[0019] -[-D-B-I-F-G-C-E-H-A-] n -

[0020] In Formula I,

[0021] A and D are galactose,

[0022] B, C, E, G and H are rhamnose,

[0023] F is N-acetylglucosamine,

[0024] I is glucose, and

[0025] n is an integer from 1 to 10.

[0026] Further, the present application provides a composition for immunomodulation containing the strain and / or the polysaccharide as an active ingredient.

[0027] Further, the present application provides a pharmaceutical composition for preventing or treating a tumor or an infectious disease containing the strain and / or the polysaccharide as an active ingredient.

[0028] Further, the present application provides a method for preventing, ameliorating or treating a tumor or an infectious disease, which comprises administering the strain and / or the polysaccharide to a subject.

[0029] Further, the present application provides the use of the strain and / or the polysaccharide for immunomodulation and / or for preventing, ameliorating or treating a tumor or an infectious disease.

[0030] Further, the present application provides the use of the strain and / or the polysaccharide for manufacturing a composition for immunomodulation.

[0031] Further, the present application provides the use of the strain and / or the polysaccharide for manufacturing a pharmaceutical composition for preventing or treating a tumor or an infectious disease.

[0032] Further, the present application provides a method for producing inflammatory T cells, which comprises priming antigen presenting cells with the Lactobacillus plantarum IMB19 strain and / or the polysaccharide having Formula I, and co-culturing the primed antigen presenting cells with T cells.

[0033] Further, the present application provides a method for producing M1 phenotype macrophages, which comprises differentiating macrophages into M1 phenotype macrophages by treating the macrophages with the Lactobacillus plantarum IMB19 strain and / or the polysaccharide having Formula I, and obtaining the differentiated M1 phenotype macrophages.

[0034] Further, the present application provides a cell therapeutic agent for preventing or treating a tumor or an infectious disease, which contains the inflammatory T cells produced by the above method and / or the Ml phenotype macrophages produced by the above method as an active ingredient. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 shows the levels of inflammatory or anti-inflammatory cytokines in splenocytes after treatment with strains derived from kimchi,

[0036] wherein a stepwise dilution of kimchi suspension was streaked on MRS-agar plates and incubated at 37°C for 48 hours, after which strains were obtained from 14 colonies and further incubated in MRS broth for 24 hours, and splenocytes were mixed with each strain at a ratio of 1:10 and incubated at 37°C and 5% CO2 for 48 hours, activation of splenocytes was evaluated by measuring cytokine levels in culture supernatants via ELISA, data are mean ± SEM values (n=2), and statistical significance to mock was calculated by one-way ANOVA, *p<0.5, **p<0.01, ***p<0.001;

[0037] Figure 2 shows the microbial and biological characteristics of Lactobacillus plantarum IMB19 strain,

[0038] Figure 2 A shows TEM images of Lactobacillus plantarum IMB19 strain, arrows indicate the capsule layer around the cell wall,

[0039] Figure 2 B shows hemolytic activity of Lactobacillus plantarum IMB19 strain after incubation in 5% sheep blood agar for 48 hours, positive control is Bacillus cereus ATCC 27348,

[0040] Figure 2 C shows gelatin hydrolysis test on Lactobacillus plantarum IMB19 strain, positive control is Bacillus cereus ATCC 11778;

[0041] Figure 3 shows a phylogenetic tree graph of Lactobacillus plantarum IMB19,

[0042] wherein a phylogenetic tree was constructed based on differences between Lactobacillus plantarum strains, average nucleotide identity (ANI) was calculated using OrthANI algorithm, and distance is directly proportional to differences in genomes;

[0043] Figure 4 shows results confirming the generation of Th 17 cells using various Lactobacillus plantarum strains,

[0044] wherein flow cytometry data showing the effect of L. plantarum IMB19, L. plantarum 3105, L. plantarum 3106 and L. plantarum 3107 on naive CD4+T cells is presented and dendritic cells primed with L. plantarum IMB19 were cultured with naive CD4+T cells, anti-CD3 (10 ng / ml) and IL-2 (100 / ml),

[0045] Figure 4 Ai shows FACS plots depicting Th17 cell production, wherein the cells were gated with live CD4+RORy+T cells,

[0046] Figure 4 Aii shows a bar graph depicting the mean cell number of Th17 cells gated with live CD4+RORy+T cells, Figure 4 B shows Th1 cells gated with live CD4+T-Bet+T cells,

[0047] Figure 4 C shows Th2 cells gated with live CD4+GATA3+T cells,

[0048] Figure 4 D shows Tregs gated with live CD4+Foxp3+regulatory T cells,

[0049] Data are presented as mean ± SD and statistical significance to mock was calculated by one-way ANOVA, **P

[0050] <0.01;

[0051] Figure 5 shows the ability of L. plantarum IMB19 to inhibit Treg cells and induce Th17 cells,

[0052] Figure 5 Ai shows representative FACS plots and bar graphs showing the effect of L. plantarum IMB19 on Th17 cell production, wherein dendritic cells primed with L. plantarum IMB19 were co-cultured with naive CD4+T cells, anti-CD3 (0.1 μg / ml), TGF-β (0.5 ng / ml), IL-6 (2 ng / ml), IL1-β (2 ng / ml), IL-2 (100 U / ml), anti-IL4 (10 μg / ml) and anti-IFNy (10 μg / ml), the cells were gated with live CD4+RORy+T cells,

[0053] Figure 5Figure A ii shows a representative FACS plot and bar chart, which illustrates the level of interleukin-17 produced by *Lactobacillus plantarum* IMB19 in Th17 cells gated with live CD4+RORγ+IL-17+ T cells.

[0054] Figure 5 B shows representative FACS plots and bar graphs, illustrating the inhibitory effect of *Lactobacillus plantarum* IMB19 on Treg cells in vitro. Dendritic cells induced by *Lactobacillus plantarum* IMB19 were co-cultured with naive CD4+ T cells, anti-CD3 (0.1 μg / ml), IL-2 (100 U / ml), and different concentrations of TGF-β. Data are presented as mean ± SEM, and statistical significance was analyzed by general one-way ANOVA. **p < 0.01, ***p < 0.001;

[0055] Figure 6 This demonstrates an anti-tumor immune response achieved through CD8+ T cell activation enhanced by Lactobacillus plantarum IMB19. Figure 6 A shows the results of ELISA analysis of cytokine levels in the supernatant of spleen cell-bacterial co-culture.

[0056] Figure 6 B shows the quantitative results of IFNγ+CD8+ T cells from a co-culture of spleen CD11c+ dendritic cells and naive CD8+ T cells induced by Lactobacillus plantarum IMB19 or Lactobacillus murineis.

[0057] Figure 6 C shows the quantitative results of IFNγ+CD8+ T cells from a co-culture of CD11b+F4 / 80+ peritoneal macrophages and naive CD8+ T cells induced by Lactobacillus plantarum IMB19.

[0058] Figure 6 D shows the quantitative results of Foxp3+CD4+ T cells from a co-culture of spleen CD11c+ dendritic cells, naive CD4+ T cells, and 2 ng / ml TGF-β induced by Lactobacillus plantarum IMB19.

[0059] Data are presented as mean ± SEM values. B was analyzed using a standard one-way ANOVA and Tukey multiple comparisons. **p < 0.01, ****p < 0.0001.

[0060] Figure 6 E schematically illustrates an in vivo assay for the cytotoxicity of Listeria monocytogenes (LM-OVA) expressed with OVA.

[0061] Figure 6 F shows CD8+ T cell specific cytotoxicity against OVA+ splenocytes mediated by L. plantarum IMB19 in LM-OVA infected mice,

[0062] Figure 6 G schematically shows a B16.F10 melanoma mouse model in vivo,

[0063] Figure 6 H shows B16.F10 melanoma growth kinetics in C57 / BI6 germ-free mice treated or not with L. plantarum IMB19 or L. murinus,

[0064] Figure 6 I shows B16.F10 melanoma growth kinetics in C57 / BI6 SPF mice treated or not with L. plantarum IMB19 or L. murinus,

[0065] Data are mean ± SEM values, Figure 6 F shows results of non-parametric two-tailed t-test, and Figure 6 H and Figure 6 I shows results of two-way ANOVA and Dunnett’s multiple comparisons, **P<0.01, ***P<0.001, ****P<0.0001;

[0066] Figure 7 shows dose-dependent effect of L. plantarum IMB19 on CD8+ T cells,

[0067] Figure 7 A and Figure 7 B shows quantification of IFNy+ CD8+ T cells in co-cultures of splenic (7A) or mLN (7B) CD11c+ dendritic cells primed with L. plantarum IMB19 at different ratios with naive CD8+ T cells,

[0068] Figure 7 C shows quantification of IFNy+ CD8+ T cells in co-cultures of splenic CD11c+ dendritic cells primed with L. plantarum IMB19 at different ratios with naive pmel TCR vaccinated CD8+ T cells in the presence of 100 ng / ml gp-100, data are presented as mean ± SEM and analyzed by typical one-way ANOVA with Tukey’s multiple comparisons, **p<0.01, ***p<0.001, ****p<0.0001;

[0069] Figure 8 shows inhibition of regulatory T cells growth by interleukin-6 production by L. plantarum IMB19,

[0070] Figure 8 A shows the quantitative results of Foxp3+CD4+ T cells in co-cultures of spleen CD11c+ dendritic cells and naive CD4+ T cells induced by Lactobacillus plantarum IMB19 at different TGF-β concentrations.

[0071] Figure 8 B shows the quantitative results of cytokines in a co-culture of spleen CD11c+ dendritic cells and naive CD4+ T cells induced by *Lactobacillus plantarum* IMB19 at a TGF-β concentration of 2 ng / ml, wherein the cells were gated with total CD4+ T cells.

[0072] Figure 8 C shows the quantitative results of Foxp3+CD4+ T cells in IL-6- / - spleen DCs and in co-cultures of spleen CD11c+ dendritic cells and naive CD4+ T cells induced by Lactobacillus plantarum IMB19 at a TGFβ concentration of 0.01 ng / ml. Data are presented as mean ± SEM and analyzed by typical one-way ANOVA and Tukey multiple comparison analysis. ***p < 0.001, ns: not significant;

[0073] Figure 9 This study demonstrated the role of *Lactobacillus plantarum* IMB19 in tumor immunity, while microbial diversity remained unchanged.

[0074] Figure 9 A shows in Figure 6 The results (%) of phylogenetic analysis of phyla in fecal samples of tumor-bearing mice fed with Lactobacillus plantarum IMB19 in H.

[0075] Figure 9 B and Figure 9 C shows that Figure 6 Results of principal coordinate analysis of α diversity (B) and bacterial β diversity in fecal samples from tumor-bearing mice fed with Lactobacillus plantarum IMB19 in H, with data indicating values ​​from two independent experiments;

[0076] Figure 10 The image shown is a transmission electron microscope (TEM) image of Lactobacillus plantarum IMB19.

[0077] Figure 10 a shows the bacterial community of *Lactobacillus plantarum* IMB19, with arrows indicating individual bacteria.

[0078] Figure 10 b shows Lactobacillus plantarum IMB19, with the arrow indicating the thick capsule layer surrounding the cell wall;

[0079] Figure 11GC-MS spectra of acetylated methyl glycosides derived from CPS extracted from Lactobacillus plantarum IMB19, where samples were dephosphorylated without (a) or with (b) HF treatment, "i" indicates impurities;

[0080] Figure 12 GC-MS spectra of acetylated 2-(-)-octyl derivatives of CPS (a), rhamnose (b), glucose (c) and galactose acetylated 2-(-)-octyl glycoside standards (d) extracted from Lactobacillus plantarum IMB19, "i" indicates impurities;

[0081] Figure 13 Proton spectra (600 MHz, 298 K) of fractions obtained by purification of crude CPS via ion-exchange chromatography are shown: CPS (a), CPS-10 (b), CPS-100 (c), CPS-200 (d), CPS-400 (e), CPS-700 (f) and CPS 7-1000 (g), the number in brackets indicates the yield (mg / mg) of each fraction compared to 28 mg of crude CPS;

[0082] Figure 14 Extensions of HSQC spectra recorded for the capsular polysaccharide of Lactobacillus plantarum IMB19 (600 MHz, 310 K) are shown: (a) extension of the anomer region, (b) extension of the methanol region, grey density corresponds to "CH2" carbons and "*" indicates minor anomer signals of repeat unit sugars linked to galactose in reduced form (Gal a and Gal b), for the structure of the repeat unit, refer to Figure 20 and for the labels, refer to Table 3;

[0083] Figure 15 GC-MS spectra of acetylated methyl glycosides of CPS-400 extracted from Lactobacillus plantarum IMB19 after dephosphorylation of the sample by HF treatment are shown, "i" indicates impurities and MurA indicates a muramic acid, which is a component of the bacterial peptidoglycan;

[0084] Figure 16 Extensions of TOCSU (black) and COZY (cyan / red) spectra recorded for the capsular polysaccharide CPS-100 of Lactobacillus plantarum IMB19 (600 MHz, 310 K) are shown, for the structure of the repeat unit, refer to Figure 20 and for the labels, refer to Table 3;

[0085] Figure 17 Extensions of NOESY (black) and COZY (cyan / red) spectra recorded for the capsular polysaccharide CPS-100 of Lactobacillus plantarum IMB19 (600 MHz, 310 K) are shown, for the structure of the repeat unit, refer toFigure 20 and for the labels, refer to Table 3;

[0086] Figure 18 An expansion of the HSQC-TOCSY (a) and HMBC (b) NMR spectra recorded for the capsular polysaccharide of Lactobacillus plantarum IMB19 (600 MHz, 310 K) is shown, for the structure of the repeating unit, refer to Figure 20 and for the labels, refer to Table 3;

[0087] Figure 19 An expansion of the H-2 proton region of the NESY (black) and COZY (cyan / red) spectra of residues B-G as part of the capsular polysaccharide CPS-100 of Lactobacillus plantarum IMB19 (600 MHz, 310 K) is shown, for the structure of the repeating unit, refer to Figure 20 and for the labels, refer to Table 3;

[0088] Figure 20 The structure of the repeating unit of the capsular polysaccharide CPS-100 of Lactobacillus plantarum IMB19 is shown, where the number 4 indicates the calculated average degree of polymerization, the phosphate group (P) attached to the oxygen of the 6' carbon residue of the substituent D is connected to the 1'carbon of A indicating the phosphodiester linkage of A and D between the repeating units of the polymeric polysaccharide;

[0089] Figure 21 HPSEC chromatograms of the individual injections of solvent (a), CPS-100 (b) and CPS-400 (c) are shown, as seen in the chromatogram of the individual injection of solvent (a), the peaks at 13.6 min and 14.74 min are solvent-induced artefacts;

[0090] Figure 22 NMR spectra recorded for CPS-400 (600 MHz, 310 K) are shown: (a) overlay of HSQC-TOCSY (black) and HSQC (cyan), (b) overlay of HMBC (black) and HSQC (cyan), (c) HSQC, (d) HSQC-TOCSY, and (e) to (h) TOCSY spectra of different regions, “*” indicates density belonging to an unidentified small motif, and the depiction of the structural units is shown in Table 4;

[0091] Figure 23 An expansion of the HSQC-TOCSY (black / gray) and HSQC (cyan / red) depicting the density of ribitol G is shown, where the HSQC-TOCSY correlations from G1 and G5 are indicated by gray dashed lines, and the depiction of the structural units is shown in Table 4 (600 MHz, 310 K);

