Obligate anaerobic human intestinal microorganisms and their uses for cancer treatment

Abstract

The present invention relates to a novel Akkermansia strain and its use. The novel Akkermansia HB03 strain (Akkermansia HB03) provided by the present invention lacks toxic genes, thus does not cause side effects in vivo, and also has excellent anti-cancer activity. Therefore, it can be widely used in the development of medicaments for preventing or treating cancer.

Description

Technical Field

[0001] The present disclosure relates to a novel Akkermansia HB03 strain belonging to the genus Akkermansia of the family Verrucomicrobiae, which is an obligate anaerobic human intestinal microorganism. Background Art

[0002] Akkermansia species strains are Gram-negative, obligate anaerobic, human intestinal mucin-degrading bacteria. Akkermansia species strains are known to be non-motile, non-spore-forming (Yuji Naito et al., A next-generation beneficial microbe: Akkermansia muciniphila, J Clin Biochem Nutr. 2018 Jul; 63(1): 33-35.) and oval. It can use mucin as the only carbon and nitrogen source and can be cultured under strict anaerobic conditions in a culture medium containing intestinal mucin. In addition, it can proliferate (colonize) in the gastrointestinal tract of various animal species.

[0003] It is well known that intestinal microorganisms play an important role in the regulation of immune responses against various types of tumors and have the potential to inhibit tumor cells and enhance immunity in the tumor ecosystem. Therefore, they can be used to treat tumors.

[0004] Cancer is one of the leading causes of death among adults worldwide. Despite improvements in treatment performance with the development of surgical techniques and the introduction of anticancer radiotherapy, approximately half of all diagnosed patients still die from cancer. Therefore, cancer is considered one of the major problems in many countries. Therefore, there is an urgent need to develop methods to treat and prevent cancer.

[0005] public

[0006] Technical issues

[0007] The inventors of the present disclosure have been working on discovering novel strains belonging to the genus Akkermansia. As a result, they discovered a novel strain in the genus Akkermansia that is different from the previously known Akkermansia muciniphila strain, and completed the present disclosure by identifying that the strain exhibits superior anticancer activity compared to the Akkermansia muciniphila strain.

[0008] Technical Solution

[0009] The present disclosure relates to providing a novel Akkermansia HB03 strain (Akkermansia sp. HB03) deposited under the deposit number KCCM12318P.

[0010] The present disclosure also provides a pharmaceutical composition for preventing or treating cancer, which contains one or more selected from the novel Akkermansia HB03 strain, its inactivated strain, its culture, its vesicles, its proteome and its metabolome.

[0011] The present disclosure also relates to providing a method for preventing or treating cancer, which comprises the step of administering the pharmaceutical composition to an individual.

[0012] The present disclosure also provides a food composition for preventing or alleviating cancer, which contains one or more selected from the novel Akkermansia HB03 strain, its inactivated strain, its culture, its vesicle, its proteome and its metabolome.

[0013] Beneficial effects

[0014] The novel Akkermansia HB03 strain provided by the present disclosure has no toxic genes, so it does not cause side effects in vivo. In addition, the composition containing the strain or vesicle, metabolome or inactivated strain derived from the strain shows excellent tumor growth inhibition effect and shows significantly excellent anticancer effect when co-administered with another anticancer agent, particularly immune checkpoint inhibitor. In addition, the protein from the strain also shows the effect of effectively inhibiting cancer cell growth and inhibiting tumor formation and progression as a metabolome. Therefore, the strain can be widely used in the development of medicaments for preventing or treating cancer. Sequence Listing <110> Health Bio Company <120> Obligate anaerobic human intestinal microorganisms for cancer treatment and their uses <130> OPA20175-PCT <150> KR 10-2019-0118268 <151> 2019-09-25 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 287 <212> PRT <213> Artificial sequence <220> <223> HB03-01 <400> 1 Ile Val Asn Ser Lys Arg Ser Glu Leu Asp Lys Lys Ile Ser Ile Ala 1 5 10 15 Ala Lys Glu Ile Lys Ser Ala Asn Ala Ala Glu Ile Thr Pro Ser Arg 20 25 30 Ser Ser Asn Glu Glu Leu Glu Lys Glu Leu Asn Arg Tyr Ala Lys Ala 35 40 45 Val Gly Ser Leu Glu Thr Ala Tyr Lys Pro Phe Leu Ala Ser Ser Ala 50 55 60 Leu Val Pro Thr Thr Pro Thr Ala Phe Gln Asn Glu Leu Lys Thr Phe 65 70 75 80 Arg Asp Ser Leu Ile Ser Ser Cys Lys Lys Lys Asn Ile Leu Ile Thr 85 90 95 Asp Thr Ser Ser Trp Leu Gly Phe Gln Val Tyr Ser Thr Gln Ala Pro 100 105 110 Ser Val Gln Ala Ala Ser Thr Leu Gly Phe Glu Leu Lys Ala Ile Asn 115 120 125 Ser Leu Val Asn Lys Leu Ala Glu Cys Gly Leu Ser Lys Phe Ile Lys 130 135 140 Val Tyr Arg Pro Gln Leu Pro Ile Glu Thr Pro Ala Asn Asn Pro Glu 145 150 155 160 Glu Ser Asp Glu Ala Asp Gln Ala Pro Trp Thr Pro Met Pro Leu Glu 165 170 175 Ile Ala Phe Gln Gly Asp Arg Glu Ser Val Leu Lys Ala Met Asn Ala 180 185 190 Ile Thr Gly Met Gln Asp Tyr Leu Phe Thr Val Asn Ser Ile Arg Ile 195 200 205 Arg Asn Glu Arg Met Met Pro Pro Pro Ile Ala Asn Pro Ala Ala Ala 210 215 220 Lys Pro Ala Ala Ala Gln Pro Ala Thr Gly Ala Ala Ser Leu Thr Pro 225 230 235 240 Ala Asp Glu Ala Thr Ala Pro Ala Ala Pro Ala Ile Gln Gln Val Ile 245 250 255 Lys Pro Tyr Met Gly Lys Glu Gln Val Phe Val Gln Val Ser Leu Asn 260 265 270 Leu Val His Phe Asn Gln Pro Lys Ala Gln Glu Pro Ser Glu Asp 275 280 285 <210> 2 <211> 127 <212> PRT <213> artificial sequence <220> <223> HB03-03 <400> 2 Ile Pro Glu Ser Ser Val Pro Ala Ala Ala Val Ser Ser Val Lys Gly 1 5 10 15 Ala Tyr Pro Asn Ala Gly Pro Ile Glu Trp Asp Ser Glu Met His Gly 20 25 30 Met Glu Arg Ile Tyr Glu Ala Glu Phe Arg Ile Asn Gly Phe Gln Phe 35 40 45 Asp Val Lys Ile Ala Pro Asp Gly Lys Ile Val Arg Ile Lys Glu Glu 50 55 60 Ile Ala Ala Ser Ser Leu Pro Glu Ala Val Arg Ala Ser Val Leu Lys 65 70 75 80 Arg Tyr Pro Gly Cys Arg Ile Lys Glu Ala Glu Lys Ile Thr Glu Gly 85 90 95 Glu Leu Val Arg Tyr Lys Val Glu Val Ala Asp Ser Arg Asp Asp Tyr 100 105 110 Asp Val Leu Ile Ala Pro Asp Gly Asn Ile Leu His Val Asp Arg 115 120 125 BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Shown are the results of searching the genome base sequences of a total of 66 Akkermansia species from NCBI and clustering based on the average nucleotide identity of the MASH genome base sequences using dRep.

[0016] Figure 2 The results of drawing an approximate maximum likelihood tree of Akkermansia strains are shown.

[0017] Figure 3 Shown are the results of extracting 120 strain-specific genes from the gene presence / absence map.

[0018] Figure 4a and Figure 4bShown are the results of checking the presence of genes by calculating the BLAST alignment score ratio (BSR) using LS-BSR (Large BLAS Alignment Score Ratio).

[0019] Figure 5 Shown are the results of identifying the presence of a pathogenicity island (PAI) in the HB03 strain.

[0020] Figure 6 The results of studying the sedimentation degree of the HB03 strain and the Akkermansia muciniphila ATCC BAA-835 strain after centrifugation of the strain culture at 7000 rpm are shown. While the HB03 strain was completely separated from the supernatant, the Akkermansia muciniphila strain was not separated.

[0021] Figure 7 Results of post-treatment (administered after tumor inoculation) of tumor-bearing mice with HB03 and HB03(P) and analysis of tumor growth differences from the control group are shown. (A) Tumor growth kinetics observed after BALB / c was inoculated with CT26 colorectal cancer cells and then treated with HB03 and HB03(P) daily from day 6 to day 18. (B) Tumor growth kinetics observed after C57BL / 6 JAX mice were inoculated with LLC lung cancer cells and then treated with HB03 and HB03(P) daily from day 6 to day 18. (C) Tumor growth kinetics observed after C57BL / 6 JAX mice were inoculated with B16F10 melanoma cells and then treated with HB03 and HB03(P) daily from day 6 to day 18. (D) Tumor growth kinetics observed after C57BL / 6 JAX mice were inoculated with Panc02 pancreatic cancer cells and then treated with HB03 and HB03(P) daily from day 6 to day 18. (E) Tumor growth kinetics observed after C57BL / 6 JAX mice were inoculated with GL261 glioma cells and then treated with HB03 and HB03(P) daily from day 6 to day 18. (F) Tumor growth kinetics observed after C57BL / 6 JAX mice were inoculated with MCA205 sarcoma cells and then treated with HB03 and HB03(P) daily from day 6 to day 18.

