Patent Application
20220111009
The present disclosure relates to a microorganism expressing VIP gene, a composition or a kit for preventing or treating a disorder causing gastrointestinal tract damage comprising the microorganism, and a method for preventing or treating a disorder causing gastrointestinal damage using the same.
Vasoactive intestinal peptide (VIP) is a peptide hormone that is vasoactive in the intestine. VIP is a peptide of 28 amino acid residues that belongs to a glucagon/secretin superfamily, the ligand of class II G protein—coupled receptors.
In humans, chromosome 6 q25 region on human genome codes for secretin family member having the length of 170 amino acids. The member composed of 170 amino acids is post-translationally cleaved to form vasoactive intestinal peptide (VIP). An active form of VIP polypeptide functions to lower blood pressure, increase vasodilation, relax smooth muscle in the respiratory system and the gastrointestinal tract tissue, reduce immune responses by not only the reduction of Th1 response but also the promotion of Th2 response, modulate innate and adaptive immune responses, or promote electrolyte secretion in the gut. In addition, VIP is reported to be active in the communication with lymphocytes in central nerve system as a neurotransmitter. The bioactivity of VIP is transduced via three known receptors: VIP1R, VIP2R, and PAC1R. These receptors are known to cause the production of intracellular calcium as well as the accumulation of cAMP. Their affinity to secretin depends on their subtypes and amino acid sequences of ligands like VIP. Human natural VIP has a short half-life of about 2 minutes in the blood stream.
U.S. Pat. No. 9,561,262 discloses a method for treating hypertension in a patient, comprising administering VIP and an anti-hypertension agent to the patient.
United States Publication No. 2016-0045557 A1 discloses Lactobacillus and Bifidobacterium strains increasing the level of VIP as probiotics for improving enteric nerve system. However, these strains were screened in the co-culture system comprising epithelial cells and enteric neuronal cells, so they are not genetically engineered strains.
Chinese Publication No. 108753670 A discloses a multifunctional complex micro-ecological formulation, nano-selenium-recombinantly expressed VIP-Lactococcus lactis, and a preparation method thereof. To express VIP which is an antibacterial peptide, Lactococcus lactis, and NICE® expression system using nisin inducible promoter and USP45 signal sequence were employed. It also discloses its use as animal feed additive.
Despite the prior arts, there is still a need for a microorganism expressing VIP gene and its use for the treatment of inflammatory intestinal disorders.
An aspect provides a recombinant microorganism genetically modified to increase the expression of VIP encoding gene.
Another aspect provides a composition for preventing or treating a disorder causing gastrointestinal tract damage in human, comprising the microorganism.
Still another aspect provides a kit for use in preventing or treating a disorder causing gastrointestinal tract damage, comprising the microorganism and a TNF-alpha blocker.
Further still another aspect provides a method for preventing or treating a disorder causing gastrointestinal tract damage.
As used herein, the term, “VIP” or “VIP protein” or “VIP polypeptide,” indicates a biologically active polypeptide having one or more biological activities as described herein. VIP as described herein includes any variant mimicking functions of natural VIP. The variant includes ones having an increased half-life and equivalent or higher biological activities as compared with natural VIP.
VIP as used herein includes, but is not limited to, human VIP, recombinant human VIP, murine VIP and/or recombinant murine VIP. The VIP may have the sequence homology of 50% or more, 60% or more, 70% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% with the amino acid sequence of SEQ ID No. 1. The VIP may have any of amino acid sequences of SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, and 9, respectively. VIP of SEQ ID No. 2 is human natural VIP. VIPs of SEQ ID Nos. 1, 3, 4, 5, 6, 7, 8, and 9 are variants of human natural VIP, i.e., VIP1, VIP2, VIP3, VIP4, VIP5, VIP6, VIP7, and VIP8, respectively.
VIP polypeptide as used herein could be isolated from various sources such as human tissues or other sources or prepared by recombination or synthetic methods. The term, “VIP polypeptide,” includes any variant of VIP polypeptide. VIP as used herein includes chimeric forms such as VIP fused with another heterologous polypeptide or amino acid sequence.
An aspect provides a recombinant microorganism genetically modified to increase the expression of VIP encoding gene.