[0092] Figure 24Results demonstrating cytokine levels are shown in order to demonstrate the immunostimulatory activity of the capsular polysaccharide in the purified fraction, wherein splenocytes were cultured in the presence of media (control), CPS fraction (CPS-400 and CPS-100, 50 pg / mL), total CPS (50 pg / mL) and lipopolysaccharide (LPS from E. coli 0111 :B4, 0.1 pg / mL), as Figure 24 demonstrated in the Examples section herein and by ELISA analysis of cell culture supernatants to assess cytokine production, the dose response curves of CPS-100 for the production of (a) IFNy, (b) IL-10, (c) TNF-a, (d) IL-6, (e) IL-12 and (f) IFNy, wherein in (f) the EC50 (half maximal effective concentration) was calculated to be 3.16 mM, data are values from two to three independent experiments with similar results, all bar graphs indicate mean ± SD, *p<0.05, ****p<0.0001 (one-way ANOVA with post-hoc Dunnett’s multiple comparison test), and ND, not detected;

[0093] Figure 25 demonstrating the tumor growth inhibition effect of Lactobacillus plantarum IMB 19 and CPS by activating CD8+ T cells and improving intratumoral infiltration,

[0094] Figure 25 A demonstrates the B16.F10 melanoma growth kinetics in C57 / Bi6 SPF mice treated or not with Lactobacillus plantarum IMB 19 or CPS,

[0095] Figure 25 B demonstrates images of tumors isolated from mice,

[0096] Figure 25 C and Figure 25 D demonstrates the proportion of tumor infiltrating CD8+ and CD4+ T cells, as determined by flow cytometry, 16 to 18 days after starting the treatment with Lactobacillus plantarum IMB 19 (C) or CPS (D),

[0097] wherein data are mean ± SEM values and data were analyzed by two-way ANOVA using Dunnett’s multiple comparisons (A) or non-parametric two-tailed t-test (C-D), *p<0.05, **p<0.01, ***p<0.001,

[0098] Figure 25 E and Figure 25 F demonstrates the percentage of tumor infiltrating IFNy+ CD8+ T cells and the mean fluorescence intensity (MFI) of tumor infiltrating CD8+ T cells when treated or not with Lactobacillus plantarum IMB 19 (E) or CPS (F), Figure 25G and Figure 25 H shows the frequency of IFNy+ CD4+ T cells when treated with or without L. plantarum IMB 19 (G) or CPS (H), where data are mean ± SEM values and data were analyzed by non-parametric two-tailed t-test, *P < 0.05, ns: not significant;

[0099] Figure 26 shows that L. plantarum IMB19 and CPS do not change the population of regulatory T cells in the tumor,

[0100] Figure 26 A and Figure 26 B shows the percentage of tumor-infiltrating CD4+ Foxp3+ regulatory T cells in tumor infiltrating lymphocytes after treatment with L. plantarum IMB 19 (A) or CPS (B), where data were analyzed by non-parametric two-tailed t-test, *P < 0.05, ns: not significant;

[0101] Figure 27 shows the tumor growth inhibition activity of L. plantarum IMB19 in EMT breast cancer,

[0102] Figure 27 A shows the growth kinetics of EMT-6 breast cancer in Balb / c SPF mice treated or not with L. plantarum IMB19, where data are mean ± SEM and analyzed by two-way ANOVA with Dunnett’s multiple comparisons, *P < 0.05, ***P < 0.001; and

[0103] Figure 28 shows the activity of CPS to improve the intratumoral infiltration of inflammatory macrophages in B16.F10 melanoma,

[0104] Figure 28 A shows the number and percentage of CD45+ CD11c+ CD11b+ tumor-infiltrating macrophages after CPS treatment 40 hours after tumor inoculation,

[0105] Figure 28 B and Figure 28 C shows the activity markers (i.e. CD11b, MHC I, MHC II, CD86 and CD40) on tumor-infiltrating macrophages (B) and dendritic cells (C) after CPS treatment,

[0106] Figure 28D shows the percentage of CD8+CD69+ T cells in tumor-draining lymph nodes after CPS treatment, where data are mean ± SEM and analyzed by two-way ANOVA with Dunnett’s multiple comparisons (A) or non-parametric two-tailed t-test (B), *P < 0.05, **P < 0.01, ***P < 0.001,

[0107] Figure 28 E and Figure 28 F shows the activation markers (i.e. MHC I, MHC II, iNOS2, CD68, CD40, CD80 and CD86) on mouse peritoneal macrophages treated in vitro with CPS (10 pg / ml) immediately after isolation (E) or after 24h of IL-4 treatment (F),

[0108] Figure 28 G shows the results of DAVID pathway analysis of Ml macrophage gene markers of CPS- or PBS-treated tumor-derived macrophages isolated 40h after tumor inoculation. DETAILED DESCRIPTION

[0109] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this application belongs. Generally, the nomenclature used herein is well-known and typical in the art.

[0110] Since commensal microorganisms are known to be closely involved in the immune regulatory response of a host such as a human, comprehensive studies on commensal microorganisms having the ability to enhance or suppress immunity (i.e. probiotics) are being conducted. Among numerous microbial species coexisting with human evolution, only a very small number of microbial species are found to have immune regulatory activity, and even in a given microbial species, the immune regulatory activity varies significantly depending on the type of strain, or even has an opposite effect. Since the activities possessed by microorganisms are very diverse and different for each microorganism, there has recently been a surge of interest in postbiotics (e.g. useful metabolites or effective molecules produced by microorganisms having immune regulatory activity). In particular, it is known that mannose, beta-glucan, and polysaccharides derived from microorganisms have various effects on the immune system of a host. However, it is reported that polysaccharides of microbial origin also have various activities depending on their type and structure, and polysaccharides such as zymosan exhibit both immune-enhancing and immune-suppressing aspects. Therefore, it is very important to elucidate the immune regulatory mechanism of each polysaccharide in order to use polysaccharides of probiotic origin for immune regulation of a subject, particularly for clinical treatment of humans. However, there are few reports on the relationship between polysaccharide structure and immune regulatory activity.

[0111] The present inventors have earnestly endeavored to elucidate the relationship between polysaccharide structure and immunomodulatory activity from the perspective of probiotics, and have revealed the relationship and mechanism of immunomodulatory activity with the structure of polysaccharides derived from various strains such as Bifidobacterium and yeast showing immunomodulatory activity, and filed their patent applications (Korean Patent Application No. 2018-0067535 (to be registered), Korean Patent Application No. 2019-0091908, etc.).

[0112] In one embodiment of the present application, the present inventors identified a new Lactobacillus plantarum IMB19 strain exhibiting high immune stimulatory activity from kimchi (Korean traditional fermented food), and deposited it at the Korean Biological Resource Center (Accession No. KCTC 14337BP).

[0113] In another embodiment of the present application, it has been confirmed that, in a co-culture system designed to include both the innate immune system and the acquired immune system, the strain increases immune stimulatory cytokines (e.g., IFN-γ), suppresses anti-inflammatory cytokines (e.g., IL-10), significantly increases effector T cells, and suppresses Treg production, thereby stimulating the immune system of the host and suppressing tumor growth and proliferation.

[0114] Accordingly, one aspect of the present application relates to a Lactobacillus plantarum IMB19 strain (Lactobacillus plantarum IMB19) having Accession No. KCTC 14337BP.

[0115] The Lactobacillus plantarum IMB19 strain of the present application can exhibit excellent immune-enhancing activity and anti-tumor activity. More specifically, the strain of the present application can exhibit various immune-enhancing and / or anti-tumor activities such as i) inducing T cell differentiation into an inflammatory phenotype, such as inducing helper T cell and suppressing Treg cell differentiation, ii) stimulating CD8+ T cells, enhancing activity, and improving intratumoral infiltration, iii) suppressing Treg cell activity, iv) increasing macrophage infiltration in tumors, and v) activating macrophages into inflammatory cells and reprogramming from M2 to M1 macrophages.

[0116] The Lactobacillus plantarum IMB19 strain of the present application (hereinafter abbreviated as Lactobacillus plantarum IMB19) is isolated from kimchi, identified as a new strain through 16S rRNA analysis, recA amplification / banding comparison, whole genome sequencing, and phylogenetic, morphological, and physiological analysis, and deposited.

[0117] In the present application, the Lactobacillus plantarum IMB19 strain can be derived from a fermented food, for example, kimchi.

[0118] In the present application, the Lactobacillus plantarum IMB19 strain can form small, smooth and circular translucent colonies.

[0119] In the present application, the Lactobacillus plantarum IMB19 strain can be amastigote.

[0120] In the present application, the Lactobacillus plantarum IMB19 strain can form rod-shaped colonies.

[0121] In the present application, the Lactobacillus plantarum IMB19 strain can have a capsule layer.

[0122] In the present application, the Lactobacillus plantarum IMB19 strain can be non-hemolytic or γ-hemolytic.

[0123] In the present application, the Lactobacillus plantarum IMB19 strain can be negative for gelatinase activity.

[0124] In the present application, the Lactobacillus plantarum IMB19 strain can not produce biogenic amines such as histamine, cadaverine, tyramine and / or putrescine.

[0125] In the present application, the Lactobacillus plantarum IMB19 strain can have kanamycin resistance.

[0126] In the present application, the Lactobacillus plantarum IMB19 strain can have immune-enhancing activity and / or anti-tumor activity. More specifically, the Lactobacillus plantarum IMB19 strain can exhibit various immune-enhancing and / or anti-tumor activities such as i) inducing T cell differentiation into inflammatory phenotypes such as inducing helper T cells and suppressing Treg cell differentiation, ii) stimulating CD8+ T cells, enhancing activity, and improving intratumoral infiltration, iii) suppressing Treg cell activity, iv) increasing macrophage infiltration in tumors, and v) activating macrophages into inflammatory cells and reprogramming from M2 to M1 macrophages, but the present application is not limited thereto.

[0127] In the present application, the Lactobacillus plantarum IMB19 strain can inhibit tumor growth.

[0128] In one embodiment of the present application, it has been confirmed that in an animal tumor model fed with the strain, significant immune-enhancing effects and anti-tumor activities are exhibited through various mechanisms such as stimulating CD8+ T cells, differentiation in macrophages, phenotype induction, suppressing Tregs, improving intratumoral infiltration of immune cells, etc.

[0129] In another embodiment of the present application, it has been confirmed that tumor growth is significantly inhibited after treatment with CPS.

[0130] Accordingly, another aspect of the present application relates to a composition for immunomodulation containing the strain and / or its culture solution as an active ingredient.

[0131] Further, another aspect of the present application relates to an immunomodulation method comprising administering the strain and / or its culture solution to a subject.

[0132] Further, another aspect of the present application relates to the use of the strain and / or its culture solution for immunomodulation.

[0133] Further, another aspect of the present application relates to the use of the strain and / or its culture solution for the manufacture of a composition for immunomodulation.

[0134] As used herein, the term "immunomodulation" means overcoming the immune imbalance in the blood and maintaining immune homeostasis. The maintenance of immune homeostasis refers to a state in which immune tolerance, which suppresses immunity, is balanced with immune response, which enhances immunity. Maintaining this state is essential for the treatment of most diseases, including tumors and cancers. In the present application, the composition is preferably used for immune stimulation.

[0135] In the present application, the composition for immunomodulation can be used for immune stimulation.

[0136] In the present application, the composition for immunomodulation can be a composition for immune stimulation or immune enhancement.

[0137] In the present application, the composition for immunomodulation can be a probiotic composition.

[0138] As used herein, the term "probiotic" means a live microorganism that is beneficial to the health of the host when administered in an appropriate amount (Nat. Rev. Gastroenterol Hepatol 11, 506-514 (2014)). Probiotics act as supplements to the host's intestinal microbiota, and can be characterized by improving intestinal barrier function and modulating the host's immune system.

[0139] In the present application, the probiotic composition can further comprise a prebiotic.

[0140] In the present application, for the prevention, improvement, or treatment of tumors, infectious diseases, and immunological diseases caused by or as symptoms of immune reduction / inhibition, the composition for immunomodulation can be used as a pharmaceutical composition. Here, the amount of use and the form of use can be appropriately adjusted according to the purpose.

[0141] In the present application, the composition for immunomodulation can be used in combination with other pharmaceutical compositions, or can be used as an adjuvant. Examples of other pharmaceutical compositions include, but are not limited to, immunotherapy-related immunotherapeutic agents, immune cell therapeutic agents, and the like.

[0142] In the present application, the composition for immunomodulation can further include other probiotic strains, compounds, adjuvants, additives, carriers, excipients, etc. in addition to the Lactobacillus plantarum IMB19 strain and / or the culture solution thereof of the present application.

[0143] As used herein, the term "culture solution thereof" can be a culture stock solution containing the Lactobacillus plantarum IMB19 strain of the present application, and can be used in a comprehensive sense including all lysates, centrifugal supernatants or precipitates, concentrates, dried products, etc. thereof. The Lactobacillus plantarum IMB19 strain of the present application can be cultured by typical methods of culturing Lactobacillus strains. The method of culturing the strain of the present application has been exemplarily described in the examples. The culture medium can include a natural culture medium or a synthetic culture medium. Examples of the carbon source of the culture medium can include glucose, sucrose, dextrin, glycerol, etc., and examples of the nitrogen source can include peptone, meat extract, yeast, soybean, ammonium salt, nitrate, and other organic or inorganic nitrogen-containing compounds, but the present application is not limited thereto. The inorganic salt included in the culture medium can include, but is not limited to, magnesium, manganese, calcium, iron, potassium, etc. In addition to the components of the carbon source, the nitrogen source, and the inorganic salt, amino acids, vitamins, nucleic acids, etc. can also be added.

[0144] In the present application, the culture solution can contain the Lactobacillus plantarum IMB19 strain and / or an active ingredient (e.g., polysaccharide) having immunomodulatory activity derived from the Lactobacillus plantarum IMB19 strain. The strain can be in a liquid state or a dried state, and the drying method can include, for example, natural drying, spray drying, and freeze drying, but is not limited thereto.

[0145] Still another aspect of the present application relates to a pharmaceutical composition for preventing or treating a tumor or an infectious disease, containing the Lactobacillus plantarum IMB19 strain of the present application or the culture solution thereof as an active ingredient.

[0146] In addition, still another aspect of the present application relates to a method of preventing or treating a tumor or an infectious disease, including administering the strain and / or the culture solution thereof to a subject.

[0147] In addition, still another aspect of the present application relates to the use of the strain and / or the culture solution thereof for preventing, ameliorating, or treating a tumor or an infectious disease.

[0148] The present application also relates to the use of the strain and / or the culture solution thereof for manufacturing a pharmaceutical composition for preventing or treating a tumor or an infectious disease.

[0149] As used herein, the term "tumor" refers to a phenomenon in which cells become autonomous and excessively proliferate by deviating from the regulatory mechanism of the body, or a neoplasm or hyperplasia resulting therefrom. The tumor includes, for example, all benign, pre-malignant, and malignant tumors, and more specific examples thereof include histiocytoma, glioma, astrocytoma, osteoma, various types of cancer such as lung cancer, small cell lung cancer, stomach 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, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, melanoma, lymphoma (Hodgkin's lymphoma, FL, MCL, MZBL, CLL, T-ALL, AML, ALL, etc.), blood cancer, leukemia, psoriasis, bone disease, fibroproliferative disorder, atherosclerosis, etc., and preferably melanoma, breast cancer, kidney cancer, lung cancer, bladder cancer, and rectal cancer, but the present application is not limited thereto.

