Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora

Definitions

• This disclosure relates to a microorganism that regulates the activation ability of Toll-Like Receptor 5 (TLR5), and a method for producing the microorganism.
• TLR5 Toll-Like Receptor 5
• ICIs immune checkpoint inhibitors
• the present disclosure provides: (Item 1) A microorganism, in which at least one gene of a group of flagella-constituting genes in the microorganism has been modified, and the modification regulates the activation ability of Toll-Like Receptor 5 (TLR5) in a host, as compared with a microorganism having the gene that has not been modified. (Item 2) The microorganism described in the preceding item, wherein the flagellum-constituting gene group includes fliC, fliD, flgE, fliF, flgK, flgM, flgN, fliT, and sigD.
• TLR5 Toll-Like Receptor 5
• the microorganism according to any one of the preceding items, wherein the modification comprises a point mutation in the gene.
• the modification includes a mutation that generates a stop codon.
• the microorganism according to any one of the preceding items, wherein the modification comprises at least two mutations in the at least one gene.
• the modification includes at least one mutation in each of at least two genes selected from the group of flagellar component genes.
• the modification includes at least two mutations in each of at least two genes selected from the group of flagellar component genes.
• the microorganism comprises enterococcus.
• TLR5 Toll-Like Receptor 5
• a method for producing a microorganism for regulating the activation ability of Toll-Like Receptor 5 (TLR5) in a host comprising: (A) modifying at least one gene of a flagella-constituting gene group in the microorganism, by converting one or more nucleotides of a target nucleic acid sequence of the gene to another nucleotide or deleting the nucleotide, or by inserting one or more nucleotides into the target nucleic acid sequence of the gene in the microorganism; (B) testing the TLR5 activation ability of microorganisms having the modified gene, and selecting microorganisms having an altered TLR5 activation ability by comparing with microorganisms having an unmodified gene; (C) repeating steps (A) and (B) as necessary
• the flagellum-constituting gene cluster comprises fliC, fliD, flgE, fliF, flgK, flgM, flgN, fliT, and sigD.
• the regulation of TLR5 activation ability comprises improving TLR5 activation ability.
• the modification increases the expression level of Flagellin in the host and/or increases the extracellular secretion level of Flagellin, compared to a microorganism having the gene without modification.
• a pharmaceutical comprising a microorganism, wherein at least one gene in a group of flagella-constituting genes in the microorganism has been modified, and the modification regulates the activation ability of Toll-Like Receptor 5 (TLR5) in a host, as compared to a microorganism having the gene that has not been modified.
• TLR5 Toll-Like Receptor 5
• the pharmaceutical described in the above item, wherein the pharmaceutical is for treating or preventing cancer or an infectious disease.
• the infectious disease includes an infectious disease caused by at least one selected from the group consisting of bacteria and viruses.
• (Item C4) The pharmaceutical agent according to any one of the preceding items, wherein the pharmaceutical agent is administered in combination with an immune checkpoint inhibitor.
• the immune checkpoint inhibitor is selected from the group consisting of agents against molecules selected from the group consisting of CTLA-4, PD-1, LAG-3, BTLA, KIR, TIM-3, PD-L1, PD-L2, B7-H3, B7-H4, HVEM, GAL9, CD160, VISTA, BTNL2, TIGIT, PVR, BTN1A1, BTN2A2, BTN3A2, and CSF-1R, and any combination thereof.
• a method for treating or preventing cancer or an infectious disease comprising administering a medicament described in any one of the preceding items.
• the present disclosure makes it possible to regulate the activation ability of Toll-Like Receptor 5 (TLR5) in a host by subjecting a microorganism to specific modifications, thereby obtaining a microorganism with improved TLR5 activation ability. Furthermore, by combining genes targeted for modification, a synergistic effect of improving TLR5 activation ability can be expected.
• TLR5 Toll-Like Receptor 5
• FIG. 1 shows the results of genome editing sites in a microorganism and its TLR5 activation ability in one embodiment of the present disclosure.
• FIG. 2-1 shows the results of genome editing sites in a microorganism and its TLR5 activation ability in one embodiment of the present disclosure.