[0022] Figure 8Results of post-treatment (administration after tumor inoculation) of tumor-bearing mice with HB03, A. muciniphila, or B. longum and analysis of tumor growth differences from the control group are shown. (A) Tumor growth kinetics observed after BALB / c mice were inoculated with CT26 colorectal cancer cells and then treated with HB03, A. muciniphila, or B. longum daily from day 5 to day 16. (B) Tumor growth kinetics observed after C57BL / 6 JAX mice were inoculated with LLC lung cancer cells and then treated with HB03, A. muciniphila, or B. longum daily from day 5 to day 16. (C) Tumor growth kinetics observed after C57BL / 6 JAX mice were inoculated with B16F10 melanoma cells and then treated with HB03, A. muciniphila, or B. longum daily from day 5 to day 14.

[0023] Fig. 9 Results of pre-treatment (administered before tumor inoculation) of tumor-bearing mice with HB03 and HB03(P) and analysis of tumor growth differences from the control group are shown. (A) Tumor growth kinetics observed after daily treatment with HB03 and HB03(P) from day 7 to day 18 before inoculation of BALB / c mice with CT26 colorectal cancer cells. (B) Tumor growth kinetics observed after daily treatment with HB03 and HB03(P) from day 7 to day 18 before inoculation of C57BL / 6 JAX mice with LLC lung cancer cells. (C) Tumor growth kinetics observed after daily treatment with HB03 and HB03(P) from day 7 to day 18 before inoculation of C57BL / 6 JAX mice with B16F10 melanoma cells. (D) Tumor growth kinetics observed after daily treatment with HB03 and HB03(P) from day 7 to day 18 prior to inoculation of C57BL / 6JAX mice with Panc02 pancreatic cancer cells.

[0024] Fig.10 Results of post-treatment (administered after tumor inoculation) of tumor-bearing mice with HB03 or Akkermansia muciniphila (AKK P2261) and analysis of tumor growth differences from the control group are shown. Tumor growth kinetics were observed after C57BL / 6 JAX mice were inoculated with MC38 colorectal cancer cells and then treated daily with HB03 or Akkermansia muciniphila (AKK P2261) from day 5 to day 14.

[0025] Fig.11Results of treating tumor-bearing mice with immune checkpoint inhibitors (aPD-1 mAb) and HB03 alone or in combination and analyzing tumor growth differences are shown. (A) Tumor growth kinetics observed after BALB / c mice were inoculated with CT26 colorectal cancer cells and mIgG or aPD-1 mAb was administered 5 times at intervals of 2 days until day 16, while control substances or HB03 were administered daily from day 5. (B) Tumor growth kinetics observed after C57BL / 6 JAX mice were inoculated with LLC lung cancer cells and mIgG or aPD-1 mAb was administered 5 times at intervals of 2 days until day 16, while control substances or HB03 were administered daily from day 5. (C) Tumor growth kinetics observed after C57BL / 6 JAX mice were inoculated with B16F10 melanoma cells and mIgG or aPD-1 mAb was administered 5 times at intervals of 2 days until day 15, while control substances or HB03 were administered daily from day 5. (D) Tumor growth kinetics observed after C57BL / 6 JAX mice were inoculated with GL261 glioma cells and administered mIgG or aPD-1 mAb 5 times at 2-day intervals until day 15, while control substances or HB03 were administered daily starting from day 5.

[0026] Fig.12 The results of treating tumor-bearing mice with immune checkpoint inhibitors (aCTLA-4 mAb) and HB03 alone or in combination and analyzing the difference in tumor growth are shown. (A) Tumor growth kinetics were observed after C57BL / 6 JAX mice were inoculated with MCA205 sarcoma cells and mIgG or aCTLA-4 mAb was administered 5 times at intervals of 2 days until day 15, while control substances or HB03 were administered daily from day 5. (B) Tumor growth kinetics were observed after C57BL / 6 JAX mice were inoculated with MC38 colorectal cancer cells and mIgG or aCTLA-4 mAb was administered 5 times at intervals of 2 days until day 15, while control substances or HB03 were administered daily from day 5.

[0027] Fig.13 The results of treating tumor-bearing mice with immune checkpoint inhibitors (aPD-L1 mAb) and HB03 alone or in combination and analyzing the difference in tumor growth are shown. Tumor growth kinetics were observed after C57BL / 6 JAX mice were inoculated with MC38 colorectal cancer cells and mIgG or aPD-L1 mAb was administered 5 times at intervals of 2 days until day 15, while control substances or HB03 were administered daily from day 5.

[0028] Fig.14The results of treating mice resistant to immune checkpoint inhibitors (aPD-1 mAb) due to antibiotic administration with immune checkpoint inhibitors (aPD-1 mAb), HB03 and Bifidobacterium longum alone or in combination and analyzing the differences in tumor growth are shown. (A) Starting 2 weeks before BALB / c mice were inoculated with CT26 colorectal cancer cells, mice were fed antibiotics and water. Only water was given from the 3rd day after inoculation, and 48 hours later (starting from the 5th day after inoculation), mIgG or aPD-1 mAb was administered 5 times at intervals of 2 days until the 16th day, while control substances or HB03 were administered every 2 days. Then, the tumor growth dynamics were observed. (B) Starting 2 weeks before C57BL / 6 JAX mice were inoculated with B16F10 melanoma cells, mice were fed antibiotics and water. Only water was given from day 3 after inoculation, and 48 hours later (from day 5 after inoculation), mIgG or aPD-1 mAb was administered 5 times at 2-day intervals until day 16, while control substances or HB03 were administered every 2 days. Then, tumor growth kinetics were observed.

[0029] Fig.15 The results of treating mice resistant to immune checkpoint inhibitors (aCTLA-4 mAb) due to antibiotic administration with immune checkpoint inhibitors (aCTLA-4 mAb) and HB03 alone or in combination and analyzing the difference in tumor growth are shown. Starting 2 weeks before inoculation of C57BL / 6 JAX mice with MC38 colorectal cancer cells, the mice were fed antibiotics and water. Only water was given from the 3rd day after inoculation, and 48 hours later (starting from the 5th day after inoculation), mIgG or aCTLA-4 mAb was administered 5 times at intervals of 2 days until the 16th day, while control substances or HB03 were administered every 2 days. Then, the tumor growth dynamics were observed.

[0030] Fig.16 The results of treating tumor-bearing mice with anticancer agents (5-FU (5-fluorouracil) or oxaliplatin) and HB03 alone or in combination and analyzing the difference in tumor growth are shown. The tumor growth kinetics were observed after C57BL / 6 JAX mice were inoculated with MC38 colorectal cancer cells and then administered PBS, 5-FU or oxaliplatin 5 times at intervals of 2 days until the 16th day, while control substances or HB03 were administered every day from the 5th day.

[0031] Fig.17 The pET-30a vector is shown.

[0032] Fig.18Results of treating tumor cells with proteins secreted by HB03 and analyzing tumor growth and apoptosis are shown. After treating CT26 colorectal cancer cells (A) and LLC lung cancer cells (B) with different concentrations of HB03-01, a protein derived from HB03, for 48 hours, cell survival was analyzed. (C) After treating LLC lung cancer cells with HB03-01 or HB03-03, a protein derived from HB03, for 24 hours, it was investigated whether apoptosis was induced based on the activation of caspase-3, a protein that regulates apoptosis.

[0033] Fig.19 The results of treating tumor-bearing mice with HB03 secreted protein and analyzing the difference in tumor growth from the control group are shown. (A) C57BL / 6 JAX mice were inoculated with MC38 colorectal cancer cells and then administered a control substance or HB03-01 (AT1) 5 times at intervals of 2 days starting from day 5 until day 16, after which the tumor growth kinetics were observed. (A) C57BL / 6 JAX mice were inoculated with MC38 colorectal cancer cells and then administered a control substance or HB03-03 (AT3) 5 times at intervals of 2 days starting from day 5 until day 16, after which the tumor growth kinetics were observed.

[0034] Best Practices

[0035] Hereinafter, the present disclosure is described in detail. The description and exemplary embodiments disclosed in the present disclosure may also be applied to other descriptions and exemplary embodiments. That is to say, all combinations of various elements disclosed in the present disclosure are included in the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the following description.

[0036] In one aspect, the present disclosure provides a novel Akkermansia HB03 strain (Akkermansia sp. HB03) with a deposit number of KCCM12318P.

[0037] In the present disclosure, the term "Akkermansia strain" refers to a class of bacteria that degrades human or animal intestinal mucin, which is Gram-negative and obligate (strict) anaerobic. Akkermansia strains can use mucin as the only carbon and nitrogen source and can be cultured under strict anaerobic conditions in a medium containing intestinal mucin. In addition, it can proliferate (colonize) in the gastrointestinal tract of various animal species.