[0150] As used herein, the term "infectious disease" refers to a pathogen-associated disease induced or exacerbated by infection caused by various pathogens. For example, the infectious disease can be a disease induced or exacerbated by infection caused by a virus, bacteria, fungus, protozoan, parasite, prion, or protein aggregate, but the present application is not limited thereto.

[0151] As used herein, the term "prevention" refers to any effect that can inhibit a disease or delay the onset of a disease by administering the pharmaceutical composition according to the present application.

[0152] As used herein, the term "treatment" refers to any effect that improves or beneficially changes the symptoms of a disease by administering the pharmaceutical composition according to the present application.

[0153] The pharmaceutical composition of the present application exhibits a prophylactic or therapeutic effect as well as an anti-inflammatory effect on various diseases through the above-described immune-enhancing effect and / or anti-tumor effect of its active ingredient.

[0154] In addition to containing the Lactobacillus plantarum IMB19 strain or a culture solution thereof of the present application, the pharmaceutical composition can further include suitable carriers, excipients, and diluents commonly used in pharmaceutical compositions.

[0155] The carriers, excipients, and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When the composition is formulated, it is generally prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrating agent, surfactant, etc.

[0156] The pharmaceutical composition according to the present application can be formulated and used in various forms according to a typical method. Suitable formulations include tablets, pills, powders, granules, sugar-coated tablets, hard or soft capsules, solutions, suspensions, emulsions, injections, oral formulations such as aerosols, external preparations, suppositories, and sterile injection solutions, but the present application is not limited thereto.

[0157] The pharmaceutical composition according to the present application can be formulated into a suitable dosage form using a pharmaceutically inert organic or inorganic carrier. Specifically, when the formulation is a tablet, coated tablet, sugar-coated tablet, or hard capsule, the formulation can include lactose, sucrose, starch or derivatives thereof, talc, calcium carbonate, gelatin, stearic acid or salts thereof. In addition, when the formulation is a soft capsule, it can include vegetable oils, waxes, fats, and semi-solid and liquid polyols. In addition, when the formulation is in the form of a solution or syrup, it can include water, polyols, glycerol, and vegetable oils.

[0158] In addition to the above carriers, the pharmaceutical composition according to the present application can further include a preservative, a stabilizer, a wetting agent, an emulsifying agent, a solubilizing agent, a sweetening agent, a coloring agent, an osmotic pressure adjusting agent, an antioxidant, and the like.

[0159] The pharmaceutical composition according to the present application is administered in a pharmaceutically effective amount. In the present application, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is determined according to various factors including the type of patient's disease, the severity of the disease, the drug activity, the sensitivity to the drug, the administration time, the administration route, the excretion rate, the treatment period, and the drugs used simultaneously, as well as other factors well known in the medical field. The pharmaceutical composition according to the present application can be administered as a sole therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered once or multiple times. It is important to administer the composition in the minimum amount capable of obtaining the maximum effect without side effects, which can be easily determined by those skilled in the art, taking into account all the above factors.

[0160] The pharmaceutical composition of the present application can be administered to a subject by any of a variety of routes. The administration mode can be, for example, subcutaneous, intravenous, intramuscular, intrauterine, epidural, or intracerebrovascular injection. The pharmaceutical composition of the present application is determined according to the type of drug as an active ingredient, taking into account various factors such as the disease to be treated, the administration route, the age, sex, and weight of the patient, and the severity of the disease.

[0161] The administration mode of the pharmaceutical composition according to the present application can be easily selected depending on the dosage form, and oral or parenteral administration is possible. Its dosage can vary depending on the age, sex, and weight of the patient, the severity of the disease, and the administration route.

[0162] The composition of the present application can be administered in combination with other therapeutic regimens or therapeutic agents. When used for the prevention or treatment of tumors, the other therapy or therapeutic agent is preferably an immunotherapy or an immune cell therapeutic agent, but is not limited thereto, and can be used in various combinations according to the judgment of a clinician.

[0163] In one embodiment of the present application, it has been confirmed that a capsular polysaccharide is an active ingredient that exhibits the immune-enhancing activity of the Lactobacillus plantarum IMB19 of the present application.

[0164] In another embodiment of the present application, it has been confirmed that the isolated capsular polysaccharide exhibits excellent immune-enhancing activity and anti-tumor activity through various mechanisms such as i) inducing T cell differentiation into an inflammatory phenotype, such as inducing helper T cell and suppressing Treg cell differentiation, ii) stimulating CD8+ T cells, enhancing activity, and improving intratumoral infiltration, iii) suppressing Treg cell activity, iv) increasing macrophage infiltration in tumors, and v) activating macrophages into inflammatory cells and reprogramming from M2 to M1 macrophages, as in the Lactobacillus plantarum IMB19 strain.

[0165] In still another embodiment of the present application, the capsular polysaccharide is structurally analyzed by NMR.

[0166] Accordingly, still another aspect of the present application relates to a capsular polysaccharide (CPS) derived from the Lactobacillus plantarum IMB19 strain, which has immune-enhancing activity.

[0167] In the present application, the capsular polysaccharide can include a polysaccharide having the following Formula I.

[0168] [Formula I]

[0169] -[-D-B-I-F-G-C-E-H-A-] n -

[0170] Here, A and D are galactose,

[0171] B, C, E, G, and H are rhamnose,

[0172] F is N-acetylglucosamine,

[0173] I is glucose, and

[0174] n is an integer of 1 or more.

[0175] In the present application, the polysaccharide having Formula I is a polymer structure of a repeating unit represented by [-D-B-I-F-G-C-E-H-A-] in Formula I.

[0176] In the present application, when n in Formula I is 2 or more, the repeating units can be connected in various ways without limitation in addition to direct covalent bonding. For example, the repeating units can be connected by chemical bonding such as glycosidic linkage or phosphodiester linkage, or can be connected via a linker.

[0177] In the present application, when n in Formula I is 2 or more, the repeating units ([ -D-B-I-F-G-C-E-H-A- ]) can be connected by a glycosidic linkage (-O-) between A and D.

[0178] In the present application, when n in Formula I is 2 or more, the repeating units ([ -D-B-I-F-G-C-E-H-A- ]) can be connected by a phosphodiester linkage.

[0179] In the present application, when n in Formula I is 2 or more, the carbon at position 1 in A of a repeating unit and the carbon at position 6 of D of another repeating unit can be connected by a phosphodiester linkage.

[0180] In the present application, at least one of A and D of Formula I can be phosphorylated, and preferably, in the case of A, the hydroxyl group of the carbon at position 1 is phosphorylated, and in the case of D, the hydroxyl group of the carbon at position 6 is phosphorylated.

[0181] In the present application, the galactose used includes all galactose or derivatives thereof that are typically present in nature. For example, in the present application, the galactose can be provided in the form of isomers in the α- or β-configuration, or the D- or L-configuration, preferably α-galactose, and more preferably α-D-galactose, but is not limited thereto.

[0182] In the present application, the rhamnose used includes all rhamnose or derivatives thereof that are typically present in nature. For example, in the present application, the rhamnose can be provided in the form of isomers in the α- or β-configuration, or the D- or L-configuration, preferably α-rhamnose, and more preferably α-L-rhamnose, but is not limited thereto.

[0183] In the present application, the N-acetylglucosamine used includes all N-acetylglucosamine and derivatives thereof.

[0184] In the present application, the glucose used includes all typical glucose or derivatives thereof. For example, in the present application, the glucose can be provided in the form of isomers in the α- or β-configuration, or the D- or L-configuration, preferably glucose having the β-configuration, and more preferably D-glucose, but is not limited thereto.

[0185] In the present application, D and B (D-B) and B and I (B-I) can be connected by an α-1,3-glycosidic linkage.

[0186] In the present invention, I and F (I-F) can be linked by a β-1,6-glycosidic linkage.

[0187] 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) can be linked by an α-1,2-glycosidic linkage.

[0188] In the present invention, H and A (H-A) can be linked by an α-1,6-glycosidic linkage.

[0189] In the present specification, a glycosidic linkage is indicated as α or β according to the direction of linkage, and the number adjacent thereto refers to the corresponding carbon number linked by a glycosidic linkage (-O-) among two monosaccharides. For example, “I and F (I-F) are linked by a β-1,6-glycosidic linkage” means that the carbon at position 1 of I and the carbon at position 6 of F are linked by a glycosidic linkage in a β configuration with respect to I.

[0190] In the present invention, the capsular polysaccharide can comprise a polysaccharide having Formula I, wherein n is an integer of 1 or more, and can comprise a plurality of polysaccharides having Formula I polymerized with various n values. Preferably, the polysaccharide having Formula I is characterized in that n is a value ranging from 1 to 10.

[0191] In one embodiment of the present invention, it has been confirmed that the average degree of polymerization of the polysaccharide having Formula I included in the capsular polysaccharide is about 4, and its average molecular weight is about 6.0 kDa. It has been confirmed that each repeating unit ([-D-B-I-F-G-C-E-H-A-]) has a molecular weight (MW) of about 1.5 kDa.

[0192] In the present invention, the “average degree of polymerization (average value of n)” of the polysaccharide having Formula I included in the capsular polysaccharide is preferably 1 to 10, and most preferably about 4.

[0193] In the present invention, the average molecular weight of the polysaccharide having Formula I included in the capsular polysaccharide can be 1.5 kDa to 15 kDa, and preferably about 6.0 kDa.

[0194] In one embodiment of the present invention, for the polysaccharide having Formula I included in the capsular polysaccharide, the capsular polysaccharide (CPS) derived from the Lactobacillus plantarum IMB19 strain is separated by ion exchange chromatography using about 100 mM NaCl as an eluent. The separation method is described in detail in the examples, but the present invention is not limited thereto. The polysaccharide can be obtained by various conventionally known purification methods based on the structure and characteristics of the polysaccharide having Formula I described in the present invention.

[0195] In the present invention, the capsular polysaccharide can further comprise teichoic acid.

[0196] As demonstrated in one embodiment of the present application, in the present application, the capsular polysaccharide can further comprise two or more teichoic acids.

[0197] In the present application, the teichoic acid can be of Gro type or Rbo type, and when the capsular polysaccharide additionally comprises two or more teichoic acids, the teichoic acids can be contained in the form of a single type or a mixture of two types.

[0198] In the present application, in addition to the polysaccharide having Formula I and / or the teichoic acid, the capsular polysaccharide can further comprise other polysaccharides or biomolecules such as lipids.

[0199] Still another aspect of the present application relates to a method of producing a polysaccharide having an immune-enhancing activity, comprising:

[0200] (a) culturing the above-mentioned strain; and

[0201] (b) obtaining a capsular polysaccharide from the cultured strain.

[0202] In the present application,

[0203] The method can further comprise (c) obtaining an effective polysaccharide fraction having an immune-enhancing activity by subjecting the obtained capsular polysaccharide to ion exchange chromatography.

[0204] The method of producing a capsular polysaccharide and the step of obtaining an effective polysaccharide fraction having an immune-enhancing activity of the present application are exemplarily described in detail in the examples of the present application, but are not limited thereto.

[0205] In one embodiment of the present application, capsular polysaccharides (CPS) derived from Lactobacillus plantarum IMB19 strain were fractionated by ion exchange chromatography using different concentrations of NaCl eluent, and the structure of the polysaccharide contained in each fraction was analyzed by NMR.

[0206] In another embodiment of the present application, among the different fractions of capsular polysaccharides (CPS) derived from Lactobacillus plantarum IMB19 strain, only CPS-100 purified using 100 mM NaCl produced significant levels of IFN-γ, TNF-α, IL-6, and IL-12, and negligible levels of IL-10, IL-17, and IL1-β, while no significant cytokine production was detected in other fractions (CPS-400 (containing teichoic acid), etc.), indicating that the effective molecule exhibiting the immune-enhancing activity of Lactobacillus plantarum IMB19 strain is a polysaccharide (Formula I) having a polymer structure with a specific repeating unit contained in CPS-100.

[0207] Thus, still another aspect of the present application relates to a polysaccharide represented by the following formula I:

[0208] [Formula I]

[0209] -[-D-B-I-F-G-C-E-H-A-] n -

[0210] In formula I,

[0211] A and D are galactose,

[0212] B, C, E, G, and H are rhamnose,

[0213] F is N-acetylglucosamine,

[0214] I is glucose, and

[0215] n is an integer of 1 or more.

[0216] In the present application, the polysaccharide having formula I is a polymer structure of the repeating unit represented by [-D-B-I-F-G-C-E-H-A-] in formula I.

[0217] In the present application, when n in formula I is 2 or more, the repeating units can be connected in various ways without limitation in addition to direct covalent bonding. For example, the repeating units can be connected by chemical bonding such as glycosidic linkage or phosphodiester linkage, or can be connected via a linker.

[0218] In the present application, when n in formula I is 2 or more, the repeating units ([ -D-B-I-F-G-C-E-H-A- ]) can be connected by a glycosidic linkage (-O-) between A and D.

[0219] In the present application, when n in formula I is 2 or more, the repeating units ([ -D-B-I-F-G-C-E-H-A- ]) can be connected by a phosphodiester linkage between A and D.

[0220] In the present application, when n in formula I is 2 or more, the carbon at position 1 in A of the repeating unit and the carbon at position 6 of D of another repeating unit can be connected by a phosphodiester linkage.

[0221] In the present application, at least one of A and D of formula I can be phosphorylated, and preferably, in the case of A, the hydroxyl group of the carbon at position 1 is phosphorylated, and in the case of D, the hydroxyl group of the carbon at position 6 is phosphorylated.

[0222] In the present application, the galactose used includes all galactose or derivatives thereof that are generally present in nature. For example, in the present application, the galactose can be provided in the form of isomers of an alpha or beta configuration, or a D or L configuration, preferably alpha-galactose, and more preferably alpha-D-galactose, but is not limited thereto.

[0223] In the present application, the rhamnose used includes all rhamnose or derivatives thereof that are generally present in nature. For example, in the present application, the rhamnose can be provided in the form of isomers of an alpha or beta configuration, or a D or L configuration, preferably alpha-rhamnose, and more preferably alpha-L-rhamnose, but is not limited thereto.

[0224] In the present application, the N-acetylglucosamine used includes all N-acetylglucosamine or derivatives thereof that are generally present in nature.

[0225] In the present application, the glucose used includes all typical glucose or derivatives thereof. For example, in the present application, the glucose can be provided in the form of isomers of an alpha or beta configuration, or a D or L configuration, preferably glucose having a beta configuration, and more preferably D-glucose, but is not limited thereto.

[0226] As used herein, the term "derivative" refers to a compound whose structure is sufficiently similar to the structure of a compound disclosed herein, and based on the similarity between them, it exhibits the same or similar activity and end use as the claimed compound; or as a precursor, a compound having a structure derived from the structure of the parent compound (e.g., a polysaccharide of Formula I described herein) that is expected to cause the same or similar activity and end use as the claimed compound. Examples of the derivative can include, but are not limited to, salts, isomers, esters, amides, salts of esters or amides, N-oxides, etc. of the parent compound.

[0227] In the present application, D and B (D-B) and B and I (B-I) of Formula I can be linked by an alpha-1,3-glycosidic linkage.

[0228] In the present application, I and F (I-F) of Formula I can be linked by a beta-1,6-glycosidic linkage.

[0229] In the present application, F and G (F-G), G and C (G-C), C and E (C-E), and E and H (E-H) of Formula I can be linked by an alpha-1,2-glycosidic linkage.

[0230] In the present application, H and A (H-A) of Formula I can be linked by an alpha-1,6-glycosidic linkage.