• FIG. 2-2 shows the results of genome editing sites in a microorganism and its TLR5 activation ability in one embodiment of the present disclosure.
• Figures 2-3 show the results of genome editing sites in a microorganism and its TLR5 activation ability in one embodiment of the present disclosure.
• Figures 2-4 show the genome editing sites of a microorganism and its TLR5 activation ability in one embodiment of the present disclosure.
• FIG. 2-5 show the results of genome editing sites in a microorganism and its TLR5 activation ability in one embodiment of the present disclosure.
• FIG. 3 is a photograph showing electrophoresis confirming the expression of Flagellin in a microorganism according to one embodiment of the present disclosure.
• FIG. 4 is a graph showing the amount of TNF ⁇ produced when human macrophage-like THP-1 cells were stimulated with a microorganism according to one embodiment of the present disclosure.
• FIG. 5 is a graph showing the amounts of TNF ⁇ and IFN ⁇ produced when human peripheral blood mononuclear cells were stimulated with a microorganism according to one embodiment of the present disclosure.
• FIG. 6 is a schematic diagram showing a test design for a simultaneous administration test with an anti-PD-1 antibody in one embodiment of the present disclosure.
• the diagram shows a case where the administration of the bacterial liquid is started from Day 0 simultaneously with the administration of the anti-PD-1 antibody.
• FIG. 7 is a graph showing the survival rate of a microorganism according to one embodiment of the present disclosure when administered alone or in combination with an anti-PD-1 antibody.
• 8 is a graph showing the survival rate when the microorganism according to one embodiment of the present disclosure was administered alone or simultaneously with an anti-PD-1 antibody. A significant difference was confirmed between the administration of the genome-edited strain alone and the administration in combination with an anti-PD-1 antibody.
• FIG. 9 is a schematic diagram showing a test design for a simultaneous administration test with an anti-PD-1 antibody in one embodiment of the present disclosure.
• a schematic diagram is shown in which administration of a bacterial liquid is started on Day -14.
• FIG. 10 is a graph showing survival rate and tumor burden when a microorganism according to an embodiment of the present disclosure is administered alone or in combination with an anti-PD-1 antibody.
• 11 is a graph showing the survival rate when the microorganism according to one embodiment of the present disclosure is administered alone or simultaneously with an anti-PD-1 antibody. A significant difference was confirmed when the genome-edited strain was administered in combination with an anti-PD-1 antibody.
• microorganisms refers to minute living organisms, including prokaryotes such as bacteria and actinomycetes, eukaryotes such as yeast and mold, lower algae, fungi, viruses, and even individual, separate cells of multicellular organisms such as animals and plants. Microorganisms also include natural microorganisms, as well as those cultured and artificially propagated, mutated microorganisms, and microorganisms artificially modified by transformation or other techniques.
• modification of a gene means that a nucleotide (e.g., dC) on a DNA strand is converted to another nucleotide (e.g., dT, dA, or dG) or deleted, or that a nucleotide or nucleotide sequence is inserted or added between certain nucleotides on a DNA strand.
• “modification” includes the substitution or deletion of one or more nucleotides at a targeted site of double-stranded DNA, or the insertion or addition of one or more nucleotides at a targeted site of double-stranded DNA.
• the double-stranded DNA to be modified is not particularly limited, but is preferably genomic DNA.
• the "targeted site" of double-stranded DNA means all or a part of the “target nucleotide sequence” that the nucleic acid sequence recognition module specifically recognizes and binds to, or the vicinity of the target nucleotide sequence (either one or both of the 5' upstream and 3' downstream), and the range can be appropriately adjusted between one base and several hundred bases in length depending on the purpose.
• the term "gene” is interpreted in the broadest sense to mean a character string of nucleic acids or a sequence of a substance that carries it (e.g., nucleotides such as DNA or RNA), and preferably a sequence or a substance that contains a sequence that exerts some function.
• it also includes adjacent transcriptional regulatory regions such as promoters and enhancers that control the timing and amount of transcription of the transcript as a transcription factor binding site, and adjacent transcriptional regulatory regions such as promoters and enhancers that control the timing and amount of transcription of the transcript as a transcription factor binding site.