[0038] The inventors of the present disclosure have isolated a novel strain of Akkermansia (Akkermansia species) from a stool sample of a healthy Korean, which is a bacterium that degrades intestinal mucin and is named Akkermansia HB03 strain (Akkermansia species HB03). In one example of the present disclosure, in order to investigate whether the isolated Akkermansia strain is a novel strain, the genome of the isolated strain was compared with the genome of a previously known Akkermansia strain. Specifically, based on the average nucleotide identity of the MASH genome base sequence, the genome base sequences of a total of 66 Akkermansia strains (Akkermansia species) were retrieved and clustered ( Figure 1 ) (Matthew R Olm et al. dRep: a tool for fast and accurate genome comparisons that enables improved genome recovery from metagenomes through de-replication. ISME J. 2017 Dec; 11(12): 2864-2868.). Only 44 strains belonging to a single species cluster (cluster 3) were subsequently analyzed. The high-speed independent pan-genome pipeline Roary was used, which uses an assembly annotated in GFF3 format prepared by Prokka (Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014 Jul 15; 30(14): 2068-9.) and calculates the pan-genome. Then, an approximate maximum likelihood tree was constructed using FastTree2 (Price MN, Dehal PS, Arkin AP. FastTree 2--approximately maximum-likelihood trees for large alignments. PLoS One. 2010 Mar 10; 5(3): e9490.) Figure 2 ). From the gene presence / absence map ( Figure 3 ) extracted 120 strain-specific genes and named the strain with this gene-specific property "Akkermansia HB03 strain (Akkermansia species HB03)". By studying whether there is a gene cluster pathogenicity island (PAI) in the whole genome of HB03 strain, it was confirmed that HB03 strain does not have a pathogenicity island ( Figure 5 ). “Akkermansia HB03 strain (Akkermansia species HB03)” was deposited in the Korea Culture Center for Microorganisms on September 11, 2018, with the deposit number KCCM12318P.

[0039] The novel Akkermansia strain provided by the inventors of the present disclosure has not been reported as a common strain or a patent strain or deposited in a microbial resource preservation and distribution institution. It was isolated from the feces of healthy Koreans for the first time and deposited as a patent strain by the inventors of the present disclosure.

[0040] It was demonstrated that the novel Akkermansia HB03 strain exhibited 2-fold or more cell productivity in the modified BTTM medium of the present disclosure compared to Akkermansia muciniphila (Table 1).

[0041] In particular, Akkermansia muciniphila has problems with centrifugation and difficulty in large-scale production because it tends to suspend rather than sediment, while the HB03 strain is easier to mass-produce and genome edit than its model strain due to its ease of centrifugation ( Figure 6 ). In addition, the HB03 strain lacks pathogenic (toxic) genes and therefore does not cause side effects in vivo. In addition, it has been confirmed that when growing in a mucin-free medium, Akkermansia muciniphila requires L-threonine, a degradation product of mucin, while the HB03 strain can grow to 1.32x10 9 The concentration of cells / mL was 1.547 cells / mL (Table 2).

[0042] This suggests that, unlike the A. muciniphila -type strains, the HB03 strain can grow by using various carbon and nitrogen sources other than mucin or L-threonine.

[0043] Since the novel Akkermansia HB03 strain exhibits excellent anticancer activity, the novel Akkermansia strain, its inactivated strain, its culture, its vesicles, its proteome, its metabolome, etc. can be used as an active ingredient for preventing or treating cancer. This will be described in detail later.

[0044] For example, the microorganism of the present disclosure can be a microorganism that has been "substantially purified". The expression "substantially purified" refers to a strain or strain mixture that has been substantially concentrated from a sample. The microorganism of the present disclosure in a sample can be substantially purified by being concentrated to 50%, 60%, 70%, 80%, 90%, 95% or more than 95%. In addition, the sample may contain 50%, 40%, 20%, 15%, 1% or less than 1% of microorganisms that lack the effects of the microorganism of the present disclosure or have undesirable effects, but is not limited thereto.

[0045] For example, the microorganisms contained in the composition of the present disclosure may be coated by a coating for oral administration. The coating may be a coating that prevents the microorganisms from being degraded in the intestine, but is not limited thereto.

[0046] For example, the microorganisms or cultures, vesicles, proteomes or metabolomes of the present disclosure can be isolated microorganisms. The term "isolated" refers to a substance that is present in a non-naturally occurring environment or does not exist naturally. It includes the basic separation of substances associated with nature and found in nature. The isolated substance can be, for example, a substance in which one or more than one substance associated with the natural state has been removed, its form has been changed or its amount has been changed, but is not limited thereto.

[0047] In another aspect, the present disclosure provides a pharmaceutical composition for preventing or treating cancer, comprising one or more selected from a novel Akkermansia HB03 strain, an inactivated strain thereof, a culture thereof, a vesicle thereof, a proteome thereof, and a metabolome thereof.

[0048] The terms used herein are the same as above.

[0049] In the present disclosure, the term "cancer" includes solid cancers and blood cancers, but is not limited thereto. Cancer includes various types of cancers, such as cancers of the head, neck, eye, mouth, throat, esophagus, chest, bone, lung, colon, rectum, stomach, prostate, breast, ovary, kidney, bladder, liver, pancreas or brain, but is not limited thereto. Unless otherwise indicated, cancer may include primary cancers and metastatic cancers. Cancer may include, for example, lung cancer, pancreatic cancer, gastric cancer, liver cancer, colorectal cancer, brain cancer, breast cancer, thyroid cancer, bladder cancer, esophageal cancer, leukemia, ovarian cancer, melanoma, head and neck cancer, skin cancer, prostate cancer, colorectal cancer, hepatocellular carcinoma, fibrosarcoma or cervical cancer.

[0050] For purposes of this disclosure, cancer may include, but is not limited to, cancers that exhibit resistance to anticancer agents.

[0051] The anticancer agent may include a chemical anticancer agent, a targeted anticancer agent, or an immune checkpoint inhibitor, but is not limited thereto.

[0052] In this disclosure, the term "chemical anticancer agent" refers to a therapeutic agent developed to inhibit the proliferation of cancer cells. The mechanism of action varies from anticancer agent to anticancer agent, such as inhibition of DNA synthesis, etc., and is used as the main treatment tool for cancers that respond well to anticancer agents, such as acute leukemia, lymphoma, testicular cancer, etc. Chemical anticancer agents are also called cytotoxic anticancer agents. Examples of chemical anticancer agents include gemcitabine, cytarabine, carboplatin (Paraplatin), cisplatin (Platinol, Platinol-AQ), crizotinib (Xalcor), cyclophosphamide (Cytoxan, Neosar), docetaxel (Tasotre), doxorubicin (Adriamycin), erlotinib (Tarceva), etoposide (Vepesid), 5-fluorouracil (5-FU), irinotecan (Capto), liposome-encapsulated doxorubicin (Doxil), methotrexate (Forlex, Mexate, methotrexate), paclitaxel (Taxol, Abraxane), topotecan (Hemisil), trabectedin (Yondelis), vincristine (Oncovin, Vincasar PFS), vinblastine (Velban), etc.

[0053] In the present disclosure, the term "targeted anticancer agent" refers to an anticancer agent that attacks cancer cells only by recognizing specific markers possessed by cancer cells and does not harm normal cells. It has several times higher killing ability against cancer cells. Examples of targeted anticancer agents include imatinib mesylate (Gleevec), cetuximab (Erbitux), gefitinib (Iressa), omotinib (Olita), osimertinib (Tagrisso), bevacizumab (Avastin), sorafenib (Nexavar), oxaliplatin (Eloxatin), sunitinib (Sutent), etc.

[0054] In the present disclosure, the term "immune checkpoint inhibitor" refers to an antibody therapeutic agent that appears as a third-generation anticancer agent after the first-generation surgery, chemotherapy (chemical anticancer agents), radiotherapy anticancer drugs and targeted therapy (second-generation anticancer drugs or targeted anticancer drugs). Its advantage is that it exhibits a strong anticancer effect by activating the immune function of immune cells and is suitable for various indications. In addition, it has almost no side effects because it only works within a given time period to prevent damage to normal cells due to excessive immune activity. Examples of immune checkpoint inhibitors include PD-1 (programmed cell death protein 1), CTLA4 (cytotoxic T lymphocyte-associated antigen 4), PD-L1 (programmed death ligand 1), PD-L2 (programmed death ligand 2), A2AR (adenosine A2A receptor), B7-J3 (B7 family molecule), B7-H4 (B7 family molecule), BTLA (B and T lymphocyte attenuator), Kirs (killer cell immunoglobulin-like receptor), LAG3 (lymphocyte activation gene 3), TIM-3 (T cell immunoglobulin and mucin domain-containing protein-3), HAVCR2 (hepatitis A virus cellular receptor 2), VISTA (V-domain Ig inhibitor of T cell activation), etc.

[0055] The inhibitor may be a monoclonal antibody (mAb) targeting the above substances, such as an antibody against PD-1 (αPD-1 mAb), an antibody against CTLA-4 (αCTLA-4 mAb), or an antibody against PD-L1 (αPD-L1 mAb), but is not limited thereto.

[0056] In the present disclosure, the term "prevention" refers to any action of inhibiting or delaying the onset of a disease by administering a pharmaceutical composition according to the present disclosure, and the term "treatment" refers to any action of improving or favorably changing disease symptoms by administering a pharmaceutical composition.

[0057] "Preventing or treating cancer" may refer to an "anti-cancer" effect.