[0231] In the present specification, the connection is expressed as a or β according to the connection direction, and the number adjacent thereto refers to the corresponding carbon number to which (-O-) is connected in two monosaccharides.

[0232] In the present invention, n in Formula I is an integer of 1 or more, preferably an integer of 1 to 10, and more preferably 4.

[0233] In the present invention, the polysaccharide can have the structure of the following Formula II.

[0234] [Formula II]

[0235]

[0236] n is an integer of 1 or more.

[0237] In the present invention, n in Formula II is preferably an integer of 1 to 10, and more preferably n is 4.

[0238] In one embodiment of the present invention, it has been confirmed that the average degree of polymerization of the polysaccharide having Formula I contained in CPS-100 (an effective fraction of capsular polysaccharide) is about 4, and its average molecular weight is about 6.0 kDa. It has been confirmed that each repeating unit ([-D-B-I-F-G-C-E-H-A-]) has a molecular weight (MW) of about 1.5 kDa.

[0239] In the present invention, the polysaccharide having Formula I can have 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, according to the degree of polymerization, the degree of phosphorylation, etc.

[0240] In the present invention, the polysaccharide can be derived from the Lactobacillus plantarum IMB19 strain having accession number KCTC 14337BP.

[0241] In the present invention, the polysaccharide having Formula I can have immune-enhancing activity and / or anti-tumor activity. More specifically, the Lactobacillus plantarum IMB19 strain can have various immune-enhancing and anti-tumor activities such as i) inducing T cell differentiation into an inflammatory phenotype such as inducing differentiation of helper T cells and suppressing Treg cells, ii) stimulating CD8+ T cells, enhancing activity, and improving intratumoral infiltration, iii) suppressing Treg cell activity, iv) increasing macrophage infiltration in tumors, and v) activating macrophages into inflammatory cells and reprogramming from M2 to M1 macrophages, but not limited to such mechanisms.

[0242] In the present invention, the polysaccharide having Formula I is capable of inhibiting tumor growth.

[0243] In the present application, the polysaccharide can be produced / isolated by those skilled in the art using the strain of the present application, and the polysaccharide of the present application can be induced or synthesized by other chemical or biological methods.

[0244] Further, it is apparent that the structure of the CPS can be modified to improve the pharmacokinetic and / or pharmacodynamic properties of the polysaccharide of the present application or its clinical formulation (e.g., solubility). For example, i) addition of at least one functional group, ii) modification of the carbon chain, iii) addition of at least one hydrogen or hydroxyl group, iv) modification of the end group (e.g., addition of a signal molecule such as a dye, etc.), and v) binding to other known sugar molecules (e.g., formulation for oral or systemic delivery, etc.) can be performed, but the present application is not limited thereto.

[0245] Accordingly, the polysaccharide of the present application is not limited to the polysaccharide having Formula I or Formula II, and is conceptually understood to include all variants, derivatives, analogs, etc. having Formula I or Formula II, as long as it exhibits the immunostimulatory and immunopotentiating effects of the present application.

[0246] Accordingly, another aspect of the present application relates to a composition for immunomodulation containing the polysaccharide having Formula I as an active ingredient.

[0247] Further, another aspect of the present application relates to a method for immunomodulation, which comprises administering the polysaccharide to a subject.

[0248] Further, another aspect of the present application relates to the use of the polysaccharide for immunomodulation.

[0249] Further, another aspect of the present application relates to the use of the polysaccharide for the manufacture of a composition for immunomodulation.

[0250] Hereinafter, unless a specific definition of a term is provided, the term should be understood to have a meaning as understood by those skilled in the art or a meaning defined according to other aspects of the present application.

[0251] In the present application, the composition for immunomodulation can be used for immunostimulation or immunopotention.

[0252] In the present application, the composition for immunomodulation can be a composition for immunostimulation or immunopotention.

[0253] In the present application, for the prevention, improvement, or treatment of tumors, infectious diseases, and immunological diseases caused by or as symptoms of immunodepression / suppression, the composition for immunomodulation can be prepared and used in the form of a pharmaceutical composition. Here, the amount of use and the form of use can be appropriately adjusted according to the purpose.

[0254] In one embodiment of the present application, the polysaccharide is confirmed to have a half maximal effective concentration (EC50) of 3.16 μM. Thus, it is preferable that the composition for immunomodulation of the present application contains the polysaccharide in an amount of 3.16 μM or more.

[0255] In the present application, examples of other pharmaceutical compositions include, but are not limited to, immunotherapeutic agents related to immunotherapy, immune cell therapeutic agents, etc.

[0256] In the present application, the composition for immunomodulation can further contain other probiotic bacterial strains, compounds, adjuvants, additives, carriers, excipients, etc. in addition to the polysaccharide of the present application.

[0257] Still another aspect of the present application relates to a pharmaceutical composition for preventing or treating a tumor or an infectious disease, containing the polysaccharide having Formula I of the present application as an active ingredient.

[0258] In addition, still another aspect of the present application relates to a method of preventing or treating a tumor or an infectious disease, comprising administering the polysaccharide to a subject.

[0259] In addition, still another aspect of the present application relates to the use of the polysaccharide for preventing, ameliorating, or treating a tumor or an infectious disease.

[0260] In addition, the present application relates to the use of the polysaccharide for manufacturing a pharmaceutical composition for preventing or treating a tumor or an infectious disease.

[0261] As used herein, the term "tumor" refers to a phenomenon in which cells become autonomous and hyperproliferate by deviating from the regulatory mechanism of the body, or a neoplasm or hyperplasia resulting therefrom. The tumor includes, for example, all benign, pre-malignant, and malignant tumors, and more specific examples thereof include histiocytoma, glioma, astrocytoma, osteoma, various types of cancer such as lung cancer, small cell lung cancer, stomach 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, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, melanoma, lymphoma (Hodgkin's lymphoma, FL, MCL, MZBL, CLL, T-ALL, AML, ALL, etc.), blood cancer, leukemia, psoriasis, bone disease, fibroproliferative disorder, atherosclerosis, etc., but the present application is not limited thereto.

[0262] As used herein, the term "infectious disease" refers to a pathogen-associated disease induced or exacerbated by infection by various pathogens. For example, the infectious disease can be a disease induced or exacerbated by infection by a virus, bacteria, fungus, protozoan, parasite, prion, or protein aggregate, but the present application is not limited thereto.

[0263] The pharmaceutical composition of the present application exhibits a prophylactic or therapeutic effect on various diseases and an anti-inflammatory effect by the above-mentioned immunopotentiating and / or antitumor effects of the polysaccharide having Formula I as an active ingredient thereof.

[0264] In addition to containing the polysaccharide having Formula I as an active ingredient thereof, the pharmaceutical composition can further comprise suitable carriers, excipients, and diluents commonly used in pharmaceutical compositions.

[0265] The carriers, excipients, and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When the composition is formulated, it is usually prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc.

[0266] The pharmaceutical composition according to the present application can be formulated and used in various forms according to typical methods. Suitable formulations include tablets, pills, powders, granules, sugar-coated tablets, hard or soft capsules, solutions, suspensions, emulsions, injections, oral formulations such as aerosols, external preparations, suppositories, and sterile injection solutions, but the present application is not limited thereto.

[0267] The pharmaceutical composition according to the present application can be formulated into suitable dosage forms using pharmaceutically inert organic or inorganic carriers. Specifically, when the formulation is a tablet, coated tablet, sugar-coated tablet, or hard capsule, the formulation can include lactose, sucrose, starch or its derivatives, talc, calcium carbonate, gelatin, stearic acid or its salts. In addition, when the formulation is a soft capsule, it can include vegetable oils, waxes, fats, and semi-solid and liquid polyols. In addition, when the formulation is in the form of a solution or syrup, it can include water, polyols, glycerol, and vegetable oils.

[0268] In addition to the above-mentioned carriers, the pharmaceutical composition according to the present application can further comprise a preservative, a stabilizer, a wetting agent, an emulsifying agent, a solubilizing agent, a sweetening agent, a coloring agent, an osmotic pressure adjusting agent, an antioxidant, etc.

[0269] The pharmaceutical composition according to the present application is administered in a pharmaceutically effective amount. In the present application, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is determined according to various factors including the type of patient's disease, the severity of the disease, the drug activity, the sensitivity to the drug, the administration time, the administration route, the excretion rate, the treatment period, and the drugs used simultaneously, as well as other factors well known in the medical field. The pharmaceutical composition according to the present application can be administered as a sole therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered once or multiple times. It is important to administer the composition in the minimum amount capable of obtaining the maximum effect without side effects, which can be easily determined by those skilled in the art, taking into account all the above factors.

[0270] The pharmaceutical composition of the present application can be administered to a subject by any of various routes. The administration mode can be, for example, subcutaneous, intravenous, intramuscular, intrauterine, epidural, or intracerebrovascular injection. The pharmaceutical composition of the present application is determined according to the type of drug as an active ingredient, taking into account various factors such as the disease to be treated, the administration route, the age, sex, and body weight of the patient, and the severity of the disease.

[0271] The administration mode of the pharmaceutical composition according to the present application can be easily selected depending on the dosage form, and oral or parenteral administration or local or systemic administration is possible. Its dose can vary depending on the age, sex, and body weight of the patient, the severity of the disease, and the administration route.

[0272] The composition of the present application can be administered in combination with other therapeutic regimens or therapeutic agents. When used for the prevention or treatment of a tumor, the other therapy or therapeutic agent is preferably an immunotherapy or an immune cell therapeutic agent, but is not limited thereto, and can be used in various combinations according to the judgment of a clinician.

[0273] In one embodiment of the present application, it has been confirmed that the strain and the polysaccharide derived from the strain according to the present application induce the expression of IFN-γ and suppress the expression of IL-10 in cells both in vivo and in vitro, thereby stimulating and enhancing the immune response.

[0274] In another embodiment of the present application, it has been confirmed that the strain and the polysaccharide derived from the strain are capable of activating CD8+ T cells via DCs, and the activation of CD+8 T cells is also significantly reduced in the absence of the MyD88 signaling system.

[0275] In still another embodiment of the present application, it has been confirmed that Th17 cells can be induced by co-culturing naive CD4+ T cells with DCs primed with the strain and the polysaccharide derived from the strain.

[0276] Accordingly, still another aspect of the present application relates to a method of producing inflammatory T cells, comprising:

[0277] (a) priming antigen presenting cells with Lactobacillus plantarum IMB19 strain and / or a polysaccharide having Formula I; and

[0278] (b) co-culturing the primed antigen presenting cells with T cells.

[0279] As used herein, the term "antigen presenting cell" refers to a cell induced to differentiate by taking up an antigen, processing it, and presenting antigen-derived fragments in association with antigen presenting molecules (such as MHC class ± molecules) to T cells. Examples of the antigen presenting cells include, but are not limited to, macrophages, B cells, dendritic cells (DCs), Langerhans cells, and the like.

[0280] In the present application, the T cells in step (b) are preferably naive T cells, more preferably naive CD8+ T cells or naive CD4+ T cells, but are not limited thereto.

[0281] As used herein, the term "inflammatory T cell" refers to a T cell that induces or enhances an immune response directly or at a proinflammatory stage. Preferably, the inflammatory T cell is a cytotoxic T cell or a helper T cell (Th cell), more preferably an IFN-γ+ CD8+ T cell or a CD4+ RORγ+ Th17 cell.

[0282] Accordingly, still another aspect of the present application relates to a cell therapeutic agent for preventing or treating a tumor or an infectious disease, containing the inflammatory T cells produced by the above-described method as an active ingredient.

[0283] In one embodiment of the present application, it has been confirmed that, when spleen macrophages or M2 phenotype macrophages are exposed to CPS, MHC I, MHC II, CD68, iNOS2, and CD40 are significantly upregulated, enabling reprogramming from immunosuppressive M2 phenotype macrophages to M1 phenotype macrophages in a tumor microenvironment.

[0284] Accordingly, still another aspect of the present application relates to a method of producing M1 phenotype macrophages, comprising differentiating macrophages into M1 phenotype macrophages by treating macrophages with Lactobacillus plantarum IMB19 strain and / or a polysaccharide having Formula I, and obtaining activated M1 phenotype macrophages.

[0285] As used herein, the term "macrophage" refers to a type of leukocyte that is capable of phagocytosis by engulfing and digesting pathogens, foreign substances, microorganisms, cancer cells, abnormal proteins, and the like, and functions as an antigen presenting cell for adaptive immunity in addition to an innate immune response.

[0286] Macrophages are classified into Ml phenotype macrophages, which are inflammatory macrophages, or M2 phenotype macrophages, which are anti-inflammatory macrophages, and are involved in the balance of immune stimulation and suppression. In particular, it is reported that M2 phenotype macrophages promote tumor growth (Ann. Oncol. 28, xii 18-xii 32 (2017); Front Oncol. 9, 421 (2019)). Ml phenotype macrophages generally refer to macrophages that are activated by IFN-g secreted by NK cells or Thl, MyD88 pathway recognized by PAMP of TLR, etc., and thus stimulate and induce immune responses such as antigen presentation, induction of inflammatory genes and secretion of inflammatory chemokines, etc. M2 phenotype macrophages (immunosuppressive macrophages) are macrophages differentiated by IL-4, IL-13, etc., exhibit immunosuppressive activity, and are involved in tissue reconstruction and wound healing.

[0287] In the present application, the macrophages in step (a) can be naive macrophages or M2 phenotype macrophages.

[0288] In the present application, Ml phenotype macrophages include, without limitation, macrophages activated to an inflammatory phenotype to stimulate or induce immune responses. Ml phenotype macrophages can be classified according to changes in gene expression profiles; for example, Ml phenotype macrophages can be characterized by up-regulation of expression of at least one selected from the group consisting of MHC I, MHC II, CD68, iNOS2, and CD40, but the present application is not limited thereto.

[0289] As used herein, the expression "differentiation of macrophages" refers to activation or transformation of macrophages from an existing gene expression profile to a phenotype exhibiting another gene expression profile. The use of the term "differentiation" is meant to include not only activation or polarization of naive macrophages, but also reprogramming of macrophages that have been activated and exhibit a specific phenotype, such as an Ml phenotype or an M2 phenotype.

[0290] As used herein, the term "up-regulation" means that the expression of a specific gene or protein is improved compared to the phenotype before the transformation of macrophages.

[0291] Accordingly, an even still another aspect of the present application relates to a cell therapeutic agent for preventing or treating a tumor or an infectious disease, which contains inflammatory macrophages produced by the above-described method as an active ingredient.

[0292] Examples

[0293] A better understanding of the present application can be obtained from the following examples. These examples are set forth to illustrate the present application and are not to be construed as limiting the scope of the present application as would be apparent to one skilled in the art.

[0294] Example 1: Materials and Methods

[0295] 1. Bacterial culture and identification

[0296] Kimchi was homogenized and its suspension was obtained. Then, serial dilution was performed, streaked on MRS broth and agar, and incubated at 37°C for 48 hours to isolate colonies, which were then further cultured for various analyses.

[0297] Lactobacillus plantarum IMB19 was cultured in MRS broth at 37°C for 24-36 hours. For TEM (Transmission Electron Microscopy), bacteria were cultured, streaked on MRS-1.5% agar (Neogen Corp., USA), and incubated for 24-30 hours. Bacterial colonies were dropped onto 2000 mesh graphene-coated copper grids. TEM imaging was performed using JEOL 1220 and Hitachi HT7700 at 80 kV.