The microorganism may comprise a genetic modification to increase the expression of VIP. The genetic modification may be one to increase the copy number of VIP encoding gene. The increased copy number could be obtained by introduction of a heterologous gene to the microorganism. The introduction could be accomplished by transformation, transduction, transfection or electroporation. The introduced heterologous gene may or may not be integrated to the genome of host cells.
The microorganism may comprise a heterologous gene encoding VIP. The gene may be operably linked to a regulatory sequence regulating its expression. The regulatory sequence may be any nucleotide sequence encoding a promoter, an operator, a terminator, or a signal peptide. The promoter may a constitutive promoter. The promoter may have a transcription initiation rate equivalent to or higher than that of an art-known promoter, for example, promoter P11, in lactic acid bacteria. The promoter may be an inducible promoter. The inducible promoter may be, for example, a nisin inducible promoter. The promoter may be PR4 derived from Lactobacillus paracasei. The PR4 promoter may have the nucleotide sequence of SEQ ID No. 10. The PR4 promoter is set forth in International Publication No. WO 2019/132231 A1 filed on Nov. 8, 2018, the content of which is incorporated hereinto in its entirety by reference. In case a nucleotide sequence encoding the signal peptide is in-frame linked to the heterologous protein gene, the signal peptide may have an increased capability to secrete the heterologous protein extracellularly compared with an art-known signal peptide, for example, signal peptide USP45. The signal peptide may be SP4 derived from Lactobacillus paracasei. SP4 may have the amino acid sequence of SEQ ID No. 11. SP4 is set forth in International Publication No. WO 2019/132231 A1 filed on Nov. 8, 2018, the content of which is incorporated hereinto in its entirety by reference. The microorganism could secrete VIP extracellularly.
The microorganism may be a bacterium. The bacterium may be a gram-positive or -negative bacterium. The gram-positive bacterium may a lactic acid bacterium. The lactic acid bacterium may belong to the genus of Lactobacillus, Lactococcus, Bifidobacterium, Streptococcus, Leuconostoc, Weissella, Pediococcus, or Enterococcus. The lactic acid bacterium may be Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus plantarum or Lactococcus lactis. The lactic acid bacterium may be Lactobacillus plantarum LMT1-9 (KCTC 13421BP), Lactobacillus paracasei LMT1-21 (KCTC 13422BP), or Lactobacillus brevis LMT1-46 (KCTC 13423BP).
Another aspect provides a composition for preventing or treating a disorder causing gastrointestinal tract damage, comprising the microorganism.
The disorder may cause gastrointestinal tract inflammation. The disorder may be one or more selected from the group consisting of inflammatory bowel disease (IBD), and colitis. The inflammatory bowel disease may be ulcerative colitis, or Crohn's disease.
The composition may further comprise one or more pharmaceutically or sitologically acceptable carriers, excipients, or diluents. The pharmaceutically or sitologically acceptable carrier includes any standard pharmaceutically or sitologically acceptable carrier such as phosphate buffered saline solution, 5% dextrose aqueous solution and emulsion (for example, oil/water or water/oil emulsion). Non-limiting examples of the excipients may include auxiliaries, binders, fillers, diluents, disintegrating agents, emulsifying agents, wetting agents, gliadants, lubricants, sweetening agents, flavors, and coloring agents. It depends on an intended administration route of an active ingredient which pharmaceutically or sitologically acceptable carrier is preferable. Typical administration route may include enteral (for example, oral) administration. The composition may be a unit dosage form. The composition may be an orally administrable formulation. The composition may be a food or a pharmaceutical composition. The composition may comprise a dried product of the microorganism. The composition may comprise a culture medium of the microorganism.
As used herein, the term, “pharmaceutically or sitologically acceptable,” means that any adverse reaction does not substantially occur upon administration to an individual. The adverse reaction may be toxicity, allergy, or an immune response.
Another aspect provides a kit for preventing or treating a disorder causing gastrointestinal tract damage comprising the recombinant microorganism and any other therapeutic agent for a disorder causing gastrointestinal tract damage. For the recombinant microorganism, the same is applied as above. The recombinant microorganism may be in the form of the above-described composition. The kit may include a written instruction to use the recombinant microorganism and any other therapeutic agent for a disorder causing gastrointestinal tract damage in preventing or treating a disorder causing gastrointestinal tract damage.