[0058] In the present disclosure, the term "anti-cancer" includes the prevention, treatment, improvement of symptoms and alleviation of cancer.

[0059] In the present disclosure, the term "anti-tumor" refers to the reduction or elimination of malignancies arising from solid cancers.

[0060] In the present disclosure, the term "inactivated strain" refers to a strain that has been non-functionalized or killed. Methods for non-functionalized or killed strains include heat inactivation, pH inactivation, chemical inactivation, etc. The inactivated strain may include cell lysates or extracellular products from the non-functionalized or killed strain.

[0061] In the present disclosure, the inactivation may be thermal inactivation by heating, but is not limited thereto.

[0062] In the present disclosure, the term "culture" may include the culture itself obtained by culturing the strain, or a concentrate or a lyophilized product of the supernatant obtained by removing the strain from the culture.

[0063] In the present disclosure, the culture refers to a product obtained by culturing the novel Akkermansia HB03 strain. In a broad sense, the culture may be the entire culture of the Akkermansia strain, the supernatant of the culture, the lysate of the culture, a fraction thereof, etc. The supernatant may be obtained by centrifuging the culture of the strain, the lysate may be obtained by physically treating or sonicating the strain, and the fraction may be obtained by centrifuging, chromatography, etc. the culture, supernatant, lysate, etc.

[0064] In the present disclosure, the term "culturing" refers to a series of actions to grow microorganisms under appropriate conditions controlled by humans.

[0065] In the present disclosure, cultivation can be understood to refer to the method of cultivating the Akkermansia strain provided by the present disclosure. Cultivation can be carried out using methods well known in the art. Specifically, cultivation can be carried out continuously by batch, batch feeding or repeated batch feeding process.

[0066] Akkermansia strains should be cultured in a medium containing mucin, suitable carbon and nitrogen sources, amino acids, vitamins, etc. under anaerobic conditions such as temperature and pH that meet the requirements of specific strains. As a carbon source, mucin or a mixture with N-acetylglucosamine, glucose and xylose can be used. In addition, sugars or carbohydrates such as galactosamine, sucrose, lactose, fructose, maltose, starch and cellulose, oils or fats such as soybean oil, sunflower oil, castor oil, coconut oil, etc., fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid can be used. These substances can be used alone or in combination. As a nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used; or organic nitrogen sources such as amino acids such as glutamic acid, methionine and glutamine, soy protein, tryptone, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation products, defatted soybean cake or its degradation products, etc. These nitrogen sources can be used alone or in combination. The culture medium can contain potassium dihydrogen phosphate, dipotassium hydrogen phosphate or its sodium salt as a phosphorus source. The phosphorus source includes potassium dihydrogen phosphate, dipotassium hydrogen phosphate or its sodium salt. In addition, inorganic compounds such as sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate can be used. Finally, substances necessary for growth, such as amino acids and vitamins, can be used. In addition, in order to prevent foaming, defoamers such as fatty acid polyethylene glycol esters can be used.

[0067] In one example of the present disclosure, the Akkermansia HB03 strain and the Akkermansia muciniphila type strain (ATCC BAA-835) of the present disclosure are cultured using a modified BTTM medium maintained at 37° C., but is not limited thereto.

[0068] In the present disclosure, the term "vesicle" refers to a structure present in cells, which is in the shape of a relatively small bag and encloses and transports chemical substances.

[0069] In the present disclosure, the term "proteome" refers to all proteins expressed by various biological entities such as specific cells, tissues, organisms, etc.

[0070] In the present disclosure, the proteome may be proteins derived from a novel Akkermansia strain such as HB03-01, HB03-02, or HB03-03, but is not limited thereto.

[0071] In the present disclosure, the term "metabolome" refers to the collection of all metabolites present in biological samples such as cells, tissues, body fluids, etc. It is usually composed of substances of 1500 Dalton or less than 1500 Dalton in size, and is divided into endogenous metabolome (amino acids, nucleic acids, fatty acids, carbohydrates, amines, glycolipids, short peptides, vitamins, hormones, etc.) and exogenous metabolome (such as drugs, foods, additives, toxic substances, etc.). Usually, target substances produced by DNA, RNA or protein degradation are also classified as metabolomes. Since the metabolome changes rapidly and dynamically every second, it immediately reflects the phenotypic changes caused by drug reactions or food intake compared to the genome or proteome (Korean Society of Biochemistry and Molecular Biology).

[0072] HB03-01 may consist of the amino acid sequence of SEQ ID NO 1, and HB03-03 may consist of the amino acid sequence of SEQ ID NO 2.

[0073] Although the HB03-01 or HB03-03 protein is defined in the present disclosure as consisting of the amino acid sequence of SEQ ID NO 1 or SEQ ID NO 2, mutations that may occur by adding insignificant sequences before or after the amino acid sequence of SEQ ID NO 1 or SEQ ID NO 2 or naturally occurring mutations or silent mutations thereof are not excluded. In addition, it is obvious to those skilled in the art that a protein having the same or equivalent activity as a polypeptide including the amino acid sequence of SEQ ID NO 1 or SEQ ID NO 2 corresponds to the protein of the present disclosure. As a specific example, the protein of the present disclosure may be a protein consisting of an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology or identity with the amino acid sequence of SEQ ID NO 1 or SEQ ID NO 2. In addition, it is obvious that a polypeptide consisting of an amino acid sequence having such homology or identity and exhibiting an efficiency equivalent to that of the protein, in which some sequences have been deleted, modified, substituted or added, is also included in the scope of the protein of the present disclosure.

[0074] That is, it is obvious that even if a "polypeptide consisting of an amino acid sequence having a specific sequence number" is described in the present disclosure, if a polypeptide has the same or equivalent activity, the polypeptide can be used, the amino acid sequence of which is composed of a polypeptide showing an efficiency equivalent to that of a polypeptide consisting of an amino acid sequence having a specific sequence number in which some of the sequence is deleted, modified, substituted or added. For example, a "polypeptide consisting of the amino acid sequence of SEQ ID NO 1" can include a polypeptide having the same or equivalent activity as that of a "polypeptide consisting of the amino acid sequence of SEQ ID NO 1".

[0075] The polypeptide can be conjugated to another sequence or linker for identification, purification or synthesis of the polypeptide.

[0076] The pharmaceutical composition may include extracts, fractions, suspensions, etc. from the novel Akkermansia HB03 strain, its inactivated strain, its culture, its vesicles, its proteome, and its metabolome, but is not limited thereto.

[0077] In the present disclosure, the term "extract" includes extracts obtained by extracting the novel Akkermansia strain provided by the present disclosure, dilutions or concentrates of the extracts, dried products obtained by drying the extracts, crude or purified products of the extracts, mixtures thereof, and the like. There is no particular limitation on the extraction method used to obtain the extract, and methods commonly used in the art can be used. Non-limiting examples of extraction methods include hot water extraction, ultrasonic extraction, filtration, reflux extraction, and the like, and they can be performed alone or in combination. There is no particular limitation on the extraction solvent used to obtain the extract, and any solvent known in the art can be used. Non-limiting examples of extraction solvents include water, alcohol, or a mixed solvent thereof, and they can be used alone or in combination.

[0078] In the present disclosure, the term "fraction" refers to a product obtained by fractionation to separate a specific component or a group of specific components from a mixture containing various components. The method for obtaining the fraction is not particularly limited, and a method commonly used in the art can be used. The fractionation solvent used to obtain the fraction is not particularly limited, and any solvent known in the art can be used. Non-limiting examples of fractionation solvents include polar solvents such as water, alcohols, etc. and non-polar solvents such as hexane, ethyl acetate, chloroform, dichloromethane, etc. They can be used alone or in combination.

[0079] In the present disclosure, the term "suspension" refers to a mixture in which fine solid particles are uniformly dispersed in a liquid without being dissolved.

[0080] In the present disclosure, the suspension may be one in which a specified amount of lyophilized strains are dispersed in water, PBS (phosphate buffered saline) or culture medium (liquid broth). The term "suspension" may be used interchangeably with "strain composition".

[0081] In the present disclosure, the pharmaceutical composition according to the present disclosure may be co-administered with an anticancer agent.

[0082] In the present disclosure, the term "co-administration" refers to the simultaneous or independent or non-independent (mixed) administration of the pharmaceutical composition of the present disclosure and an anticancer agent for treating cancer at a certain time interval for the prevention, treatment, or alleviation or reduction of the symptoms of cancer. Compared with the single administration of the pharmaceutical composition of the present disclosure or the anticancer agent, a higher anticancer activity can be obtained by co-administration. In addition, compared with the single administration of the anticancer agent, a higher anticancer activity can be obtained for cancers that show resistance to the anticancer agent.

[0083] The anticancer agent may be, for example, a chemical anticancer agent, a targeted anticancer agent, or an immune checkpoint inhibitor, but is not limited thereto. Chemical anticancer agents, targeted anticancer agents, or immune checkpoint inhibitors are the same as described above. However, any previously known anticancer agent may be used without limitation thereto.

[0084] In one example of the present disclosure, co-administration of immune checkpoint inhibitors (αPD-1 mAb, αCTLA-4 mAb or αPD-L1 mAb) and HB03 to colorectal cancer (CT26, MC38), lung cancer (LLC), melanoma (B16F10), glioma (GL261) and sarcoma (MCA205) significantly increased the anti-tumor effect compared to treatment with immune checkpoint inhibitors alone ( Figures 11 to 13 ).