• flagellum-constituting genes refers to genes or nucleic acid sequences that produce the flagellum structure or a part thereof, factors (transcription factors) that control the expression of genes that produce the flagellum structure or a part thereof, or parts thereof.
• flagellum-constituting genes include fliC, fliD, flgE, fliF, flgK, flgM, flgN, fliT, and sigD.
• TLR5 activation ability refers to the ability to activate the signal transduction pathway initiated by TLR5 activation and the resulting immune response, anti-inflammatory function, anti-infectious disease function, anti-tumor function, or anti-allergic function, and any mechanism of activation may be used.
• the method for evaluating the TLR5 activation ability is not particularly limited, and it can be measured and evaluated by a known method.
• Examples of methods for evaluating the TLR5 activation ability include a method using the expression level and/or transcription level of a factor present in the signal transduction pathway initiated by TLR5 as an index, and a method of measuring the activity of a reporter gene in vitro using a specific cell that retains a reporter gene (luciferase, alkaline phosphatase, etc.) expressed in response to TLR5 activation on the genome or as a plasmid.
• a reporter gene luciferase, alkaline phosphatase, etc.
• regulating the activation ability of TLR5 refers to enhancing, improving, or decreasing the activation ability of TLR5.
• Regulating the activation ability of TLR5" also includes achieving a desired value or level of TLR5 activation ability.
• a microorganism in which at least one gene in a group of flagella-constituting genes in the microorganism is modified, and the modification regulates the activation ability of Toll-Like Receptor 5 (TLR5) in a host, compared to a microorganism having the gene that is not modified.
• TLR5 Toll-Like Receptor 5
• the microorganism of the present disclosure is useful as a microbiome drug discovery technology that enhances the effects of cancer immunotherapy using immune checkpoint inhibitors.
• Immune checkpoint inhibitors bind to immune checkpoint molecules to release immune suppression and activate the cancer immune response.
• Representative target molecules of ICIs include cytotoxic T-lymphocyte (associated) antigen 4 (CTLA-4), programmed cell death 1 (PD-1), and PD-1 ligand 1 (PD-L1), all of which inhibit the activation of T cells in the cancer immune cycle.
• CTL-4 cytotoxic T-lymphocyte
• PD-1 programmed cell death 1
• PD-1 ligand 1 ligand 1
• PD-1 and PD-L1 also act to put the brakes on the cancer immune response.
• the activation ability of TLR5 can be regulated, and preferably improved, by modifying at least one gene in the group of flagella-constituting genes in a microorganism.
• ICI binds to the PD-1 molecule on human T cells, releasing the brakes on immune activity and indirectly killing cancer cells.
• Bacterial flagellin is known as one of the microbe-associated molecular patterns (MAMPs) and is recognized by Toll-like receptor 5 (TLR5) expressed on the surface of various host cells, such as epithelial cells, macrophages, dendritic cells, and T cells, and induces downstream immune responses.
• MAMPs microbe-associated molecular patterns
• TLR5 Toll-like receptor 5
• TLR5 interacts with flagellin from some gram-negative and gram-positive bacteria and activates the NF- ⁇ B signaling pathway via the adaptor protein MyD88.
• the flagellar component gene group may be a gene or nucleic acid sequence that produces a flagellar structure or a part thereof, a factor (transcription factor) that controls the expression of a gene that produces a flagellar structure or a part thereof, or a part thereof, and may be, for example, flagellin (fliC, Gene ID: 15140735 (EC20)), flagellar filament capping protein FliD (fliD, Gene ID: 15140737 (EC20)), flagellar hook protein FlgE (flgE, Gene ID: 15140707 (EC20)), flagellar M-ring protein FliF (fliF, Gene ID: 15140699 (EC20)), flagellar hook-associated protein FliF (fliF, Gene ID: 15140699 (EC20)), or a part thereof.