Examples of any other therapeutic agent for gastrointestinal tract damage include any agents for treating a disorder causing gastrointestinal tract damage, for example, inflammatory bowel disease. The agent for treating a disorder causing gastrointestinal tract damage may include one or more of the following agents:
(i) Steroid Anti-Inflammatory Agent
Dexamethasone, hexestrol, methimazole, betamethasone, triamcinolone, triamcinolone acetonide, fluocinonide, fluocinolone acetonide, predonisolone, methylpredonisolone, cortisone acetate, hydrocortisone, fluorometholone, beclomethasone dipropionate, estriol, paramethasone acetate, fludrocortisone acetate, clobetasol propionate, diflorasone acetate, dexamethasone propionate, difluprednate, betamethasone dipropionate, budesonide, diflucortolone valerate, amcinonide, halcinonide, mometasone furoate, hydrocortisone butyrate propionate, flumetasone pivalate, clobetasone butyrate, dexametasone acetate or the like;
(ii) 5-Aminosalicylic Acid
Sulfasalazine, mesalazine, olsalazine, balsalazide or the like;
(iii) Immunomodulator or Immunosuppressant
Methotrexate, cyclophosphamide, MX-68, atiprimod dihydrochloride, BMS-188667, CKD-461, rimexolone, cyclosporine, tacrolimus, gusperimus, azathiopurine, antilymphocyte serum, freeze-dried sulfonated normal immunoglobulin, erythropoietin, colony stimulating factor, interleukin, interferon or the like;
(iv) JAK Inhibitor
Tofacitinib, ruxolitinib or the like;
(v) TNF Inhibitor
Recombinant TNF-alpha-receptor IgG-Fc fusion protein (etanercept), infliximab, adalimumab, certolizumab pegol, golimumab, PASSTNF-α, soluble TNF-α receptor, TNF-α binding protein, anti-TNF-α antibody, CDP571 or the like;
(vi) Integrin Inhibitor
Natalizumab, vedolizumab, AJM300, TRK-170, E-6007 or the like;
(vii) Interleukin-12/23 Inhibitor
Ustekinumab, briakinumab (anti-interleukin-12/23 antibody) or the like;
(viii) Non-Steroid Anti-Inflammatory Drug (NSAID)
(a) Classical NSAID
Alcofenac, aceclofenac, sulindac, tolmetin, etodolac, fenoprofen, thiaprofenic acid, meclofenamic acid, meloxicam, tenoxicam, lornoxicam, nabumeton, acetaminophen, phenacetin, ethenzamide, sulpyrine, antipyrine, migrenin, aspirin, mefenamic acid, flufenamic acid, diclofenac sodium, ketophenylbutazone, loxoprofen sodium, phenylbutazone, indomethacin, ibuprofen, ketoprofen, naproxen, oxaprozin, flurbiprofen, fenbufen, pranoprofen, floctafenine, piroxicam, tenoxicam, epirizole, tiaramide hydrochloride, zaltoprofen, gabexate mesylate, camostat mesylate, ulinastatin, colchicine, probenecid, sulfinpyrazone, bucolome, benzbromarone, allopurinol, sodium aurothiomalate, hyaluronate sodium, sodium salicylate, salicylic acid, atropine, scopolamine, levorphanol, oxymorphone or a salt thereof or the like.
(b) Cyclooxygenase Inhibitor (COX-1 Selective Inhibitor, COX-2 Selective Inhibitor, Etc.)
Salicylic acid derivatives (e.g., celecoxib, aspirin), etoricoxib, valdecoxib, diclofenac sodium, indomethacin, loxoprofen or the like
(c) Nitric oxide-releasing NSAIDs.
In addition, chemokine inhibitors, Cluster of Differentiation (CD) inhibitors, Interleukin inhibitors, RIP kinase inhibitors, smad7 inhibitors, MadCAM inhibitors or the like may be included.
Another aspect provides a method for preventing or treating a disorder causing gastrointestinal tract damage, comprising administering a therapeutically or prophylactically effective amount of the microorganism to an individual.