[0085] In addition, in another example of the present disclosure, the co-administration of anticancer agents (5-FU (5-fluorouracil), oxaliplatin) and HB03 to a colorectal cancer (MC38) animal model significantly enhanced the anti-tumor effect compared to the administration of the anticancer agents alone ( Fig.16 ).

[0086] These results indicate that the novel Akkermansia strains of the present disclosure effectively inhibit tumorigenesis with their anticancer activity while reducing the administered dose of anticancer agents.

[0087] In the present disclosure, the pharmaceutical composition according to the present disclosure may be co-administered to an individual who exhibits resistance to an anticancer agent.

[0088] The anticancer agent may include an immune checkpoint inhibitor, a chemical anticancer agent, or a targeted anticancer agent, but is not limited thereto.

[0089] Immune checkpoint inhibitors are the same as above. 70% or more of cancer patients do not respond to immune checkpoint inhibitors or develop resistance.

[0090] In one example of the present disclosure, when an immune checkpoint inhibitor (αPD-1 mAb or αCTLA-4 mAb) and HB03 were co-administered to colorectal cancer (CT26, MC38) and melanoma (B16F10) animal models resistant to immune checkpoint inhibitors, co-administration of αPD-1 mAb and HB03 significantly increased the anti-tumor effect compared to administration of αPD-1 mAb alone, and this effect was superior to the effect of co-administration of previously known Akkermansia muciniphila strains or Bifidobacterium longum strains ( Fig.14 In addition, co-administration of αCTLA-4 mAb and HB03 significantly increased the anti-tumor effect compared with administration of αCTLA-4 mAb alone ( Fig.15 ).

[0091] This result indicates that due to the anti-cancer activity of the novel Akkermansia strains of the present disclosure, co-administration of the novel Akkermansia strains of the present disclosure effectively inhibits tumorigenesis even for cancers that exhibit resistance to immune checkpoint inhibitors.

[0092] The pharmaceutical composition of the present disclosure may include the extract in an amount of 0-0001 wt % to 50 wt %, specifically 0.01 wt % to 10 wt %, based on the total weight of the composition, but is not limited thereto.

[0093] According to the pharmaceutical composition of the present disclosure, it is also possible to contain suitable carriers, excipients or diluents that are usually used to prepare pharmaceutical compositions. Specifically, the pharmaceutical composition can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external dosage forms, suppositories or sterilized injections according to common methods. In the present disclosure, the carrier, excipient or diluent that can be included in the pharmaceutical composition can be lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talcum powder, magnesium stearate and mineral oil. This dosage form is made of commonly used diluents or excipients such as fillers, extenders, adhesives, wetting agents, disintegrants, surfactants, etc. Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and solid dosage forms are prepared by mixing extracts or parts thereof with at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talcum powder are also used. Liquid dosage forms for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water or liquid paraffin, various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. may also be contained. Dosage forms for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized dosage forms, and suppositories. For non-aqueous solutions or suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, esters for injection such as ethyl oleate, etc. may be used. As the matrix of suppositories, semi-synthetic fatty acid esters (witepsol), polyethylene glycol, Tween 61, cocoa butter, laurel fat, glycerin gelatin, etc. may be used.

[0094] In one example of the present disclosure, as a result of pre-treatment (administered before tumor inoculation) or post-treatment (administered after tumor inoculation) of the strain composition HB03 or HB03(P), wherein the strain composition HB03 is prepared by resuspending a lyophilized novel Akkermansia HB03 strain in liquid culture medium and HB03(P) is prepared by heat-inactivating HB03 for a predetermined time in tumor animal models of colorectal cancer (CT26), lung cancer (LLC), pancreatic cancer (Panc02), melanoma (B16F10), glioma (GL261) and sarcoma (MCA205), tumor growth was significantly reduced ( Figure 7 and Fig. 9), and the antitumor effect was superior to that when the muciniphilic Akkermansia strain (ATCC BAA-835 strain) or the longum Bifidobacterium strain (ATCC BAA-999 strain) was administered ( Figure 8 Furthermore, when the anticancer effects were compared after administration of AKK p2261 or HB03, administration of HB03 further increased the anticancer effects ( Fig.10 ). This confirmed that HB03 had a superior anticancer effect compared to the other 32 strains.

[0095] In another example of the present disclosure, HB03-01 is a protein derived from HB03 that significantly reduces the proliferation of colorectal cancer and lung cancer cells ( Fig.18 A and Fig.18 B) and together with another protein HB03-03, activates the apoptosis-regulating enzyme (caspase-3) in lung cancer cells ( Fig.18 C) The results indicate that the novel Akkermansia strains disclosed herein can be used to prevent or treat cancer due to their anti-cancer activity.

[0096] In another aspect, the present disclosure provides a method for preventing or treating cancer, comprising the step of administering the pharmaceutical composition to an individual.

[0097] The terms used below are the same as those mentioned above.

[0098] In the present disclosure, the term "subject" refers to any animal that exhibits or has a risk of exhibiting symptoms of cancer. The individual can be effectively treated by administering the pharmaceutical composition of the present disclosure to an individual suspected of having cancer. The pharmaceutical composition of the present disclosure can be applied to any individual to prevent or treat cancer without particular limitation. For example, the individual can be a human, a non-human mammal such as a monkey, a dog, a cat, a rabbit, a guinea pig, a rat, a mouse, a cow, a sheep, a pig, a goat, etc., a bird, a fish, etc. In addition, it can be a non-human animal, but is not limited thereto.

[0099] In the present disclosure, the term "administering" refers to introducing a substance into an individual by a suitable method.

[0100] The pharmaceutical composition of the present disclosure can be applied in a pharmaceutically effective amount. In the present disclosure, the term "pharmaceutically effective amount" refers to an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dose level can be determined according to the severity of the disease, the activity of the drug, the age, weight, health status and gender of the patient, the sensitivity of the patient to the drug, the time of application, the route of administration, the excretion rate, the duration of treatment, the drug used simultaneously with the composition of the present disclosure, and other factors well-known in the medical field. The pharmaceutical composition of the present disclosure can be applied as an independent therapeutic agent or in combination with another therapeutic agent, and can be applied successively or simultaneously with another therapeutic agent. It can also be applied as a single dosage form or multiple dosage forms. Taking all of the above factors into account, it is important to apply the composition in a minimum amount that can show the maximum effect without causing side effects.

[0101] Specifically, for mammals, the dosage of the pharmaceutical composition according to the present disclosure can be, for example, about 0.0001 mg / kg to 100 mg / kg per day, particularly 0.001 mg / kg to 10 mg / kg. The daily dose of the pharmaceutical composition of the present disclosure can be administered once or several times a day, but is not particularly limited thereto. The above dosage does not limit the scope of the present disclosure in any way.

[0102] The route of administration or the mode of administration of the pharmaceutical composition of the present disclosure is not particularly limited, and any route of administration or mode of administration can be adopted, as long as the composition can be delivered to the desired target position. Specifically, the composition can be administered orally or by various parenteral routes. Non-limiting examples of routes of administration include oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal or intranasal routes. For local treatment, it can be administered by a suitable method including intralesional administration if necessary. In addition, the pharmaceutical composition can be administered by any device capable of delivering the active substance to the target cell.

[0103] On the other hand, the present disclosure provides a food composition for preventing or alleviating cancer, comprising one or more than one selected from the novel Akkermansia HB03 strain, its inactivated strain, its culture, its vesicle, its proteome and its metabolome.

[0104] The terms used herein are the same as above.

[0105] In the present disclosure, the term "alleviation" refers to any effect of improving or favorably altering the symptoms of a disease by administering a composition. In the present disclosure, the disease refers to cancer.

[0106] In the present disclosure, the term "food" includes any food in the ordinary sense, such as meat, sausage, bread, chocolate, candy, snacks, sweets, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, soup, beverages, tea, drinks, alcoholic beverages, vitamin complexes, functional health foods, health foods, etc.

[0107] Functional health food is the same as food for special health purposes (FoSHU), which refers to food with high medical effect that has been processed and can effectively play a biological regulatory function in addition to nutrition. Here, the term "functional" refers to the effect of obtaining nutrients that are beneficial to the regulation of human body structure and function or hygienic purposes such as physiological effects. The food disclosed in the present invention can be prepared by methods commonly used in the art, or by adding raw materials and ingredients commonly used in the art. In addition, the food can be prepared into any dosage form that is considered to be a food, but is not limited to this. The food composition disclosed in the present invention can be prepared into various dosage forms, and its advantage is that it does not have side effects that may occur during long-term use because it contains food raw materials different from general medicines and has excellent portability. Therefore, the food disclosed in the present invention can be taken as a supplement to enhance the effect of preventing or alleviating cancer.

[0108] Health food refers to food that has a positive effect on maintaining or improving health compared to ordinary food, and refers to health food or food used for health. In some cases, the terms functional health food, health food and food used for health can be used interchangeably.

[0109] Specifically, functional health food is a food made by adding the composition of the present disclosure to food raw materials such as beverages, teas, spices, chewing gum, candies, etc., or a food made into capsules, powders, suspensions, etc. When ingested, it has a special effect on health. However, unlike general drugs, it uses food as a raw material, so it has the advantage of not having side effects that may occur when using drugs for a long time.