• flagellin flagellin
• FliC flagellar filament capping protein
• FliD flagellar filament capping protein
• FliD flagellar filament ca
• proteins include, but are not limited to, associated protein FlgK (flgK, Gene ID: 15140733 (EC20)), flagella biosynthesis anti-sigma factor FlgM (flgM, Gene ID: 15140731 (EC20)), flagella synthesis protein FlgN (flgN, Gene ID: 15140732 (EC20)), flagella biosynthesis regulatory protein FliT (fliT, Gene ID: 15140738 (EC20)), and RNA polymerase sigma-28 factor SigD (sigD, Gene ID: 15140716 (EC20)). All Gene IDs indicate the Gene IDs assigned to E. cassliflavus EC20, which is used as the Ref_seq for E. casseliflavus. In one embodiment, the gene encoding flagellin may be called hag in some bacterial species (e.g., Gene ID: 936742 for Bacillus subtilis).
• the expression level of Flagellin in a host can be increased and/or the amount of extracellular secretion of Flagellin can be increased compared to a microorganism having the unmodified gene.
• Flagellin is a major structural subunit of flagella produced by motile bacteria, and is highly conserved in both gram-negative and gram-positive bacteria. In mammals, TLR5 receives various flagellins and causes activation of innate and acquired immunity.
• flagellin (FliC) possessed by Salmonella typhimurium is a 494 amino acid protein, and is known to upregulate the production of chemokines and antibacterial substances in human epithelial cells and monocytes, and to induce maturation of dendritic cells.
• the flagellin protein which has a specific amino acid sequence, activates human TLR5. It is known that the expression of the flagellin protein is controlled in conjunction with the expression of various flagellar component factors, and that the expression of the flagellin protein increases or decreases with the dysfunction of these genes. For example, it is known that when the FliD protein, which forms a cap structure at the tip of the flagellum, is deleted, Flagellin is unable to form a flagellar structure and is secreted outside the cell.
• base editing is used to induce dysfunction of flagellum-related genes related to the expression and/or secretion of Flagellin in a microorganism, thereby increasing the amount of Flagellin expression or the amount of Flagellin secreted extracellularly, thereby regulating and/or enhancing the ability of the microorganism to activate TLR5 in a host.
• the microorganisms used in the present disclosure include enterococci, such as E. gallinarum and E. casseliflavus.
• enterococci such as E. gallinarum and E. casseliflavus.
• E. gallinarum and E. casseliflavus are known to have flagella and are motile.
• E. gallinarum and E. casseliflavus have very few reports of being associated with hospital infections and are found only in special and rare situations where the immune system is weakened, making them highly useful as bacterial preparations.
• the microorganism used in the present disclosure may be a flagellated microorganism, and examples of such microorganisms include those of the genus Listeria (Listeria monocytogenes), Pseudomonas (Pseudomonas aeruginosa PAO1), Shewanella (Shewanella oneidensisMR-1), Salmonella (Salmonella Typhimurium str. LT2), Clostridioides (Clostridioides difficile 630), Enterococcus (Enterococcus saccharolyticus 310, Enterococcus sp. HSIEG1, E. gallinarum MR ⁇ 0518, E. casseliflavus DSM20680, E.
• Listeria Listeria monocytogenes
• Pseudomonas Pseudomonas aeruginosa PAO1
• Shewanella Shewanella oneidensisMR-1
• Salmonella Salmonella Typhimurium str. LT2
• Enterococcus sp.6D12 DIV0197 E. gallinarum DSM100110, Enterococcus sp.8G7 MSG3316, Enterococcus sp.RIT-PI-f, Enterococcus sp.6C8 DIV0013, Lactobacillus genus (Lactobacillus capillatus, Lactobacillus sucicola DSM 21376, Lactobacillus hordei, Lactobacillus satsumensis DSM 16230, Lactobacillus oeni, Lactobacillus aquaticus, Lactobacillus uvarum DSM 19971, Lactobacillus cacaonum DSM 21116, Lactobac illus mali DSM 20444), Vagococcus (Vagococcus penaei, Vagococcus fluvialis), Carnobacterium (Carnobacterium funditum, Carnobacterium mobile, Carnobacterium pleistocenium), Marin
• NSP9.1 Bacillus sonorensis
• Anoxybacillus genus (Anoxybacillus flavithermus), Halalkalibacillus genus (Halalkalibacillus halophilus), Marinococcus genus (Marinococcus halophilus), Oxobacter genus (Oxobacter pfennigii), Clostridium genus (Clostridium sp.