The method may further comprise administering a therapeutically or prophylactically effective amount of a TNF-alpha blocker to an individual. The TNF-alpha blocker may be administered simultaneously with, prior to, or following the administration of the microorganism. The TNF-alpha blocker may be administered at the same route as or different route from the microorganism. For example, the TNF-alpha blocker may be administered orally or parenterally. The parenteral administration may be injection such as intravenous, intraperitoneal or subcutaneous injection. For example, the TNF-alpha blocker may be administered intraperitoneally, while the microorganism may be administered orally. The TNF-alpha blocker can be intraperitoneally administered after the microorganism is orally administered. The TNF-alpha blocker can be administered at a single dose of 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 40 mg, 10 mg to 50 mg, or 20 mg to 50 mg.
In the above-described method, the disorder may be one that causes gastrointestinal tract inflammation. The disorder may be one or more selected from the group consisting of inflammatory bowel disease, autoimmune disease, radiation induced gastrointestinal tract damage or graft versus host disease (GVHD), inflammatory bowel disease (IBD), and colitis such as chronic colitis. The inflammatory bowel disease may be ulcerative colitis, or Crohn's disease.
As used herein, the term, “administering” or “administration,” indicates a behavior to give the microorganism, the composition comprising the microorganism or a therapeutic treatment to a physiological system (e.g. an individual, or in vivo, in vitro or ex vivo cells, tissues or organs). Accordingly, in the method, the microorganism can be in the form of above-described composition. Examples of acceptable routes to human body may include oral, or mucosal administration, e.g. to enteral mucous, oral mucosa or buccal. The administration may be performed in combination with an additional therapeutic agent. The combinational administration includes administration in any order among simultaneous and sequential ones.
As used herein, the term, “treatment,” indicates prophylactic treatment or therapeutic treatment. In a specific embodiment, “treatment” indicates administration of the microorganism or the composition to an individual for therapeutic or prophylactic purpose.
“Therapeutic” treatment is an administration to an individual showing pathological signs or symptoms for reducing or removing the signs or symptoms. The signs or symptoms may be biochemical, cellular, histological, functional or physical, subjective or object ones.
“Prophylactic” treatment is an administration to an individual showing no or only early pathological signs for reducing a risk of developing pathology. The microorganism or the composition as described herein can be provided for a prophylactic treatment to reduce the likelihood of pathology development or, if pathology has been developed, minimizing its severity.
As used herein, “therapeutically effective amount” indicates an amount sufficient to accomplish the described purpose. The effective amount can be determined experimentally. The effective amount can be determined depending on the extracellular secretion rate of VIP by the microorganism. For example, the effective amount may be the number of microorganisms which is capable of secreting 0.01 to 300 mg, or 0.5 to 100 mg of VIP daily for a human of 60 kg body weight. For example, it may be 1×107 to 1×1011 cfu, 1×107 to 1×1010 cfu, 1×108 to 1×1011 cfu, 1×108 to 1×1010 cfu, 2.5×108 to 7.5×109 cfu, 5.0×108 to 5.0×109 cfu, 7.5×108 to 2.5×109 cfu, or about 1×109 cfu each administration to each individual.
In the above-described method, the individual may be a mammal. The mammal may be human, horse, pig, cow, dog, cat, monkey, chimpanzee, sheep, or goat. The individual may be human. The individual may be a non-human mammal.
The recombinant microorganism according to one aspect can produce and secrete VIP.
The composition or the kit according to another aspect can be used for preventing or treating a disorder causing gastrointestinal tract damage.
The method according to still another aspect can efficiently prevent or treat a disorder causing gastrointestinal damage.
Hereunder, detailed descriptions are provided with reference to examples. However, the examples are provided only for illustrative purpose, but not intended to limit the scope of the present disclosure in any manner.
In the present example, VIP gene was introduced to lactic acid bacterial cells, and it was tested if the gene was expressed and extracellularly secreted. Furthermore, the lactic acid bacterial cells transformed with VIP gene were orally administered to an individual and tested for improvement in symptoms to show their efficacy for treating IBD. As VIP, a human VIP variant consisting of the amino acid sequence of SEQ ID No. 1 was used. The VIP variant has 4 amino acid substitutions in human natural VIP consisting of the amino acid sequence of SEQ ID No. 2, thereby to have an increased half-life in the human bloodstream.