[0110] The food composition of the present disclosure can be very usefully used because it can be taken routinely and thus a high effect of preventing or alleviating cancer can be expected.

[0111] The food composition may further comprise a physiologically acceptable carrier. The type of carrier is not particularly limited, and any carrier commonly used in the art may be used.

[0112] In addition, the food composition may contain additional ingredients commonly contained in food compositions for improving smell, taste, visual appearance, etc. For example, it may contain vitamins A, C, D, E, B1, B2, B6 or B 12, niacin, biotin, folic acid, pantothenic acid, etc. In addition, it may also contain minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), etc. In addition, it may also contain amino acids such as lysine, tryptophan, cysteine, and valine.

[0113] In addition, the food composition may include food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder, advanced bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), dyes (tar pigments, etc.), colorants (sodium nitride, sodium nitrite, etc.), bleaching agents (sodium sulfite), flavor enhancers (monosodium glutamate, etc.), sweeteners (sweetness, sodium cyclamate, saccharin, etc.), flavoring agents (vanillin, lactones, etc.), expanders (alum, D-potassium tartrate, etc.), enhancers, emulsifiers, thickeners, coating agents, gum bases, defoamers, solvents, improvers, etc. Additives can be selected according to the type of food and used in appropriate amounts.

[0114] According to common methods, the composition of the present invention can be added alone or together with another food or food ingredient. The mixed amount of active ingredients can be appropriately determined according to the purpose of use (prevention, health care or treatment). Usually, in order to prepare food or beverage, the addition amount of the food composition of the present invention can be 50 weight parts or less than 50 weight parts, particularly 20 weight parts or less than 20 weight parts based on food or beverage. However, if it is taken in for a long time for the purpose of health or sanitation improvement, a less amount can be used. And, since there is no safety problem, the active ingredient can be used in large quantities.

[0115] For example, the food composition of the present disclosure can be used as a health drink composition, in which case it can also include various flavoring agents, natural carbohydrates, etc. as ordinary beverages. Natural carbohydrates can be monosaccharides such as glucose or fructose, disaccharides such as maltose or sucrose, polysaccharides such as dextrin or cyclodextrin, or sugar alcohols such as xylitol, sorbitol, erythritol, etc. As sweeteners, natural sweeteners such as thaumatin or stevia extract or synthetic sweeteners such as saccharin or aspartame can be used. Per 100mL of the health drink composition of the present disclosure, the ratio of natural carbohydrates is generally about 0.01g to 0.04g, specifically about 0.02g to 0.03g.

[0116] In addition, the health drink composition may include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectin acid, pectinate, alginic acid, alginates, organic acids, protective colloid thickeners, pH control agents, stabilizers, preservatives, glycerol, alcohols, carbonating agents, etc. In addition, it may include pulp for preparing natural fruit juice, fruit juice beverages or vegetable beverages. These ingredients may be used alone or in combination. The proportion of these additives is not important, but is generally 0.01 to 0.1 parts by weight per 100 parts by weight of the health drink composition of the present disclosure.

[0117] The content of active ingredients in the food composition of the present disclosure can vary, as long as the effect of preventing or alleviating cancer can be achieved. For example, based on the gross weight of the food composition, the content of the strain according to the present disclosure can be 0.00001 wt % to 100 wt % or 0.01 wt % to 80 wt %, but is not limited thereto.

[0118] In another aspect, the present disclosure provides use of a novel Akkermansia HB03 strain (Akkermansia sp. HB03) with a deposit number of KCCM12318P, an inactivated strain thereof, a culture thereof, a vesicle thereof, a proteome thereof, or a metabolome thereof for preventing or treating cancer.

[0119] The terms used herein are the same as above.

[0120] Embodiments of the invention

[0121] Hereinafter, the present disclosure is described in more detail by examples. However, these examples are provided only to illustrate the present disclosure and the scope of the present disclosure is not limited by these examples.

[0122] Example 1: Identification of novel strains by genome base sequencing

[0123] A total of 66 Akkermansia species were searched from NCBI and clustered based on the average nucleotide identity (ANI) of the MASH genome base sequences using dRep ( Figure 1 (Matthew R Olm et al. dRep: a tool for fast and accurate genome comparisons that enables improved genome recovery from metagenomes through de-replication. ISMEJ. 2017 Dec; 11(12): 2864-2868.) Only 44 strains belonging to a single species cluster (cluster 3) were subsequently analyzed.

[0124] The high-speed independent pan-genome pipeline Roary was used, which uses the assembly of annotations in the GFF3 format prepared by Prokka (Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014 Jul 15; 30(14): 2068-2069.) and calculates the pan-genome. Then, an approximate maximum likelihood tree was constructed using FastTree2 (Price MN, Dehal PS, Arkin AP. FastTree 2--approximately maximum-likelihood trees for large alignments. PLoS One. 2010 Mar10; 5(3): e9490.) ( Figure 2 ). 120 strain-specific genes were extracted from the gene presence / absence map ( Figure 3 ). Most of them are hypothetical proteins with unknown functions. The strain specific for these genes was named "Akkermansia HB03 (Akkermansia species HB03)".

[0125] Since Roary relies on protein sequences translated from active CDS (coding sequences), genomic base sequences with erroneous or defective annotations were excluded from the analysis. Therefore, a search of genomic sequences (not protein coding sequences) was performed for HB03 strain-specific genes predicted by Roary and their absence was confirmed. The presence of genes was investigated by calculating BLAST alignment score ratios (BSRs) using LS-BSR (Large Scale BLAS Alignment Score Ratios). Figure 4a and Figure 4b ).

[0126] By studying whether there is a pathogenicity island (PAI) of the gene group necessary for expressing pathogenicity (toxicity) in the whole genome of the HB03 strain, it was confirmed that the HB03 strain does not have a pathogenicity island ( Figure 5 ).

[0127] As a result of whole genome comparison with the Akkermansia muciniphila type strain (ATCC BAA-835), the HB03 strain showed 8% difference. Even for the genome matching portion, the ANI value was only 97%, indicating that the strain was a new strain different from the type strain.

[0128] Example 2: Cultivation characteristics of new strains

[0129] The HB03 strain was cultured in a modified BTTM medium (Nat. Med. 2017.23.107-113) (the content of N-acetylglucosamine in BTTM was reduced to 1 / 10, and the content of L-threonine was reduced to 1 / 5) at 37°C under anaerobic conditions.

[0130] As a result, as shown in Table 1, the HB03 strain showed a cell yield twice or more and a growth rate twice or more than that of Akkermansia muciniphila.

[0131] [Table 1]

[0132]

[0133] In addition, Akkermansia muciniphila has problems with centrifugation and large-scale production because it tends to suspend rather than settle, while the HB03 strain is easier to mass-produce and genome edit than its model strain due to its ease of centrifugation ( Figure 6 ).

[0134] In addition, when the amino acid L-threonine was absent from the medium, the Akkermansia muciniphila strain did not grow, whereas the HB03 strain grew to 1.0x10 9 The concentration of cells / mL was 1.37 cells / mL (Table 2). This means that unlike the Akkermansia muciniphila type strain, the HB03 strain can grow even in the absence of mucin or L-threonine, a degradation product of mucin, by using various carbon sources and nitrogen sources other than mucin or L-threonine as nutrients.

[0135] [Table 2]

[0136]

[0137] Thus, it was confirmed that the strain of the present invention has various industrially useful characteristics different from the Akkermansia muciniphila strain. The strain "Akkermansia HB03 (Akkermansia species HB03)" of the present invention was deposited in the Korea Culture Center for Microorganisms on September 11, 2018, with the deposit number KCCM12318P.

[0138] Example 3: Preparation of tumor animal model and strain composition

[0139] 3-1. Tumor animal model

[0140] Male C57BL / 6J JAX and BALB / c mice aged 6 to 8 weeks were purchased from Daehan Biolink. Each mouse was housed in a cage and kept in a controlled environment (12-hour light / dark cycle, lights off at 7 p.m.) with free access to food and water. Tumors were induced by injecting pancreatic cancer cells (Panc02), melanoma cells (B16F10), glioma cells (GL261), sarcoma cells (MCA205), colorectal cancer cells (MC38), and lung cancer cells (LLC) into C57BL / 6 JAX mice and injecting colorectal cancer cells (CT26) into BALB / c mice. Specifically, 1x10 7 cells / 100μLPanc02, 2x10 5 cells / 100μL B16F10, 5x10 5 cells / 100μL GL261, 5x10 5 cells / 100μL MCA205, 5x10 5 cells / 100μL MC38, 5x10 5 cells / 100 μL LLC or 5x10 5 CT26 tumor cells were injected subcutaneously into mice. Tumor size was measured at 2-day or 3-day intervals until the end of the experiment, and tumor volume was calculated as length x width. 2 x0.5. To investigate the changes in tumor size and immune environment due to HB03 and its fragments, the intestinal contents were recovered, immersed in liquid nitrogen, and then kept at −80 °C. All animal experimental procedures were approved by the Animal Care and Use Committee of the Korea Institute of Bioscience and Biotechnology.