• Clostridium cochlearium Clostridium cochlearium
• Bacillaceae genus Bacillaceae bacterium EAG3
• Pontibacillus genus Pontibacillus halophilus, Pontibacillus yanchengensis
• Halobacillus genus Halobacillus sp.
• Novibacillus genus Novibacillus genus (Novibacillus thermophilus), B Revibacillus genus (Brevibacillus brevis), Paenibacillus genus (Paenibacillus napythalenovorans), Desulfotomaculum genus (Desulfotomaculum hydrothermale, Desulfofundulus thermocisternus), Mycobacterium genus (Mycobacterium tuberculosis), Acetobacterium genus (Acetobacterium wieringae), Virgibacillus genus (Virgibacillus pantothenticus), Paenibacillus genus (Paenibacillus sp.
• P1XP2 Sporosarcina genus (Sporosarcina sp. D27), Lysinibacillus genus (Lysinibacillus Examples of bacteria that may be of concern include bacteria from the genus Virgibacillus sinibacillus boronitolerans, Brevibacillus laterosporus, Sediminibacillus halophilus, Terribacillus saccharophilus, Oceanobacillus iheyensis, Oceanobacillus massiliensis, Virgibacillus alimentanus, Lentibacillus sediminis, and Virgibacillus dokdonensis.
• ATCC700327 can be used as a parent strain for E. casseliflavus
• JCM8728 can be used as a parent strain for E. gallinarum, to perform base editing to regulate and/or enhance the TLR5 activation ability of the present disclosure.
• the amount of Flagellin secreted extracellularly can be increased by causing fliD dysfunction by base editing.
• Deficiency of the flagellar cap component FliD promotes extracellular secretion of Flagellin monomers.
• the interaction site with TLR5 is not exposed, and so the ability to activate TLR5 is lost.
• the Flagellin monomer is in a free state, it has high TLR5 activation ability.
• FliD is a protein with a total length of 433 amino acids, and there are two sites at which stop codons can be introduced, up to the 150th position on the N-terminus. If stop codons are introduced at these positions by base editing, most of FliD is deleted, preventing proper folding and resulting in dysfunction.
• the expression level of Flagellin can be increased by causing a functional loss of the transcriptional repressor FlgM of the Flagellin component gene fliC by base editing.
• fliC is negatively controlled by the transcriptional repressor FlgM until the final stage of flagellum formation, so dysfunction of FlgM induces constitutive expression of the fliC gene.
• FlgM has a total length of 91 amino acid residues, and there are sites at positions 64 and 84 at the N-terminus where stop codons can be introduced, but introduction of a stop codon at position 64 is expected to cause a functional loss.
• multiple editing is performed on at least two genes selected from the group of flagellum-constituting genes, such as fliC, fliD, flgE, fliF, flgK, flgM, flgN, fliT, and sigD, thereby achieving a further increase in the expression or extracellular secretion of Flagellin.
• flagellum-constituting genes such as fliC, fliD, flgE, fliF, flgK, flgM, flgN, fliT, and sigD
• genes related to TLR5 activation can be modified.
• examples of such genes include mprA (emrR) (DNA-binding transcriptional regulator), hemK (N5-glutamine methyltransferase), and yjeA (Elongation factor P Lys34 lysyltransferase).
• mprA emrR
• hemK N5-glutamine methyltransferase
• yjeA Elongation factor P Lys34 lysyltransferase
• the modification of at least one gene in the microorganism of the present disclosure can be achieved by standard molecular biology techniques.
• the modification can include a point mutation in the gene, and a method for site-specifically and precisely modifying a target double-stranded polynucleotide can be, for example, a method of contacting a target double-stranded polynucleotide with a Cas protein and a guide RNA, or a method of contacting a target double-stranded polynucleotide with a complex of a Cas protein and a nucleic acid base conversion enzyme and a guide RNA.