1. Construction of VIP Gene-Containing Vector
DNA synthesized by Macrogen, Inc. (South Korea) was used as VIP gene consisting of the nucleotide sequence of SEQ ID No. 14. The synthesized VIP gene fragment and Lactic Acid Bacteria (LAB)-E. coli shuttle vector pMT54-PR4-SP4 (SEQ ID No. 13) were cleaved with restrictive endonucleases SalI and XhoI, respectively. The cleaved gene fragment and vector in the reaction mixture were purified using a gel purification kit (GeneAll Biotechnology, Co., Ltd.), and then, were incubated in the presence of an alkaline phosphatase to obtain dephosphorylated cleaved VIP gene fragment and cleaved pMT54-PR4-SP4 vector.
The vector DNA of 1 μl, the VIP gene DNA of 3 μl, 0.5 μl of T4 DNA ligase (Takara), 1 μl of a buffer solution, and 5.5 μl of distilled water were mixed together in a test tube to make a total volume to 10 μl, and then, the mixture was incubated at 16° C. for 12 hours to link the VIP gene with the vector fragment to obtain the VIP gene-containing vector. E. coli competent cells (TOP10 Competent Cells) were transformed with the vector according to the method of Sambrook et al. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edn, 1989).
The transformed E. coli was spread onto an LB (Luria-Bertani)-agar plate containing 10 μl/ml of chloramphenicol, and then, incubated. As a result, formed colonies were selected, cultivated, and the vector was isolated from the culture. The vector was designated as pMT447. The pMT447 vector comprises the VIP gene operably linked to PR4 promoter and SP4 signal sequence. Therefore, the cells transformed with pMT447 vector expresses and secretes the VIP gene extracellularly in a high efficiency.
2. Introduction of Recombinant Vector to Lactic Acid Bacterial Cells
The recombinant vector pPM447 constructed in the above 1 were introduced to Lactobacillus plantarum LMT1-9 (KCTC 13421BP), Lactobacillus paracasei LMT1-21 (KCTC 13422BP), and Lactobacillus brevis LMT1-46 (KCTC 13423BP), respectively, and then, were tested for the secretion of VIP protein.
Each strain was cultivated to reach OD600 of 0.5 in a flask containing 50 mL of MRS medium (Difco Co., USA), and then, centrifuged at 4° C., 7,000 rpm for 10 minutes. Cell pellets were washed with 25 mL of cold EPS (ice-cold electroporation solution) (containing 1 mM K2HPO4, 1 mM KH2PO4, pH 7.4, 1 mM MgCl2, and 0.5 M sucrose) twice. MRS is also called as De Man, Rogosa and Sharpe agar medium.
After washing, the cells were re-suspended in 1 ml of cold EPS to prepare competent cells for electroporation and kept in a deep freezer of −80° C. The competent cells of 40 μl and the respective vector DNA (1 μg/μl) of 1 μl were added to a cuvette, and allowed to stand in ice for 5 minutes. Electric pulse was imposed on the cells under the condition of 25 pf, 8 kV/cm, 400 ohms, and then, the cells were immediately added to 1 ml of MRS liquid medium and cultivated at 37° C. for one hour. Then, the cultivated cells were spread onto MRS medium (also known as ‘MRS-CM’) containing 10 μg/ml of chloramphenicol and cultivated at 37° C. for 48 hours.
The obtained pMT447 vector-introduced strains were statically cultivated in MRS liquid medium at 37° C. for 16 hours, respectively. The culture was inoculated to 3% (v/v) in MRS liquid medium, and then, statically cultivated for 8 hours at the same temperature. The culture of 1 ml was centrifuged at 7,000 rpm for 5 minutes to take its supernatant. To the supernatant of 1 ml was added trichloroacetic acid of 100 μl to obtain a mixture and the mixture was allowed to stand at 4° C. for one hour to concentrate the culture. The culture was centrifuged at 13,000 rpm for 4° C., and the pellets were washed with 1 ml of cold acetone, dried at room temperature for 10 minutes, and eluted with Tris-HCl buffer (pH 8.8) of 100 μl.