[0141] 3-2. Strain composition

[0142] Strain compositions HB03 (composition of Akkermansia species HB03), Akkermansia muciniphila (composition of Akkermansia muciniphila ATCC BAA-835), AKKp2261 (composition of Akkermansia muciniphila p2261), and Bifidobacterium longum (composition of Bifidobacterium longum ATCC BAA-999) were prepared by culturing each strain (Akermansia species HB03, Akkermansia muciniphila ATCC BAA-835, Bifidobacterium longum ATCC BAA-999, or Akkermansia muciniphila p2261), which were maintained in 20% glycerol at -70°C and resuspended in 5.0 x 10 9CFU / mL liquid culture medium. In the following, unless otherwise specified, "A. muciniphila" refers to the model strain A. muciniphila BAA-835, and "AKK p2261" refers to A. muciniphila p2261. HB03 and A. muciniphila were cultured using the modified BTTM of Example 2, and AKK p2261 was cultured using COS medium (Columbia medium + 5% sheep blood (Biomrieux TM ); Columbia medium: polypeptone 17.0 g / L, tryptone 3.0 g / L, autolyzed yeast extract 3.0 g / L, corn starch 1.0 g / L, sodium chloride 5.0 g / L), Bifidobacterium longum was cultured using MRS medium (De Man, Rogosa and Sharpe (MRS) medium).

[0143] A pasteurized strain composition (HB03(P)) was prepared by boiling HB03 at 70°C for 30 minutes.

[0144] Example 4: Application of strain composition

[0145] 100 μL each of the strain composition HB03, HB03(P), Akkermansia muciniphila, AKK p2261 or Bifidobacterium longum of Example 3-2 was administered by oral gavage to the animal models of colorectal cancer (CT26, MC38), lung cancer (LLC) and melanoma (B16F10) of Example 3-1 from the 7th day before tumor inoculation or from the 5th day or 6th day to the 18th day, to the animal model of glioma (GL261) from the 7th day before tumor inoculation or from the 5th day or 6th day to the 22nd day, to the animal model of sarcoma (MCA205) from the 7th day before tumor inoculation or from the 5th day or 6th day to the 16th day, and to the animal model of pancreatic cancer (Panc02) from the 7th day before tumor inoculation or from the 6th day to the 30th day.

[0146] Example 5: Effect of administering a strain composition to a tumor animal model on inhibiting tumor growth

[0147] After post-treatment (administration after tumor inoculation) of the tumor animal model of Example 3-1 with HB03 or HB03(P), the difference in tumor growth was compared with that of the control group. The results are shown in Figure 7 .

[0148] After post-treatment (administration after tumor inoculation) of the tumor animal model of Example 3-1 with HB03, Akkermansia muciniphila or Bifidobacterium longum, the difference in tumor growth was compared with that of the control group. The results are shown in Figure 8 .

[0149] After the tumor animal model of Example 3-1 was pretreated with HB03 or HB03(P) (administered before tumor inoculation), the difference in tumor growth was compared with that of the control group. The results are shown in Fig. 9 .

[0150] After post-treatment (administration after tumor inoculation) of the tumor animal model of Example 3-1 with HB03 or AKK p2261, the difference in tumor growth was compared with that of the control group (PBS). The results are shown in Fig.10 .

[0151] like Figure 7 and Fig. 9 As shown, when HB03 or HB03(P) was administered for a certain period of time before or after tumor cell transplantation, tumor growth was significantly reduced.

[0152] In addition, if Figure 8 As shown in FIG. 1 , when the anticancer effects after administration of Akkermansia muciniphila, Bifidobacterium longum, or HB03 were compared, administration of HB03 further increased the anticancer effect. Fig.10 As shown, when the anticancer effects after administration of AKK p2261 or HB03 were compared, administration of HB03 further increased the anticancer effect. Thus, it was confirmed that HB03 exhibited a higher anticancer effect than the other three strains.

[0153] Since the results indicate that the intestinal microorganism HB03 or HB03(P) inhibits tumor growth as a cancer therapeutic agent, the strain HB03 of the present disclosure can be used to treat cancer.

[0154] Example 6: Combined administration of immune checkpoint inhibitors and bacterial strain compositions inhibits tumor growth in tumor animal models Long effect

[0155] After immune checkpoint inhibitors (aPD-1 mAb) and HB03 were co-administered to the tumor animal model of Example 3-1, the difference in tumor growth with the control group not co-administered was compared. MIgG (150 μg / mouse) and PBS (1001 μL / mouse) were applied to the control group, and αPD-1 mAb (150 μg / mouse) and PBS (100 μL / mouse) or αPD-1 mAb (150 μg / mouse) mice) and HB03 (100 μL / mouse) were applied to the test group. After tumor inoculation, 100 μL of mIgG or αPD-1 mAb was intravenously injected into animal models of colorectal cancer (CT26), lung cancer (LLC), melanoma (B16F10) and glioma (GL261) 5 times, 2 days apart, from the 5th day to the 16th day, and 100 μL of PBS (control, control group) or HB03 was administered daily by oral gavage. Comparison of tumor size on day 16 Fig.11 shown.

[0156] To compare the difference in tumor growth between the combined administration group of another immune checkpoint inhibitor (αCTLA-4 mAb) and HB03 and the non-combination administration control group, mIgG (25 μg / mouse) and PBS (100 μL / mouse) were administered to the control group, and αCTLA-4 mAb (25 μg / mouse) and PBS (100 μL / mouse) or αCTLA-4 mAb (25 μg / mouse) and HB03 (100 μL / mouse) were administered to the test group. After tumor inoculation, 100 μL of mIgG or αCTLA-4 mAb was intravenously injected 5 times from day 5 to day 16 at intervals of 2 days into the sarcoma (MCA205) and colorectal cancer (MC38) animal models, and 100 μL of PBS (control, control group) or HB03 was administered daily by oral gavage. The comparison results of tumor size on day 16 are shown in Figure 2. Fig.12 shown.

[0157] In addition, in order to compare the difference in tumor growth between the combined administration group of another immune checkpoint inhibitor (αPD-L1 mAb) and HB03 and the non-combination administration control group, mIgG (100 μg / mouse) and PBS (100 μL / mouse) were administered to the control group, and αPD-L1 mAb (100 μg / mouse) and PBS (100 μL / mouse) or αPD-L1 mAb (100 μg / mouse) and HB03 (100 μL / mouse) were administered to the test group. After tumor inoculation, 100 μL of mIgG or αPD-L1 mAb was intravenously injected 5 times from day 5 to day 16 at intervals of 2 days to the colorectal cancer (MC38) animal model, and 100 μL of PBS (control, control group) or HB03 was administered daily by oral gavage. The comparison results of tumor size on day 16 are shown in Figure 2. Fig.13 shown.

[0158] like Figures 11 to 13 As shown, the anti-tumor effect was significantly increased when the immune checkpoint inhibitors αPD-1 mAb, αCTLA-4 mAb, or αPD-L1 mAb were co-administered with HB03 compared with when the immune checkpoint inhibitors were administered alone.

[0159] The results suggest that the gut microbiome HB03 provides an advantage in inhibiting tumor growth during cancer treatment by reducing the concentration of immune checkpoint inhibitors, thereby increasing the anti-tumor effect while reducing side effects and drug resistance.

[0160] Example 7: Combined administration of immune checkpoint inhibitors and bacterial strain compositions inhibits tumor growth in drug-resistant animal models Long effect

[0161] Ampicillin (1 μg / mL), streptomycin (5 μg / mL) and colistin (1 μg / mL) were dissolved in drinking water and applied to the animal model of Example 3-1 for 2 weeks, and then the tumor was inoculated, after which the drinking water was replaced with ordinary drinking water. After 48 hours, the immune checkpoint inhibitor (αPD-1 mAb) was co-administered with HB03 or Bifidobacterium longum, and the difference in tumor growth with the non-co-administered group was compared. After tumor inoculation, mIgG or αPD-1 mAb was intravenously injected into colorectal cancer (CT26) and melanoma (B16F10) animal models 5 times from day 5 to day 16 at intervals of 2 days, and 100 μL of PBS (control, control group), HB03 or Bifidobacterium longum was administered by oral gavage every day. The comparison results of tumor size on day 16 are as shown in Fig.14 shown.

[0162] In addition, ampicillin (1 μg / mL), streptomycin (5 μg / mL) and colistin (1 μg / mL) were dissolved in drinking water and applied to the animal model of Example 3-1 for 2 weeks, and then the tumor was inoculated, after which the drinking water was replaced with ordinary drinking water. After 48 hours, the immune checkpoint inhibitor (αCTLA-4 mAb) was co-administered with HB03 to compare the difference in tumor growth with the non-co-administered group. After tumor inoculation, the colorectal cancer (MC38) animal model was intravenously injected with mIgG or αPD-1 mAb 5 times from day 5 to day 16 at intervals of 2 days, and 100 μL of PBS (control, control group) or HB03 was administered daily by oral gavage. The comparison results of tumor size on day 16 are as shown in Fig.15 shown.

[0163] like Fig.14 As shown, when the immune checkpoint inhibitor αPD-1 mAb was co-administered with HB03 to mice that had become resistant to the immune checkpoint inhibitor due to antibiotic administration, the anti-tumor effect was significantly increased compared with the immune checkpoint inhibitor alone, and this effect was superior to the effect of co-administration with Akkermansia muciniphila or Bifidobacterium longum.