• a complex is formed between the Cas9 protein and the guide RNA, and the complex binds to the target double-stranded polynucleotide.
• the Cas9 protein modifies the base sequence in the target polynucleotide by not cleaving the target double-stranded polynucleotide or by cleaving only one strand, i.e., without causing a double-stranded cleavage.
• the modification is preferably performed in single-base units.
• the specific and precise modification of the single base unit is preferably performed using a nucleic acid base conversion enzyme in the complex.
• the nucleic acid base conversion enzyme include deaminases.
• deaminases that can be used include cytosine deaminase, cytidine deaminase, adenosine deaminase, and the like.
• the complex in one embodiment may contain an Indel formation inhibitor such as uracil DNA glycosylase inhibitor (UGI) to inhibit Indel formation.
• UBI uracil DNA glycosylase inhibitor
• the specific and precise modification of the single base unit can also utilize a method using a complex of a nucleic acid sequence recognition module and DNA glycosylase.
• a complex of a nucleic acid sequence recognition module and DNA glycosylase is expressed from an expression vector or RNA molecule introduced into a cell
• the nucleic acid sequence recognition module specifically recognizes and binds to a target nucleotide sequence in a double-stranded DNA of interest (e.g., genomic DNA)
• the action of the DNA glycosylase linked to the nucleic acid sequence recognition module causes an abasic reaction in the sense strand or antisense strand of the targeted site (which can be appropriately adjusted within a range of several hundred bases including all or part of the target nucleotide sequence or their vicinity), resulting in an abasic site (AP site) in one strand of the double-stranded DNA.
• the base excision repair (BER) system in the cell is activated, and first, an AP endonuclease recognizes the AP site and cuts the phosphate bond of one strand of DNA, and an exonuclease removes the abasic nucleotide. Next, a DNA polymerase inserts a new nucleotide using the opposite strand DNA as a template, and finally, a DNA ligase repairs the splice. When a repair error occurs at any stage of this BER, various mutations are introduced.
• the CRISPR-Cas system recognizes the sequence of a double-stranded DNA of interest by using a guide RNA complementary to the target nucleotide sequence, so any sequence can be targeted simply by synthesizing an oligo-DNA capable of specifically hybridizing with the target nucleotide sequence, and since the double-stranded DNA is unwound at the targeted site to generate a single-stranded region and an adjacent region having a loosened double-stranded DNA structure, DNA glycosylase can be made to act efficiently in a targeted site-specific manner without combining factors that change the structure of the double-stranded DNA.
• a CRISPR-Cas system that does not have at least one DNA cleavage ability of Cas (CRISPR-mutant Cas) or a CRISPR-Cas system that does not have both DNA cleavage abilities of Cas (CRISPR-mutant Cas) can be preferably used as the nucleic acid sequence recognition module.
• the nucleic acid sequence recognition module of the present disclosure using CRISPR-mutant Cas is provided as a complex of an RNA molecule consisting of a guide RNA complementary to a target nucleotide sequence and a tracrRNA required for recruiting the mutant Cas protein, and the mutant Cas protein.
• the modification can be performed in any environment, in vivo or in vitro. In one embodiment, the modification can also be performed outside the body, i.e., ex vivo or in vitro.
• the modification of at least one gene in the microorganism of the present disclosure can include a mutation that generates a stop codon or a mutation that generates an amino acid substitution by the above-mentioned method. This can regulate and/or improve the activation ability of TLR5 in the host.
• the modification of at least one gene in the microorganism of the present disclosure can include at least two, three, four, five, six, seven, or eight mutations in a gene.
• the ability to activate TLR5 in a host can be regulated and/or improved.
• modification of at least one gene in a microorganism is preferably performed by base editing, preferably single base editing.
• the modification of at least one gene in the microorganism of the present disclosure can include at least one mutation in each of at least two genes selected from the flagellar component gene group, and the at least two genes can be independently selected from the flagellar component gene group.