[0298] For identification, the cell morphology of selected isolated strains was observed using a microscope, their genetic characteristics were analyzed using genomic DNA, and 16s rRNA sequencing was performed by Macrogen (Korea).

[0299] 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'.

[0300] 2. Primary cell-based test

[0301] 2-1. In vitro splenocyte stimulation

[0302] All animal experiments and procedures were performed in accordance with the ethical regulations and approval of POSTECH Animal Care and Use Committee (POSTECH). C57BL / 6 mice were housed and bred in a pathogen-free animal barrier facility and used at 6-8 weeks of age. Spleens were harvested therefrom and gently ground to release splenocytes. Cells were suspended, subjected to RBC lysis with ammonium chloride buffer, and resuspended in complete RPMI medium (Welgene, Korea) containing 10% FBS (Hy-Clone, Australia). Cells were plated at a density of 200 k / well in 96-well plates in 200 μL medium / well containing 10 ng / mL anti-CD3 (Bio-xCell, USA) and 2.5 ng / mL GM-CSF (Peprotech, USA). Fractionated CPS-100, unfractionated total CPS (tCPS), LPS (lipopolysaccharide derived from E. coli 0111:B4, Invivogen, USA), and medium were added as needed, followed by incubation at 37°C and 5% CO2 for 48 hours. After centrifugation, the supernatant was collected and frozen for cytokine estimation by enzyme-linked immunosorbent assay (ELISA, e-Bioscience, Ready-SET-Go! ELISA kit) according to the manufacturer's instructions.

[0303] 2-2. Immune cell co-culture system

[0304] Isolation of splenocytes was performed as described above. CD11c+APC (Miltenyi Biotec) and naive CD4+T cells (Stem Cell Technologies) were isolated according to the manufacturer's protocol. APCs were cultured with probiotic strains for 18-20 hours at 37°C and 5% CO2. Then, the probiotic strains were washed and the primed APCs and CD4+T cells were co-cultured with them under specific conditions.

[0305] 3. Safety evaluation

[0306] 3-1. Hemolysis test

[0307] Lactobacillus plantarum IMB19 was grown under optimal growth conditions, streaked on 5% sheep blood agar (Hanil, Komed), and then incubated for 48 hours. Alpha (a) 2 hemolysis was considered as partial degradation of hemoglobin in red blood cells (not representing actual hemolysis), beta (b) hemolysis (observed as clear zones on agar plates) was considered as complete degradation of hemoglobin in red blood cells, and gamma (g) hemolysis was considered as no hemolysis. Bacillus cereus ATCC 27348 was used as a positive control.

[0308] 3-2. Gelatin degradation test

[0309] The basic protocol was performed according to ASM scientific recommendations (Dela Cru et al., 2012). Lactobacillus plantarum IMB19 grown under optimal growth conditions was inoculated with a loop in gelatin medium and incubated at 30°C for up to 5 days and checked daily for gelatin liquefaction and bacterial growth. Gelatin usually liquefies at temperatures equal to or higher than 28°C. The test tubes were stored in the refrigerator for 30 minutes to determine if the liquefaction was due to gelatinase activity. Then, the test tubes were tilted to observe if the gelatin was degraded. When the gelatin was degraded, it remained liquid even after exposure to low temperatures. Bacillus cereus ATCC 11778 was used as a positive control.

[0310] 3-3. Biogenic amine analysis

[0311] Lactobacillus plantarum IMB19 was grown under optimal conditions and streaked on a special medium containing precursors of histamine, cadaverine, tyramine and putrescine according to Bover-cid and Holzapfel (Bover-cid et al., 1999) followed by incubation at 37°C, 30°C and 23°C for 4 days. Then, the results of the analysis were determined to be positive or negative by observing the change in the color of the medium. Escherichia coli ATCC 25922 was used as a positive control. The basic medium containing any of ornithine, lysine, tyrosine and histidine and the bromocresol purple indicator was used for the culture on agar.

[0312] 3-4. Antibiotic resistance test

[0313] The basic protocol was performed according to ISO recommendations (ISO-10932, 2010). The minimum inhibitory concentration (MIC) of the strains to antibiotics was evaluated using the broth dilution method.

[0314] In the culture microdilution, the test organisms were cultured alone in the medium and all organisms were washed with 1X PBS. The bacterial solution washed with PBS was adjusted to an optical density (OD) of 0.01-0.02 at 600 nm. 10 μL (1 x 10 5 -2 x 10 5 CFU) of the strain was inoculated into a 96-well plate containing 200 μL LSM broth medium and antibiotics.

[0315] The strain was considered susceptible when it was inhibited at a specific antibiotic concentration equal to or lower than the cutoff value set according to the parameters established by the European Food Safety Authority (EFSA, 2018) and resistant when it was not inhibited at a specific antibiotic concentration higher than the cutoff value established by the regulations.

[0316] 4. Isolation of the rough capsular polysaccharide (CPS)

[0317] Isolation of crude CPS was performed with a modified version of the method routinely described (Verma et al., 2018). The bacterial culture was centrifuged and sonicated using a Branson Digital Sonifier at 10% amplitude and 10 sec pulse for 15 min. The supernatant was treated with trichloroacetic acid (0.5% w / v) at 4°C overnight. The sample was centrifuged at 6000 rpm for 20 min and 100% ethanol was added to the supernatant at a ratio of 3: 1 to precipitate crude polysaccharides at -20°C. The precipitate was resuspended in Tris buffer (100 mM, Sigma Aldrich, USA) in endotoxin-free distilled water containing magnesium chloride (20 mM, Sigma Aldrich) and calcium chloride (20 mM, Sigma Aldrich) and then 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. Pronase (0.3 mg / mL, Sigma-Aldrich, USA) was added to it to degrade protein contaminants followed by overnight incubation at 4°C. The sample was treated with trichloroacetic acid (1-2% w / v) at 37°C for 30 min to remove total proteins including the added enzymes. All the polysaccharides in the sample from which proteins were removed were reprecipitated by ethanol precipitation. The precipitate 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 freeze-drying with a final yield of 20 mg / liter of culture.

[0318] 5. GC-MS analysis conditions

[0319] All chemical derivatives were analyzed by gas-liquid chromatography (GLC-MS) using an Agilent 7820A (Santa Clara, CA, USA) equipped with a mass selective detector 5973N and a Zebron ZB-5 capillary column (Phenomenex, 30 m x 0.25 mm i.d, film thickness: 0.25 pm, flow rate: 1 mL / min, and He as carrier gas). The 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: 150 °C for 5 min, 10 °C / min from 150 °C to 300 °C, and 300 °C for 12 min.

[0320] 6. NMR acquisition parameters

[0321] For structural analysis of the isolated polysaccharides, NMR spectra were recorded in D2O using a Bruker 600 MHz spectrometer equipped with a cryogen-free inverse probe tilted along the Z axis. The spectra were measured at 298 K or 310 K, corrected with acetone (δH 2.225 ppm; δc 31.45 ppm) as internal standard, acquired using Topspin 2.0 software (Bruker) and processed and investigated with Topspin 3.6. 1 H2.225 ppm; 13 C 31.45 ppm) as internal standard, acquired using Topspin 2.0 software (Bruker) and processed and investigated with Topspin 3.6. 1 H- 1 H DQ-COSY (double quantum COZY spectra, hereafter COSY), TOCSY and NOESY spectra were collected with 2048 x 512 points (t1 x t2) data sets and TOCSY and NOESY spectra were collected with 24 scans and mixing times of 100 ms and 200 ms, respectively. Heteronuclear 1 H- 13 C HSQC, HMBC and HSQC-TOCSY spectra were acquired in 1 H- detection mode using 2048 x 512 points data sets. In the selection step, HSQC and HSQC-TOCSY were performed under multiple editing in order to discriminate the CH2 densities from the other ones. A low-pass J filter was used to suppress single-bond correlations, HMBC was optimized for long-range coupling constants and a 60 ms delay was used for evolution of long-range correlations. For HSQC-TOCSY, the mixing time was set to 100 ms. In all two-dimensional experiments, the data matrix was extended to 4092 x 2048 points and transformed by applying a qsine or sine window function.

[0322] 7. Animal and mouse tumor models

[0323] C57BL / 6 & Balb / c mice were purchased and housed in POSTECH animal facility. Pmel-1 TCR transgenic, MyD88- / - and IL-6- / - mice were purchased from Jackson Lab and maintained in POSTECH animal facility. C57BL / 6-derived melanoma cell line B16.F10 and Balb / c-derived breast cancer cell line EMT-6 were obtained from ATCC and maintained according to the provided protocol. Syngeneic tumor models were obtained by subcutaneous injection of 200,000 B16.F10 tumor cells or 500,000 EMT-6 cells. Tumor size was measured every two days until endpoint and tumor volume was calculated using length x width 2 x 0.5. For initial infiltration analysis of innate immune cells, tumor cells were injected subcutaneously at 5 ml / mouse and tumor cells were analyzed after 40 hours. All experimental animal procedures were performed under the approval of the Institutional Animal Care and Use Committee (IACUC) of POSTECH.

[0324] 8. Cell-based in vitro assay method

[0325] Total cells harvested from spleen and / or lymph nodes were used for whole splenocyte culture or subjected to magnetic bead isolation (Miltenyi Biotec) in order to enrich CD11c+dendritic cells, naive CD8+T cells or naive CD4+T cells. Total splenocytes (200,000 cells / well) were cultured with bacteria at a 1 : 1 ratio in 96 plates and supernatants were harvested at 48 hours and used for ELISA (eBioscience Ready-SET-Go! kit). CD11c+dendritic cells (200,000 cells / well) were primed with bacteria at the appropriate ratio for 18-20 hours, washed, T cells (200,000 cells / well) were added and cultured for 72 hours. As indicated, 0.01 pg / ml anti-CD3 (BioXCell) and 2.5 ng / ml GM-CSF (Peprotech) were added to stimulate CD8+T cells and 0.1 pg / ml anti-CD3, 10 ng / ml GM-CSF, 100 U / ml IL2 and TGFp (Peprotech) were added to stimulate CD4+T cells.

[0326] For peritoneal macrophages, cells were harvested 5 days after intraperitoneal injection of 2% Biogel (Bio-Rad) and then cultured in vitro with 10 ng / ml recombinant murine MCSF (Peprotech). For polarization of selectively activated macrophages, IL-4 (Peprotech) was added to the cells for 24 hours before treatment with CPS, LPS (lipopolysaccharide derived from E. coli 0111:B4, Invivogen) or Pam3CSk4 (Sigma).

[0327] 9. Metagenomic analysis and whole-genome sequencing

[0328] Genomic analysis of fecal particles from tumor-bearing SPF mice was outsourced to Macrogen (Korea). Whole-genome sequencing of bacteria was performed on the Illumina platform (Macrogen (Korea),) and bacterial culture samples were sent directly for analysis. Bioinformatics analysis was also outsourced to Macrogen.

[0329] 10. Classification and gene expression profile analysis of tumor-infiltrating macrophages

[0330] 500 μg of CPS was injected into mice twice at 24-hour intervals, followed by subcutaneous inoculation with 5 x 10⁵ CPS. 6 B16.F10 tumor cells were inoculated. Forty hours post-inoculation, intact tumors, including infiltrating immune cells, were digested into single-cell suspensions in liberase. Samples were collected from 5–10 mice in the same treatment group and stained with FixableViability-ef506 (eBioscience), CD45-AF488 (BioLegend, 30-F11), CD3-ef450 (eBioscience, 145-2C11), CD19-PB (eBioscience, 1D3), IA / IE-PECy7 (BioLegend, M5 / 114.15.2), CD11c-PE (eBioscience, N418), and CD11b-PerCpCy5.5 (BD, M1 / 70). Live CD45 cells were then stained. + CD3 - CD19 - MHCII hi CD11c + CD11b + Macrophages were placed in FBS-supplemented medium, centrifuged, and stored in TRIzol (Sigma). Gene expression profiles of the samples were analyzed in Macrogen, and the mean fold change in gene transcription levels between treatment groups was calculated. Genes with a fold change of 1.5 or greater between two comparisons were entered into DAVID v6.7 (a database for annotation, visualization, and comprehensive discovery, v6.7) for pathway analysis. Genes identified with significantly enhanced immune function were displayed on a heatmap (p < 0.05).

[0331] 11. In vivo cytotoxicity assay of Listeria monocytogenes

[0332] The analysis was performed by means of methods conventionally known. Specifically, Listeria monocytogenes expressing OVA peptide (LM-OVA) was injected at 5000 CFU / mouse into C57 / B16 mice. The mice were fed with Lactobacillus plantarum IMB19 strain every two days. On day 6, the spleen cells of naive C57 / bl6 mice were pulsed with OVA peptide and the cells pulsed or not with peptide were stained with CFSE or CTV, mixed at a 1 : 1 ratio and intravenously administered to the infected mice. Two hours later the mice infected with LM-OVA were sacrificed, the spleen cells / lymph nodes were harvested and the cell death rate of the peptide-pulsed spleen cells was detected using flow cytometry.

[0333] 12. Purification using chromatography

[0334] The isolated total capsular polysaccharide (tCPS: 28 mg) was purified by anion exchange Q-Sepharose fast flow (GE Healthcare; V = 4.4 mL, flow rate: 16 mL / h). The resin was packed, washed in 1 M NaCl and equilibrated with 10 volumes of 10 mM NaCl. Then, the total capsular polysaccharide (tCPS) was dissolved in 10 mM NaCl (5 mL) and adsorbed to the resin. Elution was performed stepwise by adding 16 mL of NaCl (10 mM, 100 mM, 200 mM, 400 mM, 700 mM and 1000 mM) sequentially. The eluates eluted with each concentration of NaCl were collected, desalted by dialysis (cut-off 1 kDa) and freeze-dried. The six fractions eluted at the respective NaCl concentrations were labelled CPS-X (X is the NaCl concentration used for elution, mM).

[0335] Molecular weight measurements of CPS-100 were extrapolated using an Agilent 1100 HPLC system and a TSK G-5000PWXL size exclusion column (30 cm x 7.8 mm) equilibrated with 50 mM NH4HCO3 as eluent (flow rate = 0.8 mL / min) and a refractive index detector to monitor the eluate. The column was calibrated by injecting dextran standards (50 μL of a 1 mg / mL solution) with known molecular weights (12 kDa, 50 kDa, 150 kDa 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 (LogPM = -0.811 mL + 11.7; R2= 0.98).

[0336] 13. Statistical analysis

[0337] Tumor growth curves were analyzed by two-way ANOVA with Sidak’s post-hoc test for comparison between two groups, Dunnett’s post-hoc test for comparison between multiple groups and control, or Tukey’s post-hoc test for comparison between two or more groups. For other comparisons, unpaired Student’s t-test was used when comparing two groups, and one-way ANOVA with Bonferroni correction for multiple testing was used when comparing two or more 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 data were analyzed using Flow-jo software v10.1.

[0338] Example 2: Isolation and identification of Lactobacillus plantarum IMB19

[0339] Lactobacillus plantarum IMB19 was isolated from homemade kimchi fermented using microorganisms derived from raw materials. To form colonies of Lactobacillus, serially diluted kimchi suspension was streaked on MRS broth plates (De Man, Rogosa, and Sharpe broth, Becton-Dickinson, USA). Single colonies were isolated and further cultured in MRS broth. Since it was not possible to distinguish strains from each other based on colony morphology, 14 isolated bacteria were subjected to PCR and 16s rRNA sequencing, and sequence similarity was confirmed using BLAST from NCBI. It was found that all isolated bacteria were lactic acid bacteria (LAB). Among the isolated bacteria, strains with at least 99% similarity to Lactobacillus plantarum were identified, and it was also confirmed that many of the isolated bacteria had 99% similarity to Weissella koreensis. These results are consistent with the conventional report that Lactobacillus plantarum and Weissella koreensis are dominant species in kimchi.