[0164] In addition, if Fig.15 As shown, when the immune checkpoint inhibitor αCTLA-4 mAb was co-administered with HB03 to mice that had become resistant to the immune checkpoint inhibitor due to antibiotic administration, the anti-tumor effect was significantly increased compared to the immune checkpoint inhibitor alone.

[0165] The results indicate that intestinal microorganism HB03 overcomes the reduced anti-cancer effect caused by resistance to immune checkpoint inhibitors during cancer treatment, thereby improving the anti-tumor effect and inhibiting tumor growth.

[0166] Example 8: Effect of anticancer drugs and bacterial strain combination on inhibiting tumor growth in tumor animal models

[0167] After the chemical anticancer agent (5-FU: 5-fluorouracil) and the targeted anticancer agent (oxaliplatin) were co-administered with HB03 to the tumor animal model of Example 3-1, the difference in tumor growth was compared with the non-co-administered group. Physiological saline (100 μL / mouse) and PBS (100 μL / mouse) were administered to the control group, and 5-FU (2 mg / kg), oxaliplatin (20 mg / kg) and saline (100 μL / mouse) or 5-FU (2 mg / kg), oxaliplatin (20 mg / kg) and HB03 (100 μL / mouse) were administered to the test group. After tumor inoculation, 100 μL of 5-FU or oxaliplatin was intravenously injected 5 times from the 5th day to the 16th day to the colorectal cancer (MC38) animal model at intervals of 2 days, and 100 μL of PBS (control, control group) or HB03 was administered daily by oral gavage. The comparison results of tumor size on the 16th day are as shown in Figure 2. Fig.16 shown.

[0168] like Fig.16 As shown, when the anticancer agents 5-FU and oxaliplatin were co-administered with HB03 to a colorectal cancer animal model, the antitumor effect was significantly increased compared to when the anticancer agents were administered alone.

[0169] Example 9: Effects of administering a bacterial strain composition to tumor cells on inhibiting tumor growth and inducing cell apoptosis

[0170] Genes HB03-01 and HB03-03 encoding proteins produced by the HB03 strain were amplified from the genomic DNA of the HB03 strain by PCR. The amplified genes were then cloned into the pET-30a vector ( Fig.17 ) and overexpressed in E. coli. After centrifugation, the strain was resuspended in PBS buffer and the bacteria were lysed by ultrasound. After centrifugation again and separation of only the supernatant, HB03-01 (SEQ ID NO 1) and HB03-03 (SEQ ID NO 2) proteins were purified from the supernatant using a His-taq column.

[0171] Then, whether the purified HB03-01 and HB03-03 proteins affect the proliferation or apoptosis of cancer cells was studied. Specifically, after treating colorectal cancer cells (CT26) and lung cancer cells (LLC) with HB03-derived protein HB03-01 at a concentration of 0 μg / mL (untreated), 0.1 μg / mL, 1.0 μg / mL or 10 μg / mL or with 10 μg / mL of HB03-03, the activity of the cancer cell proliferation and apoptosis-regulating enzyme caspase-3 was studied.

[0172] MTT assay was performed to study the changes in cancer cell proliferation. DMEM medium (Gibco, USA) was used to culture 1×10 cells / mL in a 96-well plate in a 5% CO2 incubator at 37°C. 4 Cancer cells were cultured at 10 cells / well for 24 hours. Low glucose was maintained by treatment with 2-deoxyglucose (2-DG) to a final concentration of 20 mM. After 6 hours of culture, HB03-01 was treated with 0 μg / mL, 0.1 μg / mL, 1.0 μg / mL, or 10 μg / mL for 48 hours. After adding 10 μL of MTT solution, the culture was continued for 2 hours. The culture medium was removed from the 96-well plate and the formazan formed on the plate was dissolved with 100 μL of DMSO. Afterwards, the absorbance was measured at 540 nm using an ELISA reader (Model 680, BioRad). The control group was treated with 0 μg / mL HB03-01 (untreated), and the measured values ​​were expressed as relative cell viability (%) relative to the cell number of the control group being 100%.

[0173] Western blotting is carried out to study the activity level of caspase-3. The lung cancer cells treated with 10 μ g / mL HB03-01 or HB03-03 for 24 hours are homogenized in the ice-cold 20mM Tris-HCl buffer (pH 7.5) comprising 2mM EDTA (ethylenediaminetetraacetic acid), 0.5mM EGTA (ethylene glycol tetraacetic acid), 300mM sucrose and 2mM PMSF (phenylmethylsulfonyl fluoride). After 50 μ g of protein are electrophoresed on 15% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel, they are transferred to PVDF (polyvinylidene fluoride) membrane using semi-dry transfer system (Hoefer, Holliston, MA, USA). Non-specific binding is blocked by incubation with 5% skim milk powder. After the membrane was incubated overnight at 4°C with the primary antibody diluted to 1:500, it was incubated with the secondary antibody by binding to HRP (horseradish peroxidase) diluted to 1:1000. The immune complex was detected using an ECL detection kit (Amersham Biosciences Korea, Seoul, Korea) and autoradiography was performed on X-ray film. The antibodies used for Western blotting were actin and caspase-3. They were purchased from Santa Cruz Biotechnolgy (Santa Cruz, CA).

[0174] like Fig.18 A and Fig.18 As shown in B, HB03-01, a derivative protein of HB03, significantly reduced the proliferation of colorectal cancer and lung cancer cells. Fig.18As shown in Figure C, it, together with other HB03-derived proteins, HB03-03, activated apoptosis-regulating enzymes (caspase-3) in lung cancer cells.

[0175] This demonstrates that the proteins or metabolome derived from the intestinal microorganism HB03 creates an environment that is favorable for cancer treatment or inhibition of tumor growth.

[0176] Example 10: Effect of administering a strain composition to a tumor animal model on inhibiting tumor growth

[0177] After post-treatment (administration after tumor inoculation) of the tumor animal model of Example 3-1 with HB03-01 (AT1) or HB03-03 (AT3), the difference in tumor growth was compared with that of the control group. The results are shown in Fig.19 .

[0178] The results demonstrated that HB03-01(AT1) and HB03-03(AT3) significantly inhibited tumor growth compared to the control group not treated with HB03-01(AT1) or HB03-03(AT3).

[0179] This result indicates that the protein derived from the intestinal microorganism HB03 is advantageous as a cancer therapeutic agent in inhibiting tumor growth. Therefore, the protein of the HB03 strain of the present disclosure can be used to treat cancer.

[0180] The results of the above examples indicate that the presence or increased levels of useful intestinal microorganisms and the levels of activation or inactivation of microorganisms affect cancer treatment and tumor growth.

[0181] Specifically, the present disclosure has demonstrated that the administration of specific microorganisms, i.e., the novel beneficial microorganisms of the present disclosure, regulates intestinal microorganisms by reducing or inhibiting other immune responses, promotes the treatment of tumors, and inhibits tumor growth. Specifically, the novel beneficial microorganisms disclosed in the present disclosure create an environment that is conducive to cancer treatment and inhibit tumor growth by secreting metabolites (such as amino acids, nucleic acids, fatty acids, sugars, amines, glycolipids, short peptides, vitamins, hormones, etc. present in cells, tissues, or body fluids) or proteomes.

[0182] Data in tables and graphs are expressed as mean ± SEM. P values ​​for studying statistical differences in tumor growth measurements were estimated by t-test (Excel software). P < 0.05 was considered statistically significant: *p < 0.05, **p < 0.01, ***p < 0.001.

[0183] It should be understood by those skilled in the art that the present disclosure can be implemented in different forms without changing the technical ideas or basic features. In this regard, it should be understood that all of the above embodiments are illustrative rather than restrictive. It should be understood that all changes and modifications derived from the attached claims and their equivalents are included within the scope of the present disclosure.

[0184]

[0185] The above translation proves that there is no difference with the original content.

[0186] September 25, 2019

[0187] Patent Attorney Sun Min

Claims

1. A species of Akkermansia ( Akkermansia sp.) HB03 strain, characterized in that: It was deposited in the Korea Center for the Culture of Microorganisms with the accession number KCCM12318P.

2. A pharmaceutical composition comprising one or more than one selected from the Akkermansia strain according to claim 1, its inactivated strain and its proteome, wherein the inactivation is thermal inactivation performed by heating, and wherein the proteome is one or more than one protein selected from HB03-01 shown in SEQ ID NO 1 and HB03-03 shown in SEQ ID NO 2.

3. Use of the pharmaceutical composition according to claim 2 in the preparation of a medicament for preventing or treating a cancer selected from colorectal cancer, lung cancer, melanoma, glioma, sarcoma or pancreatic cancer. The use according to claim 3, wherein the cancer is a cancer that exhibits resistance to anticancer agents.

5. The use according to claim 4, wherein the anticancer agent is an immune checkpoint inhibitor.

6. Akkermansia spp. deposited in Korea Center for the Culture of Microorganisms with the accession number KCCM12318P ( Akkermansia sp.) HB03 strain, its inactivated strain or its proteome in the preparation of a medicament for preventing or treating a cancer selected from colorectal cancer, lung cancer, melanoma, glioma, sarcoma or pancreatic cancer, wherein the inactivation is thermal inactivation by heating, and wherein the proteome is one or more than one protein selected from HB03-01 shown in SEQ ID NO 1 and HB03-03 shown in SEQ ID NO 2.

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