• the modification of at least one gene in the microorganism of the present disclosure can include at least two mutations in each of at least two genes selected from the flagellar component gene group.
• the microorganisms of the present disclosure can reside in the gut, oral cavity, and/or skin of a subject, e.g., are of a genus that constitutes at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, or at least about 40% or more of the total culturable microorganisms in the subject's feces.
• the microorganisms in the subject's gut or feces can be analyzed by any technique known in the art, including 16S ribosomal sequencing.
• the microorganisms of the disclosure are capable of stably colonizing the human gut, oral cavity, and/or skin.
• the microorganisms of the disclosure are capable of colonizing the gut of a subject with, for example, increased abundance, stability, or ease of initial colonization in the gut compared to the same or similar microorganisms that are not modified.
• the microorganism of the present disclosure may further include a gene for therapeutic use.
• a gene for therapeutic use may be a gene that the microorganism originally possesses, or a gene that exerts a desired effect may be introduced as is or with a partial modification.
• the gene for therapeutic use may be a type 1 fimbrin D-mannose specific adhesin (fimH) or the like.
• the microorganism of the present disclosure may further include a gene for diagnostic use. Such a gene for diagnostic use may be a gene that the microorganism originally possesses, or a gene that exerts a desired effect may be introduced as is or with a partial modification.
• the gene for diagnostic use may be a bacterial actin-like cytoskeleton protein (cell shape-determining protein (mreB) or the like.
• the microorganism of the present disclosure may further include a gene for colonization.
• the gene for colonization may be a DNA-binding transcriptional activator (DNA-binding transcriptional activator) flhD or the like.
• the transgene can inhibit functional expression of the target protein by the effect of a stop codon introduced as a result of single-base editing.
• the microorganisms of the present disclosure may be utilized as therapeutic preparations, and such therapeutic preparations may, for example, comprise a therapeutically effective amount of the microorganisms of the present disclosure, for example, at least about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, or less by weight of the microorganism.
• the disease treatable using the microorganism of the present disclosure can include cancer.
• cancer can include bladder cancer (including aggressive and metastatic bladder cancer), breast cancer (e.g., estrogen receptor positive breast cancer, estrogen receptor negative breast cancer, HER-2 positive breast cancer, HER-2 negative breast cancer, triple negative breast cancer, inflammatory breast cancer), colon cancer (including colorectal cancer), kidney, liver, lung cancer (including small cell lung cancer and non-small cell lung cancer (including adenocarcinoma, squamous cell carcinoma, bronchioloalveolar carcinoma and large cell carcinoma)), genitourinary tract cancer, such as ovarian cancer (including fallopian tube, endometrial and peritoneal cancer), cervical cancer, prostate cancer (e.g., hormone refractory prostate cancer) and testicular cancer, lymphatic system cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic cancer), stomach cancer (e.g., gastroesophageal cancer, upper gastric or lower gas
• bladder cancer including aggressive and meta
• the microorganism of the present disclosure can function as a vaccine enhancer or can have the function of improving the effect of a vaccine enhancer.
• a method for producing a microorganism for regulating the activation ability of Toll-Like Receptor 5 (TLR5) in a host comprising: (A) modifying at least one gene of a group of flagella-constituting genes in the microorganism, by converting one or more nucleotides of a target nucleic acid sequence of the gene to another nucleotide or deleting the gene, or by inserting one or more nucleotides into the target nucleic acid sequence of the gene; (B) testing the TLR5 activation ability of the microorganism having the modified gene, and selecting a microorganism having an altered TLR5 activation ability by comparing it with a microorganism having an unmodified gene; and (C) repeating steps (A) and (B) as necessary if a microorganism having an improved TLR5 activation ability cannot be selected.
• the method of the present disclosure may have any of the features
• TLR5 Toll-Like Receptor 5
• the method of the present disclosure can include any of the features described elsewhere herein.
• Short Protocols in Molecular Biology A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F. M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M. A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F. M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, J. J. et al. (1999).
• gene synthesis and fragment synthesis services such as GeneArt, GenScript, Integrated DNA Technologies (IDT) can be used, and other references include, for example, Gait, M. J. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M. J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R. L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. (1994).