[0340] The analyzed Lactobacillus plantarum IMB19 16S rRNA sequence information is as follows.

[0341] - Lactobacillus plantarum IMB19 16S rRNA (785 forward) (SEQ ID NO: 3)

[0342] AGCGCTGGGATGATGCTAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTA

[0343] AGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCA

[0344] CAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATA

[0345] CTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTG

[0346] TCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCA

[0347] ACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCG

[0348] GAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTAC

[0349] AATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAG

[0350] TTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCA

[0351] TGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAA

[0352] CACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGGACAGATGATTAGG

[0353] GTGAAGTCGTAACAGGGTAAAACCGTAAAGATGTTCAACCCGCCACATCTGTCGCGTCTCCGT

[0354] CGTAGATATAAGAAAGCCAAAGGGCCTTTCTTCCATGGCTGGGTGTTCATGCAATAACATCGA

[0355] CCGGTTATCCACGACACAAGAAAGGATTACGTTGGTCCTGGTTGTGCGCTCAGGTTTTATAGT

[0356] GACAGCGGGCCTATTTGTATGGTGTAAACCGGAGTGCTAACAATCTTCTACAAGAAACAGCCT

[0357] GTACATAAATTTACGGCATATATATACCGGAACGTGGCTTGGCCACGTATGTTATTAACGCGG

[0358] GCTGGCAGGAACTTACTAGGCCGTGCCATTCCGGTGTCAAATCCGACCGAATCCGGGGACTCG

[0359] TCTCGCGGAAATGTGTTTCTTTTTAGAGACATGGATTCTTACAAACCGAGACCCTGTCATGCC

[0360] CGGGATGAGGGTCTGCCACTAACAACTTTCCGAACATGATGGGAAGAACCCCCTAACGGGCGC

[0361] CCACCTGGAGGAATTTGGGCCGGGGCACCACCGCCCGAGGTGGGGCGGAAAACCCCCTCCAGG

[0362] GGTCCCATCCTCAATTTTTCCGGGGGGGACCCCCCTCCCCCCCAAAATGAGGGAAAACCCCCG

[0363] GGGGGGCACCCCCAAAAGAAGGAGAGCCCCCCACCCTCACTCTTCCCGCCCGGCGTGCGGGGG

[0364] CGGGTTTTTTTTTCTGTCAAAATAAATTTTGTGTTGTTTGTGTGTTCCTCCCCCCCCCGCCGC

[0365] GGGGGCGGGGTTGTACTTTTTTCCCTCTCCATCCCCCCCCCACCACAAAAGAAAAGGAGGGGA

[0366] CGACACCCACAGTGGGTGTGTTTTT

[0367] - Lactobacillus plantarum IMB191 16S rRNA (907 reverse) (SEQ ID NO: 4)

[0368] TTGACGGGGGGGTCTCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAA

[0369] CCCCCCAACACTTAGCATTCATCGTTTACGGTATGGACTACCAGGGTATCTAATCCTGTTTGC

[0370] TACCCATACTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTT

[0371] CTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAA

[0372] GTTTCCCAGTTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAAAAAA

[0373] CCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCG

[0374] GCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCAATACCTGAACAGTTACT

[0375] CTCAGATATGTTCTTCTTTAACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCACGCGG

[0376] CGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTT

[0377] TGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCA

[0378] TGGTGAGCCGTTACCCCACCATCTAGCTAATACGCCGCGGGACCATCCAAAAGTGATAGCCGA

[0379] AGCCATCTTTCAAACTCGGACCATGCGGTCCAAGTTGTTATGCGGTATTAGCATCTGTTTCCA

[0380] GGTGTTATCCCCCGCTTCTGGGCAGGTTTCCCACGTGTTACTCACCAGTTCGCCACTCACTCA

[0381] AATGTAAATCATGATGCAAGCACCAATCAATACCAGAGTTCGTTCGACTTGCATGTATTAGGC

[0382] ACGCCGCCAGCGTTCGTCCTGACAGAGAGAAAAAAAAAAAAAAAAAAGGGCCGGGGGGATCGG

[0383] GGGGGGGGGGGGGGGGGGTGAGGGGTTGAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG

[0384] GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG

[0385] GGGGGGGGGGGGGGGGGGGGGGGGTGTGTGGGGGGGGGGGGGTTGTTGTTTTTGTTTGGGGGG

[0386] GGGGTTGTTTTTTGTGTGTGTTTTGTTGTTTGTTTGGGGGTGTGTTTTGTTGTGGGGTGGGGT

[0387] GTTGGGGGGGTTGGGGGGGGGGTGTTGTTTGGGGGGGGTGGGGGGGGGGGTTTTTTTGTTGTT

[0388] GTGTGGTTGTGTGTTGTGTGGTGGGTGGGGGGGGTGGTGTGTGTGTGGGGGTGGGGGGTGTTT

[0389] GGTGGGGGGGGGGTTGTTGTGGGGGGGTGGTGTTTGTTTTTTGTTTTTTTTTGTGTGTGGGGG

[0390] GGGGGGGTGGGGGGTGGTTTGTGGGGTGTTGTTTGTGTGTGGTTGGTGGTGGTGTGTGGGGGG

[0391] GTTGGGGGGGGGGGGGTTGTCTTTTTTGTTGGTGTTGGGTGTTTGTTGGTGTTGGTGTGTGGT

[0392] GGGGTGGTGTGGTGGGTGGGTGCTTGTTGTGTGTGTGGTGTGT

[0393] Example 3: Selection of Lactobacillus plantarum IMB19

[0394] To confirm the immunostimulatory effect of the isolated bacteria on immune cells, the effect of the bacterial culture isolates on murine spleen cells was tested, thus identifying bacteria having a stimulating effect on immune cells. After 48 hours of incubation of total spleen cells with the isolated bacteria, the cytokine levels in the culture supernatant were measured. Using IFN-γ as a well-known marker of inflammation and IL-10 as 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 was selected that induced negligible levels of IL-10 and very high levels of IFN-γ, and was named Lactobacillus plantarum IMB19 (colony 1). Figure 1

[0395] Example 4: Analysis of microbiological and biochemical properties of Lactobacillus plantarum IMB19

[0396] The colony morphology of Lactobacillus plantarum was small, smooth, round, and translucent. Cryosection TEM confirmed the amastigote and rod-shaped microbial colony of Lactobacillus plantarum (A). A thick capsule layer of individual bacteria was clearly observed. When cultured on sheep blood agar, neither a transparent zone nor a green zone was formed, thus the corresponding strain was confirmed to be non-hemolytic or γ-hemolytic, like other lactobacilli (B). Gelatinase activity was negative for Bacillus cereus ATCC 11778 used as a positive control within 5 days (C). In addition, the production of four biogenic amines was tested in a specialized medium (histamine, cadaverine, tyramine, and putrescine) for the strain. Lactobacillus plantarum IMB19 was found not to produce biogenic amines, unlike Escherichia coli ATCC 25922 (Table 1). Figure 2 Figure 2 Figure 2 ​​​​

[0397] [Table 1]

[0398] Production of biogenic amines activity of L. plantarum IMB19 and E. coli ATCC 25922

[0399]

[0400]

[0401] As shown in Table 2 below, the results of the antibiotic sensitivity test confirmed the sensitivity to most of the clinically relevant antibiotics, except kanamycin, as most of the Lactobacillus species (Appl. Environ. Microbiol. 85, (2019)).

[0402] [Table 2]

[0403] Antibiotic resistance test of L. plantarum IMB19

[0404]

[0405] *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.

[0406] Example 5: Analysis of the genetic properties of L. plantarum IMB19

[0407] The identification of L. plantarum was confirmed by sequence similarity comparison based on 16S rRNA sequencing. Moreover, it was distinguished from other genetically closely related species such as L. pentosus and L. paraplantarum by recA gene analysis using PCR (Appl. Environ. Microbiol. 67, 3450-3454 (2001)).

[0408] DNA isolation, Pac-Bio & Illumina Hi-seq sequencing and bioinformatics-based analysis were performed (Macrogen, Korea).

[0409] L. plantarum IMB19 was distinguished from other genetically closely related species by amplifying recA gene using recA gene-derived primers and comparing their bands. Since several L. plantarum strains are known to have similarities in the whole genome, phylogenetic analysis was performed based on average nucleotide index (OrthoANI) and L. plantarum IMB19 was identified as a unique strain (Figure 3 ). The presence of putative virulence genes was confirmed using VirulenceFinder 2.0 based on homology of virulence gene sequences to four known strains. Using a 90% nucleotide cutoff, no significant hits for virulence genes were found. As can be seen from ResFinder (J. Clin. Microbiol. 52, 1501-1510 (2014)), it was confirmed that there were no antibiotic resistance genes in the whole genome of Lactobacillus plantarum IMB19.

[0410] As disclosed in the above examples, Lactobacillus plantarum IMB19 was found to be a novel strain, which is characterized differently from conventionally reported Lactobacillus plantarum strains, and was deposited with the Korean Federation of Science and Technology Societies (KOFST) Culture Collection (KCTC) on October 21, 2020, under accession number KCTC 14337BP.

[0411] Example 6: Immunostimulatory effect of Lactobacillus plantarum IMB19 on murine T cells

[0412] Several strains belonging to Lactobacillus plantarum are well known for their beneficial effects on host immunity and health (Biomed. Res. Int. 2018, 9361614 (2018)), and it was confirmed that the Lactobacillus plantarum IMB19 of the present application directly affects murine immune cells. The inventors predicted that the uptake of bacterial antigens by antigen-presenting cells (APCs) makes it possible to change the activation status, thereby triggering other immune cells and thus modulating the immune system. Therefore, a co-culture system containing both APCs and CD4+ T cells, which represent the innate immune system and the adaptive immune system, respectively, was designed. CD11c+ APCs were exposed to bacteria for 20 hours (APC:bacteria = 1:100). These APCs were co-cultured with naive CD4+ T cells under suboptimal external stimulation that does not skew the immune phenotype. In order to confirm the differentiation of T cells into Th1, Th2, Th17, or regulatory T cells (Tregs), other transcription factors such as Tbet, GATA3, RORγ, and Foxp3 were analyzed. Lactobacillus plantarum IMB19 significantly induced the production of CD4+ RORγ+ Th17 cells compared to the other three strains without a special external influence Figure 4 A). The significant production of other Th cell subsets was uncertain and similar to other Lactobacillus plantarum strains Figure 4 B).

[0413] In order to verify the above results, the Th17 cell production by Lactobacillus plantarum IMB19 was tested under conditions where Th17 cell production was minimal. Lactobacillus plantarum IMB19 greatly increased the production of CD4+ RORγ+ Th17, which produced a significant amount of IL-17 Figure 5 A).

[0414] On the other hand, L. plantarum IMB19 did not induce significant amounts of Foxp3 Figure 5 B), thus, based on the results demonstrating whether L. plantarum IMB19 is able to generate Tregs from naive CD4+ T cells in the presence of sufficient concentrations of TGF-β, the generation of Tregs was significantly inhibited upon increasing concentrations of TGF-β.

[0415] Example 7: Demonstration of the in vivo role of L. plantarum IMB19 in promoting anti-tumor immune responses

[0416] L. plantarum IMB19 strain and various Lactobacillus strains (14 types) as controls were incubated with splenocytes to compare whether the production of cytokines by immune cells was altered. As a result, L. plantarum IMB19 showed significantly higher levels of IFN-γ and significantly lower levels of IL-10 than other Lactobacillus strains Figure 6 A). As a reference, another strain, L. murinus, exhibited the highest ability to induce IL-10 and low ability to produce IFN-γ Figure 6 A).

[0417] Since CD8+ T cells are the main anti-tumor effector cells, the ability of two isolated strains to stimulate CD8+ T cells was demonstrated. In the co-culture of dendritic cells (DCs) and CD8+ T cells primed with bacteria, the level of IFN-γ was increased when treated with L. plantarum IMB19 and significantly increased, in particular, compared to the case of treatment with L. murinus Figure 1 B). The activation of CD8+ T cells after co-culture with splenic and mesenteric lymph node antigen-presenting cells was changed in a bacterial concentration-dependent manner Figure 7 A and Figure 7 B).

[0418] L. plantarum IMB19 did not directly stimulate CD8+ T cells in the absence of dendritic cells. TCR-bearing CD8+ T cells against the mouse melanoma-specific antigen Pmel-1 were similarly activated in the presence of the antigen gp-100 Figure 7 C). In addition to DCs, macrophages are known to be APCs that play an important role in tumor growth and suppression, and thus the effect of L. plantarum IMB19 on CD8+ T cell stimulation through CD11b+F4 / 80+ peritoneal macrophages was examined. Similar to dendritic cells, L. plantarum IMB19 greatly increased the proportion of IFN-γ+ CD8+ T cells during co-culture of macrophages and CD8+ T cells Figure 6 C).

[0419] CD4+Foxp3+regulatory T cells (Tregs) accumulate in tumors and increase tumor progression by suppressing effector functions of CD8+T cells and other immune cells. Moreover, several bacteria have been reported to promote Treg production (Nature 453, 620-625 (2008); Sci. Immunol. 3, (2018)). Therefore, the inventors confirmed whether Lactobacillus plantarum IMB19 affects the induction of Tregs during CD11c+DC and CD4+T cell co-culture. Lactobacillus plantarum IMB19 significantly inhibited the production of Tregs in the presence of TGF-beta under strong Treg-skewing culture conditions Figure 6 D). This effect persisted even at lower TGF-beta levels and no Treg production Figure 8 A) was observed under any of the tested conditions. On the other hand, the levels of interleukin-17A (IL-17A) increased Figure 8 B), indicating the production of T-helper-17 cells (Th17). Interleukin-6 (IL-6) is required for Th17 production and inhibits Treg production in the presence of TGF-beta (Eur. J. Immunol. 40, 1830-1835 (2010)). Therefore, IL-6-deficient DCs produced Tregs during co-culture with CD4+T cells. Thus, the inhibition of IL-6 production by DCs elicited by Lactobacillus plantarum IMB19 under Treg-skewing culture conditions inhibited the production of Tregs.

[0420] To test whether CD8+T cells activated by Lactobacillus plantarum IMB19 were functional cytotoxic, the cytotoxicity was tested using an acute monocytosis Listeria monocytogenes infection model expressing the OVA antigen (LM-OVA) Figure 6 E). LM-OVA infected mice fed with Lactobacillus plantarum IMB19 showed a significant increase in the lysis of OVA-pulsed CFSE-targeted cells in vivo Figure 6 F). Moreover, the effect of Lactobacillus plantarum IMB19 on SPF (specific pathogen free) mice and GF (germ free) mice subcutaneously inoculated with B16.F10 melanoma was evaluated Figure 6 G). Lactobacillus plantarum IMB19 showed a significant inhibition of melanoma growth in both SPF and GF mice compared to Lactobacillus murinus Figure 6 H and Figure 6 I). To evaluate whether Lactobacillus plantarum IMB19 induced any dysbiosis leading to the anti-tumor effect, 16s ribosomal RNA sequencing was performed on fetal samples from tumor-bearing animals fed with Lactobacillus plantarum IMB19. However, there was no significant change in the microbial diversity in the feces of mice fed with Lactobacillus plantarum IMB19 compared to PBS control (Figure 9 A- Figure 9 C). This means that the anti-tumor immune modulation mediated by L. plantarum IMB19 corresponds to a L. plantarum IMB19 specific effect and is not a consequence of dysbiosis. Therefore, the data in this example indicate that L. plantarum IMB19 is a positive modulator of the cytotoxic T cell-mediated anti-tumor immune response in vivo.