To the elute were added 4× loading buffer (Thermo) and 10× reducing agent (Thermo), and electrophoresis was performed on SDS-PAGE gel. The gel was transferred onto nitrocellulose membrane with Trans blot semi-dry cell (bio-rad) to perform western blotting. Specifically, the membrane was blocked with TBST buffer containing 1% skim milk for one hour, reacted with anti-HA antibody (Santa cruz) at room temperature for 2 hours, and then, washed with TBST for 5 minutes three times and detected with ECL. In pMT447 vector, VIP gene was operably liked with hemagglutinin (HA) gene at its 3′ terminal, so was expressed in HA tagged form.
As shown in
3. Efficacy of VIP-Secreting Lactic Acid Bacteria for IBD in DSS Induced Model
The transformed L. plantarum LMT1-9, L. paracasei LMT1-21, and L. brevis LMT1-46 as described in 2 were orally administered to mouse colitis model induced with dextran sulfate sodium salt (DSS), and the survival rate and disease activity index (DAI) score of mice were measured to evaluate the therapeutic efficacy of the strains on intestinal inflammation.
(1) Administration of Recombinant Microorganisms Alone
Specifically, the transformed strains were statically cultivated primarily in a flask containing MRS medium of 10 ml containing 10 μg/ml of chloramphenicol at 37° C. for 24 hours. On the next day, the cultivated strains were inoculated on 300 to 600 ml of MRS CM medium to reach OD600 of 0.01, and secondarily cultivated under the same condition as the primary cultivation. After 16 to 18 hours, when OD600 of the culture reached 2 to 3, the culture was centrifuged at 7,000 rpm for 10 minutes to discard the supernatant, and cells were suspended in 1×PBS so as to be administered at 1×109 cfu as a single dose per mouse. This cell-containing PBS solution was administered orally (1×109 cfu as a single dose per mouse) with drinking water once a day for 16 days.
First, male Balb/c mice with the age of 8 to 10 weeks (20 to 25 g, 10 mice per group) (ORIENTBIO) were used as experimental animals. The day when strains were administered initially was set Day −7, and DSS diluted to 2% (v/v) in distilled water was supplied as drinking water on Day 0 to Day 6 to all groups but PBS group. Mice took drinking water voluntarily, but at an amount of 6 ml or less a day generally. The mice were sacrificed on Day 9.
After DSS was administered, survival rate was measured every other day and body weight, hair-raising, movement, and diarrhea were checked to obtain DAI score.
Disease activity index (DAI) scoring was performed with reference to Ameho, Gut 1997; 41:487˜493 and Wallace, Gastroenterology 1989; 96: 29˜36. The results were shown in
In
As shown in
As shown in
Specifically, as shown in
(2) Effects of the Combination of the Recombinant Microorganism and a TNF-Alpha Blocker
The initial administration day was set Day −7 to start the therapeutic efficacy evaluation test. DSS was diluted to 2% (v/v) in distilled water, which was supplied to all groups but PBS group as drinking water over Day 0˜Day 6. On Day 9, mice were euthanized. The survival rate was checked every other day since DSS treatment, and body weight, hair raising, animal's movement, diarrhea were checked to calculate DAI score. To evaluate the efficacy of the combination with Etanercept (TNF blocker; Enbrel), Enbrel was intraperitoneally administered to the group at a dosage of 10 mg/kg on Day 0, 2, 4, and 6.
As shown in
Enbrel used in this experiment is only an illustrative agent for treating inflammatory bowel disease, and other agents for treating inflammatory bowel disease could be used in combination with VIP expressed from transformed LAB strains.
(3) Histopathology Test
The animals were euthanized on the 9th day after starting the experiment. Two parts with the distance of 2 cm in the distal area of the colon were extracted at the length of 0.5 cm, respectively, and were fixed in 10% formalin solution and paraffin embedded. The embedded tissue was cut with a microtome to pieces having the length of 5 μm to prepare slides and stained with H&E. The stained tissue was tested for severity with referring to the evaluation method of Erben et al., Int J Clin Exp Pathol 2014; 7(8):4557-4576, which was performed three, or four or more random parts per sample.
The evaluation method may be briefly described as below. Intestinal epithelial cells damage and inflammatory cell infiltration was separately evaluated and two scores were summed to obtain scores ranging from 0 to 8.
Inflammatory cell infiltration was evaluated according to the following standard:
Epithelial cell damage was evaluated according to the following standard:
As shown in
Specifically, as shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2018-0173063 | Dec 2018 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2019/018596 | 12/27/2019 | WO | 00 |