[0421] Example 8: Analysis of the chemical characteristics of the crude polysaccharide fraction derived from L. plantarum IMB19

[0422] Based on the results of the analysis using transmission electron microscopy (TEM), it was confirmed that the cells of L. plantarum IMB19 are surrounded by a capsule material containing a carbohydrate composition composed of rhamnose, galactose, glucose and glucosamine, as well as small amounts of glycerol and ribitol Figure 10 and Figure 11 A). These two polyols are usually associated with the presence of teichoic acids, which are interconnected to each other via phosphodiester linkages (Tomita, Tanaka and Okada, 2017). Usually, the detection of these polyols is very difficult because the methanolysis does not cleave the phosphodiester linkages completely. Therefore, before methanolysis and acetylation, the GC-MS analysis was repeated by dephosphorylating the sample with aqueous HF and the presence of teichoic acids was confirmed based on the increase in the amount of both polyols. For the monosaccharides, rhamnose has the absolute L configuration and glucose and galactose have the D configuration Figure 12 ). On the other hand, for glucosamine, the D configuration is assumed based on the exclusive presence of the stereoisomer.

[0423] Example 9: Purification of the crude polysaccharide

[0424] The chemical analysis of the carbohydrate components indicates that the CPS can be a mixture of polymers. Therefore, the six fractions obtained by purifying the CPS using ion exchange chromatography are denoted as CPS-X, "X" indicating the concentration of the eluent used. The yields obtained by purification are as follows: CPS-10 11%, CPS-100 13%, CPS-200 9.0%, CPS-400 51%, CPS-700 7.0% and CPS-1000 7.9%. The use of 1 The H NMR analysis compares the spectral profile of each fraction with that of the original mixture (CPS) Figure 13 ).

[0425] In particular, the individual fractions are as follows Figure 13 ).

[0426] CPS-10 Figure 13b) : CPS-10 shows a rather inhomogeneous spectrum. The anomeric region of CPS-10 comprises two major strong signals at 4.2 ppm and 1.3 ppm, which are related to material different from carbohydrates.

[0427] CPS-100 Figure 13 c) : The anomeric region (5.6 ppm - 4.5 ppm) of CPS-100 comprises nine major signals with a doublet at 5.5 ppm indicating an a-configuration of the residue 3 J H1,H2 = 3.4 Hz) and phosphorylation 3 J H1,P = 7.0 Hz). Figure 14 In addition, an acetyl group (methyl at 2.06 ppm) and several methyl groups including deoxy residues (1.3 ppm) were observed, which are consistent with the presence of N-acetylglucosamine and rhamnose units, respectively.

[0428] CPS-200 Figure 13 d) : CPS-200 appears as a mixture with CPS-100 and CPS-400, showing four rather broad signals in the anomeric region and intense methyl signals at 1.6 ppm (a value not consistent with methyl groups in rhamnose) and in the crowded methanol region (4.4 ppm - 3.2 ppm).

[0429] The integration indicates a ratio of anomeric protons to methanol protons of 1.0 : 12, and the typically expected ratio is 1 : 6 or less, indicating that CPS-400 does not have a typical polysaccharide structure, and the increase in the ratio is due to the presence of ribitol and glycerol units, as confirmed by chemical analysis Figure 15 ). In addition, glucose is the most abundant monosaccharide, followed by small amounts of two monosaccharides, glucosamine (GlcN) and muramic acid (MurA), which are characteristic of peptidoglycan.

[0430] This observation indicates that CPS-400 is teichoic acid (TA).

[0431] CPS-700 and CPS-1000 Figure 13 f and Figure 13 g) : The anomeric region contains only some of the signals found in CPS-400, and the ratio of anomeric protons to methanol protons in both fractions is atypical. Further signals of non-carbohydrate material were observed.

[0432] CPS-10, CPS-700, and CPS-1000 are not abundant in CPS, so only small amounts of them were obtained.

[0433] Example 10: NMR analysis of CPS-100

[0434] By analyzing the entire dataset recorded at 310K 1 H- 1 H homonuclear (COSY, TOCSY, NOESY) and 1 H- 13 The structure of the capsular polysaccharide was determined by C heteronuclear (HSQC, HMBC, HSQC-TOCSY) 2D NMR spectroscopy (Table 3).

[0435] [Table 3]

[0436] The protons of the repeating unit of CPS-100 ( 1 H, static) NMR chemical shift and carbon ( 13 C (italicized) NMR chemical shift

[0437]

[0438] *C-3 with overlapping C and E, and its exact chemical shift cannot be definitively determined.

[0439] **C-5 with overlapping C, E, G, and H atoms, and whose exact chemical shift cannot be definitively determined.**

[0440] Temperature from 297K ( Figure 13 c) Increased to 310K ( Figure 14 This reduces the overlap between the three anodic signals at approximately 5.15 ppm and simplifies the NMR characteristics of the associated residues.

[0441] HSQC spectrum ( Figure 14 ) Displayed 1 The nine main anodic densities at H 5.6-4.5 are derived from... Figure 14 a and the capital letter AI in Table 3 indicate that the corresponding protons are all present in similar proportions. NMR analysis began at H⁻¹ (5.51 ppm) for A, showing three correlations in the TOCSY spectrum, similar to 3.85 ppm in the COSY spectrum. Figure 16 Therefore, this density was assigned to H-2, and H-3 (3.91 ppm) and H-4 (4.04 ppm) were assigned in a similar manner. Since there was no further correlation with H-1, A was identified as the galactose unit. Due to the strong H-4 / H-5 correlation, H-5 was identified in the NOESY spectrum. Figure 17 ), and two H-6 ( ) were identified through corresponding COZY-related methods. Figure 16 and Figure 17 ).

[0442] HSQC and HSQC-TOCSY spectra ( Figure 18a) the high chemical shift (67.1 ppm) based on C-6 defines all carbon chemical shifts of A, which is the alpha-galactose unit linked to O-6. Since the TOCSY spectrum of H-1 (5.12 ppm) derived from D has the same pattern as A, D is galactose, based on 3 J H1,H2 (3.9 Hz) value is in the alpha configuration, and the chemical shifts of H-6 / C-6 (4.04 ppm - 4.02 ppm / 65.6 ppm) imply phosphorylation at this position, which is in agreement with the data in the literature (Sechenkova et al., 2004). Therefore, D is 6P-alpha-Gal, and based on the high field values of all other carbon chemical shifts, it is determined that D has no branching anymore.

[0443] For B, H-1 (5.24 ppm) shows two strong TOCSY correlations with H-2 (4.25 ppm), which is in agreement with the COZY density ( Figure 16 ). As a result of reading TOCSY from this second proton ( Figure 16 ), all other protons of a methyl-containing unit are confirmed at 1.28 ppm, which is a general diagnostic of the pattern of rhamnose residues. Therefore, B is rhamnose in the alpha-configuration at the anomeric center, which is based on the similarity of its C-5 value (about 70.5 ppm) to the reference glycoside (69.4 ppm or 73.6 ppm for alpha or beta methyl glycoside, Bock & Pedersen, 1983), and is 3-substituted as defined by the glycosylation shift experienced on the corresponding carbon. The TOCSY pattern of residues C, E, G, and H (H-1 at 5.14 ppm, 5.10 ppm, 4.97 ppm, and 4.88 ppm, respectively) is similar to B, therefore they are alpha-rhamnose units, and the low field value of C-2 (77.8 ppm - 79.6 ppm) compared to the reference value (71.0 ppm, Bock & Pedersen, 1983) implies substitution at O-2.

[0444] For F, H-1 (5.01 ppm) has four TOCSY correlations ( Figure 16This is attributed to H-2 to H-5 in the COSY spectrum and is a pattern of glucose conformation residues. HSQC-TOCSY analysis of H-5 confirmed a correlation with the density at 69.5 ppm, attributed to C-6, and in turn correlated with H-6 (4.14 ppm and 3.96 ppm). Therefore, F was determined to be N-acetylglucosamine based on the C-2 (54.9 ppm) and H-2 (3.93 ppm) values ​​and the substitution at C-6 (69.4 ppm). The α-configuration was inferred from the shape of the anolyte signal (broad singlet) and the C-3 value (71.8 ppm), which was very similar to the reference α-glycoside (72.0 ppm, Bock & Pedersen, 1983). Finally, H-1 (4.50 ppm) of I had a TOCSY pattern representing all protons of the unit. Therefore, based on 13 C chemical shift, I was determined to be glucose substituted at C-3 (82.8 ppm) and based on 3 J H1,H2 The value (7.9 Hz) is in β-configuration.

[0445] By analyzing the HMBC spectrum ( Figure 5 b) and NOESY spectrum ( Figure 4 ), and deduce the sequence between residues. First, the correlation between the HMBC linked by H-1 of B and C-3 of I and the corresponding density is represented as B1I3( Figure 18 b). Other correlations were found in the same manner, such as C1E2, D1B3, F1G2, H1A6, and I1F6. Furthermore, the H-1 of E and the H-1 of G showed long-range correlations with carbon at approximately 79 ppm (a value similar to the C-2 of C and H). The precise allocation of these densities to E1H2 and G1C2 was inferred by analyzing the NOESY spectra correlated with the H-1 of E and the H-2 of H, as well as with the H-1 of G and the H-2 of C. Figure 17 These properties were confirmed by observing the detailed inverse correlations in H-2 proton amplification of different rhamnose units. Figure 19 ).

[0446] Finally, information regarding phosphorylation of A and D via phosphodiester linkages at O-1 and O-6, respectively, explains why no HMBC linkages occur between the H-1 of A and the C-6 of D, or between the H-6 of D and the C-1 of A, or why these protons show no NOE correlation between residues. In fact, HMBC spectroscopy cannot detect any correlation between the two units because the number of linkages (5 linkages) between the H-1 (or C-1) of A and the C-6 (or H-6) of D exceeds the sequence limit (3 linkages). Similarly, the density of the phosphate moiety between A and D keeps the protons of these units very far apart to provide detectable NOE action. Therefore, the repeating unit structure of CPS-100 is non-sugar, as... Figure 20 As reported in China.

[0447] In addition, a small number of NMR signals were studied to understand their characteristics. In the HSQC spectrum ( Figure 14 In ), the carbon chemical shifts of indicative densities such as Galα and Galβ (respectively...) 1 H / 13 C 5.26ppm / 93.5ppm and 4.57ppm / 97.8ppm) show α or β residues in their free reduced form. The TOCSY spectrum of H-1 ( Figure 16 The results show a typical pattern for sugars with a galactose conformation (i.e., for Galα, the third density almost overlaps with the COZY density). These findings can be explained by considering that the procedures used to separate CPS include sonication and trichloroacetic acid treatment, both of which are able to induce some cleavage of phosphodiester linkages, particularly the anomeric phosphate of galactose A, due to the extreme instability of phosphodiester linkages. Therefore, a small signal adjacent to a strong signal is expected ( Figure 14 (Indicated by "*") belongs to the first repeating residue and is linked to galactose in its free reduced form. However, its low intensity and dense methanol region hinder accurate identification of its properties. Finally, in the HSQC spectrum ( Figure 14 In a), for the anodic density integral, the sample has an average degree of polymerization of 4 and an average MW of approximately 6 kDa (MW of repeating units is 1500 Da), of which 11 kDa (calculated based on HPSEC) appears to be a slightly high estimate. Figure 21 ).

[0448] Example 11: NMR Analysis of CPS-400

[0449] The structural features of CPS-400 are analyzed in a manner similar to that described in the above embodiments (Table 4).

[0450] [Table 4]

[0451] NMR data of CPS-400 (600 MHz, 310 K). The 1 -position of the ribitol (or glycerol) points to the right; the column “motif” depicts the structure of the unit and in case of substituted Rbo and Gro units, it indicates the nature of the substituent and designates its position.

[0452]

[0453] *For all these residues, H-6 / C-6 is consistent.

[0454] **The assignment can be reversed.

[0455] First, the anomeric region of the HSQC spectrum ( Figure 22 a and Figure 22 b) shows several residues, where in the monosaccharide residues appear 1 signals at H 5.3 ppm - 5.1 ppm, while the signals at 1 H / 13 C 5.39 ppm / 75.5 ppm are not anomeric, but represent C-2 of the glycerol unit (Gro) of A and are shifted to lower field due to acylation. The substituent at O-2 is alanine (Ala), which is identified by 1 H / 13 C methyl at 1.64 / 16.5 ppm and Ha / Ca at 4.30 ppm / 50.2 ppm. Furthermore, H-1 / C-1 and H-3 / C-3 of A are equivalent and are identified based on the corresponding HSQC-TOCSY ( Figure 22 A) and TOCSY ( Figure 22 G) correlations at 4.11 ppm / 65.0 ppm.

[0456] Finally, the C-1 (or C-3) values indicate the identification of a second 1,3-diphosphorylated Gro unit (B) and a 1,5-diphosphorylated ribitol unit (Rbo, C) in the HSQC analysis, for which reference is made to Gerlach et al., 2018, which indicates that A is phosphorylated at both ends, as in the case of Gro-type teichoic acids, and the last residue suggests the presence of another teichoic acid based on a ribitol-phosphate backbone. For the monosaccharide units, the analysis focused on the strongest signals (D, E, F’ and F), which were identified as a- glucose units based on the efficient propagation of magnetization up to H-6 in the anomeric signals of the TOCSY spectrum. Based on 13 the similarity of the C chemical shifts, these Glc units are no longer substituted (Bock & Pedersen, 1983).

[0457] The positions of these units were inferred through HMBC spectral analysis and comparison with reference data. Indeed, E is connected to the O-2 junction of the Gro unit (H) (Shashkov, Potekina, Senchenkova, and Kudryashova, 2009), while F' is connected to the O-4 junction of the Rbo unit (I) (Streshinskaya et al., 2011). For D, H-1 shows a long-range correlation with carbon at 78.3 ppm (…). Figure 18 b), where the proton (4.30 ppm) is linked to "CH2" at 69.9 ppm in the HSQC-TOCSY spectrum. Figure 23 This new unit is denoted as G, and will be... 1 H / 13 Densities at C 4.30ppm / 78.3ppm and 4.14ppm / 66.9ppm were assigned to G4 and G5 ( Figure 18 c and Figure 23 G was identified as ribitol, and other signals were detected by HSQC-TOCSY spectral analysis. In fact, in 1 H / 13 The "CH2" density at C 4.16 ppm / 67.7 ppm has three correlations with the signal indicating G4, and this density is denoted as G1. Furthermore, C-1 (67.7 ppm) of G represents the unglycosylated but phosphorylated carbon at an adjacent position, as reported for I. This information leads to the assignment of the remaining two HSQC-TOCSY correlations to C-2 (70.5 ppm) and C-3 (80.6 ppm), and conversely, the corresponding H-2 (4.15 ppm) and H-3 (3.97 ppm) are identified in the HSQC spectrum. Figure 18 , Figure 23 (and Table 4). Therefore, G is Rbo glycosylated at O-3 and O-4, and the two units F and D ( Figure 22 (HMBC in b) is connected to it.

[0458] This type of substitution was identified in other L. plantarum strains, but their NMR data reported Rbo units without phosphoesters, so these chemical shifts cannot be compared with the data of the present application (Tomita et al., 2017). However, the results of the present application are similar to those of the phosphomurans of Bifidobacteria (Valueva et al., 2013), which indicate a reverse substitution pattern of ribitol with glucose units attached at C-2 and C-3 (Valueva et al., 2013). Interestingly, the NMR data reported by Valueva et al. (2013) for the dephosphorylated form are in agreement with those reported by Tomita et al. (2009) for 3,4-diglucosylated ribitol. Therefore, the substitution pattern of the ribitol units (2,3 or 3,4) has not been defined unambiguously. Thus, the NMR data of the present application indicate that CPS-400 is a mixture of both Gro- and Rbo-type phosphomurans, each showing the presence of several substituents in non-stoichiometric amounts. For the Gro-type TA, the non-stoichiometric substituents are alanine and a-glucose. For the Rbo-type TA, a-glucose does not appear at both O3 and O4 of ribitol, at O4 alone, or at any position. Attempts to separate both TAs by size-exclusion chromatography failed, as CPS-400 appeared as a symmetric peak of about 45 kDa ( Figure 21 ), and could not be further separated.

[0459] Example 12: Immunostimulatory activity of CPS-100 and CPS-400

[0460] The activity of CPS-100 and CPS-400 on the immune response was confirmed.

[0461] The effect of CPS on the immune system was confirmed using spleen cells in which all the immune cells are mixed in physiological ratios. The endpoint analysis was performed by testing different cytokines via ELISA. Cytokines are a group of secreted peptide / glycoproteins involved in cell signaling that mediate and regulate inflammatory or tolerogenic immune responses in vivo. Therefore, a shift in cytokines in the immune cell pool when exposed to CPS implies a similar effect in vivo. To confirm the immune response generated by CPS-100 and CPS-400, interferon-gamma (IFN-g) was used as an inflammatory marker and interleukin-10 (IL-10) as a regulatory cytokine for the analysis. The results of the study indicate that CPS-100 is immunostimulatory, as shown by high IFN-g production and negligible IL-10 production ( Figure 24 a and Figure 24 b). On the other hand, no levels of IFN-g were detected in CPS-400 (TA fraction) Figure 24a). Other cytokines were evaluated under similar conditions, including TNF-a (tumor necrosis factor-a), IL-6 (interleukin 6), IL-12 (interleukin 12), IL-17 (interleukin 17) and IL1-β (interleukin 1 beta).

[0462] CPS-100-stimulated cells produced very high levels of TNF-a, IL-6 and IL-12 Figure 24 C to Figure 24 E), while no IL-17 and IL1-β were detected. On the other hand, CPS-400 did not show a significant increase in any of the measured cytokines Figure 24 c and Figure 24 e). Since IFN-γ is a major immune stimulatory marker produced by various types of immune cells, to confirm the specificity of the immune stimulatory response of CPS, it was evaluated whether the production of IFN-γ was dependent on the concentration of CPS-100. During the induction of IFN-γ for 48 hours, the half maximal effective concentration (EC50) of CPS-100 was 3.16 μΜ Figure 24 f), indicating that CPS-100 can be used as an effective immune stimulator.

[0463] Therefore, CPS-100 exhibits similar immune stimulatory properties to the Lactobacillus plantarum IMB19 strain, which means that the effective molecule that exhibits the immune-enhancing activity of the Lactobacillus plantarum IMB19 strain is CPS-100.

[0464] Example 13: Improvement of CD8+ T cell function and anti-tumor immune action by Lactobacillus plantarum IMB19 and CPS

[0465] It was evaluated whether the activity of CPS as an in vitro immune stimulator could lead to in vivo tumor inhibitory activity. It was confirmed that the growth of subcutaneous melanoma was significantly reduced in the treatment group orally administered with Lactobacillus plantarum IMB19 and in the treatment group intraperitoneally administered with CPS Figure 25 A and Figure 25 B). The tumor growth delay in both groups was associated with CD8+ T cell infiltration Figure 25 C). The increase in IFN-γ production and frequency by tumor-infiltrating CD8+ T cells meant that the cytotoxic activity of CD8+ T cells was greatly increased due to the above administration Figure 25 E and Figure 25 F). Intratumoral CD4+ T cells also showed upregulation of IFN-γ production in both treatment groups Figure 25 G and Figure 25 H). On the contrary, there was no difference in the intratumoral Treg population compared to the PBS administration group Figure 26 A and Figure 26B). Oral administration of Lactobacillus plantarum IMB19 modulates tumor growth in EMT-6 breast carcinoma Figure 27 Therefore, these data indicate that CPS and Lactobacillus plantarum IMB19 enhance anti-tumor immune activities that suppress cancer growth.

[0466] Example 14: Increased intratumoral macrophage infiltration by CPS Figure 28 A). The number of macrophages was very high in mice administered with CPS was confirmed by flow cytometry Figure 28 A). In the same conditions, activation markers of CD11c+CD11b+macrophages and CD11c+dendritic cells were identified. Surprisingly, the activation status of CPS-treated dendritic cells did not show significant differences with the control group Figure 28 C). However, macrophages were more strongly activated and showed higher expression of CD11b, MHC I, MHC II, CD86 and CD40 Figure 28 B). To confirm the activation of the systemic adaptive immune system in the same mice, CD69 was identified in the draining lymph nodes as a marker of early activation of CD8+T cells. CD69 was significantly and markedly upregulated due to CPS administration compared to the control group Figure 28 D). These data indicate that CPS plays an important role in the activation of macrophages and is able to limit tumor growth.

[0467] Example 15: Macrophages are differentiated into inflammatory macrophages by CPS and macrophages are reprogrammed from M2 to M1 phenotype

[0468] To characterize the effect of CPS on macrophages, the change in phenotype was confirmed when macrophages were exposed to CPS. Peritoneal CD11b+F4 / 80+macrophages showed an activated phenotype when treated with CPS and a significant upregulation of MHC I, MHC II, CD68, iNOS2 and CD40 occurred in CPS-treated macrophages compared to LPS- or Pam3CSK4-treated macrophages Figure 28 E), thus indicating an M1 phenotype or inflammatory phenotype of macrophages. In particular, TLR2 was upregulated after CPS treatment similarly to Pam3CSK4, indicating that CPS can be a TLR2 ligand. However, in contrast to the in vivo experiments, the expression of CD80 and CD86 was not changed Figure 28 E).

[0469] Alternatively, activated macrophages or M2 phenotype macrophages significantly promote immune suppression and enhance tumor growth (On Oncol. 28, xii18-xii32 (2017)) (Front Oncol. 9, 421 (2019)). Thus, these results indicate that CPS in tumors is able to reprogram M2 macrophages to M1 phenotype. In fact, the number of IL-4-induced M2 phenotype peritoneal macrophages was significantly increased when using CPS compared to when using LPS and Pam3CSK4 ( Figure 28 F). In addition, the inflammatory macrophage marker iNOS2 was significantly upregulated independently of MHC I, MHC II, CD40, and CD68 ( Figure 5 F). These data indicate that CPS treatment generates inflammatory macrophages and reprograms immunosuppressive macrophages to an immunostimulatory phenotype.

[0470] While specific embodiments of the present application have been disclosed in detail herein, it will be apparent to those skilled in the art that the description is merely exemplary in nature and that the scope of the application is not limited to the specific embodiments described. Thus, the scope of the present application should be defined by the appended claims and equivalents thereof.

[0471] Accession No.

[0472] Name of Depository Institution: Korea Research Institute of Bioscience and Biotechnology

[0473] Accession No.: KCTC 14337BP

[0474] Date of Deposit: October 21, 2020

[0475] Industrial Applicability

[0476] According to the present application, the novel Lactobacillus plantarum IMB19 strain and polysaccharides derived from the strain exhibit strong ability to stimulate CD8+ T-cell activity and superior Treg cell inhibitory activity, and stimulate and enhance anti-tumor immune responses by various mechanisms such as CPS increasing macrophage infiltration and macrophage differentiation / reprogramming to an inflammatory (M1) phenotype within tumors. Thus, the strain and polysaccharides derived from the strain according to the present application can be used for immunomodulation, particularly for immune enhancement, in a subject, and are capable of inducing and improving anti-tumor immune responses, thereby inhibiting tumor growth. The novel strain and polysaccharides derived from the strain according to the present application can be used for preventing, ameliorating, or treating, for example, tumors, infectious diseases, and various immunological diseases caused by or as symptoms of immune dysfunction.

[0477] SEQUENCE LIST FREE TEXT

[0478] Electronic file attached. SEQUENCE LISTING <110> EMINOBIOM LTD <120> Novel lactobacillus plantarum strains, polysaccharides derived from the strains and uses thereof <130> PF-B2760-CN <140> PCT / KR2021 / 000351 <141> 11 January 2021 <150> KR 10-2020-0003493 <151> 10 January 2020 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 16S rRNA primer (forward) <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 16S rRNA primer (reverse) <400> 2 tacggytacc ttgttacgac tt 22 <210> 3 <211> 1522 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus plantarum IMB19 16S rRNA_785 forward <400> 3 agcgctggga tgatgctagt gttggagggt ttccgccctt cagtgctgca gctaacgcat 60 taagcattcc gcctggggag tacggccgca aggctgaaac tcaaaggaat tgacgggggc 120 ccgcacaagc ggtggagcat gtggtttaat tcgaagctac gcgaagaacc ttaccaggtc 180 ttgacatact atgcaaatct aagagattag acgttccctt cggggacatg gatacaggtg 240 gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 300 acccttatta tcagttgcca gcattaagtt gggcactctg gtgagactgc cggtgacaaa 360 ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg 420 tgctacaatg gatggtacaa cgagttgcga actcgcgaga gtaagctaat ctcttaaagc 480 cattctcagt tcggattgta ggctgcaact cgcctacatg aagtcggaat cgctagtaat 540 cgcggatcag catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 600 catgagagtt tgtaacaccc aaagtcggtg gggtaacctt ttaggaacca gccgcctaag 660 gtgggacaga tgattagggt gaagtcgtaa cagggtaaaa ccgtaaagat gttcaacccg 720 ccacatctgt cgcgtctccg tcgtagatat aagaaagcca aagggccttt cttccatggc 780 tgggtgttca tgcaataaca tcgaccggtt atccacgaca caagaaagga ttacgttggt 840 cctggttgtg cgctcaggtt ttatagtgac agcgggccta tttgtatggt gtaaaccgga 900 gtgctaacaa tcttctacaa gaaacagcct gtacataaat ttacggcata tatataccgg 960 aacgtggctt ggccacgtat gttattaacg cgggctggca ggaacttact aggccgtgcc 1020 attccggtgt caaatccgac cgaatccggg gactcgtctc gcggaaatgt gtttcttttt 1080 agagacatgg attcttacaa accgagaccc tgtcatgccc gggatgaggg tctgccacta 1140 acaactttcc gaacatgatg ggaagaaccc cctaacgggc gcccacctgg aggaatttgg 1200 gccggggcac caccgcccga ggtggggcgg aaaaccccct ccaggggtcc catcctcaat 1260 ttttccgggg gggacccccc tcccccccaa aatgagggaa aacccccggg ggggcacccc 1320 caaaagaagg agagcccccc accctcactc ttcccgcccg gcgtgcgggg gcgggttttt 1380 ttttctgtca aaataaattt tgtgttgttt gtgtgttcct cccccccccg ccgcgggggc 1440 ggggttgtac ttttttccct ctccatcccc cccccaccac aaaagaaaag gaggggacga 1500 cacccacagt gggtgtgttt tt 1522 <210> 4 <211> 1554 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus plantarum IMB1916S rRNA_907 reverse <400> 4 ttgacggggg ggtctccagg cggaatgctt aatgcgttag ctgcagcact gaagggcgga 60 aaccccccaa cacttagcat tcatcgttta cggtatggac taccagggta tctaatcctg 120 tttgctaccc atactttcga gcctcagcgt cagttacaga ccagacagcc gccttcgcca 180 ctggtgttct tccatatatc tacgcatttc accgctacac atggagttcc actgtcctct 240 tctgcactca agtttcccag tttccgatgc acttcttcgg ttgagccgaa ggctttcaca 300 tcagacttaa aaaaccgcct gcgctcgctt tacgcccaat aaatccggac aacgcttgcc 360 acctacgtat taccgcggct gctggcacgt agttagccgt ggctttctgg ttaaataccg 420 tcaatacctg aacagttact ctcagatatg ttcttcttta acaacagagt tttacgagcc 480 gaaacccttc ttcactcacg cggcgttgct ccatcagact ttcgtccatt gtggaagatt 540 ccctactgct gcctcccgta ggagtttggg ccgtgtctca gtcccaatgt ggccgattac 600 cctctcaggt cggctacgta tcattgccat ggtgagccgt taccccacca tctagctaat 660 acgccgcggg accatccaaa agtgatagcc gaagccatct ttcaaactcg gaccatgcgg 720 tccaagttgt tatgcggtat tagcatctgt ttccaggtgt tatcccccgc ttctgggcag 780 gtttcccacg tgttactcac cagttcgcca ctcactcaaa tgtaaatcat gatgcaagca 840 ccaatcaata ccagagttcg ttcgacttgc atgtattagg cacgccgcca gcgttcgtcc 900 tgacagagag aaaaaaaaaa aaaaaaaagg gccgggggga tcgggggggg gggggggggg 960 ggtgaggggt tgaggggggg gggggggggg gggggggggg gggggggggg gggggggggg 1020 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 1080 gggggggggg ggggtgtgtg gggggggggg ggttgttgtt tttgtttggg gggggggttg 1140 ttttttgtgt gtgttttgtt gtttgtttgg gggtgtgttt tgttgtgggg tggggtgttg 1200 ggggggttgg ggggggggtg ttgtttgggg ggggtggggg ggggggtttt tttgttgttg 1260 tgtggttgtg tgttgtgtgg tgggtggggg gggtggtgtg tgtgtggggg tggggggtgt 1320 ttggtggggg gggggttgtt gtgggggggt ggtgtttgtt ttttgttttt ttttgtgtgt 1380 gggggggggg ggtggggggt ggtttgtggg gtgttgtttg tgtgtggttg gtggtggtgt 1440 gtgggggggt tggggggggg ggggttgtct tttttgttgg tgttgggtgt ttgttggtgt 1500 tggtgtgtgg tggggtggtg tggtgggtgg gtgcttgttg tgtgtgtggt gtgt 1554

Claims

1. A type of Lactobacillus plantarum ( Lactobacillus plantarum The IMB19 strain has the accession number KCTC14337BP.

2. A composition for immune stimulation comprising the strain according to claim 1 as an active ingredient.

3. A pharmaceutical composition for treating tumors, comprising the strain according to claim 1 as an active ingredient, wherein the tumor is melanoma or breast cancer.

4. A method for generating inflammatory T cells, comprising: (a) Initiating antigen-presenting cells using the strain according to claim 1; as well as (b) Co-culture the antigen-presenting cells with T cells. The inflammatory T cells mentioned therein are CD4+RORγ+Th17 cells and / or CD8+ T cells.

5. The method according to claim 4, wherein the antigen-presenting cells are selected from macrophages, B cells, dendritic cells (DCs), and Langerhans cells.

6. A method for generating M1 phenotype macrophages, comprising: Macrophages were differentiated into M1 phenotype macrophages by treating them with the strain according to claim 1. as well as The differentiated M1 phenotype macrophages were obtained.

Images