• Example 1 Measurement of TLR5 activation ability of genome-edited strains
• the TLR5 activation ability of each strain was measured using genome-edited strains prepared by modifying the target genes and their editing sites as shown in Figures 1 and 2.
• the target genes to be edited were fliC, fliD, flgE, fliF, flgK, flgM, flgN, fliT, and sigD, each of which was used alone.
• the gRNA for each target gene was designed mainly to introduce a stop codon at the 5' end, which induces a complete loss of protein function, and to induce amino acid substitutions in active sites, etc., predicted from protein structure prediction.
• TLR5 activation ability was measured as follows. The strains to be evaluated were cultured overnight (12-18 hours) at 37°C in Brain Heart infusion medium, washed with Opti-MEM medium, and the turbidity (660 nm) was measured. The turbidity was adjusted to 1.0 with Opti-MEM medium, and then a bacterial solution was prepared by diluting 104 times with Opti-MEM. The following three types of plasmids were introduced into human embryonic kidney cells HEK293T to construct cells for evaluating TLR5 activation ability.
• the three types of plasmids are a plasmid that forcibly expresses human TLR5, a plasmid that places the NanoLuc (registered trademark) gene under the control of an activation-responsive sequence to evaluate the activation ability of TLR5, and a plasmid that constitutively expresses the firefly luciferase gene that functions as an internal standard.
• the three types of plasmids were introduced into HEK293T cells, and after 16 to 20 hours, the diluted bacterial solution prepared above was added in an amount of 1/10 of the medium in which the plasmid-introduced cells grew, and the cells were co-cultured at 37°C for 4 hours.
• the TLR5 activation ability was measured using the activity of NanoLuc as an index using Promega's Nano-Glo (registered trademark) Dual-Luciferase (registered trademark) Reporter Assay System. Following the recommended protocol, the activity of firefly luciferase, which is the internal standard, was measured, and then the activity of NanoLuc, which reflects TLR5 activation, was measured. NanoLuc activity was standardized with firefly luciferase activity to evaluate the TLR5 activation ability of each strain.
• Example 2 Confirmation of Flagellin expression
• MTM medium 1% w/v Bacto Peptone, 0.5% w/v NaCl, 0.3% w/v Beet extract
• the culture was heat-treated at 60°C for 20 minutes.
• the mixture was centrifuged at 2,900 x g for 10 minutes, and the supernatant was filtered through a 0.22 ⁇ m filter. 15 mL of the filtrate was concentrated to 1 mL using an Amicon-15 (MWCO 10k, Millipore).
• the agarose beads were precipitated by centrifugation at 1,000 ⁇ g for 2 minutes, the supernatant was removed, and the beads were suspended in 500 ⁇ L PBS. This centrifugation and suspension procedure was repeated three times in total, and then the agarose beads were suspended in 45 ⁇ L of 1 ⁇ Laemmli dye and heat-treated at 95 ° C. for 5 minutes to elute the bound protein from the beads. After centrifugation at 1,000 ⁇ g for 2 minutes, the supernatant was collected and used as a TLR5-binding protein sample.
• Flagellin-derived bands were detected in the wild-type strain and the flgN-, fliD-, and flgK-edited strains, but no Flagellin bands were detected in the fliC- and fliF-edited strains ( Figure 3).
• Example 3 Other Genetic Modifications By rendering mprA, a transcription factor, dysfunctional by base editing as in Example 1, the master regulatory factor of the flagellum-related gene group is activated, which is expected to result in increased expression of flagellin and enhanced activation ability of TLR5.
• Example 4 Evaluation of the antitumor effect of Enterococcus alone or in combination with immune checkpoint inhibitors using a tumor-bearing mouse model
• a tumor-bearing mouse model was created using a mouse colon cancer cell line, and the E. casseriflavus wild-type strain or its genome-edited strain was administered alone or in combination with an anti-PD-1 antibody (anti-mouse PD-1 [CD279], clone: RMP1-14, Bio X Cell), which is an immune checkpoint inhibitor, to evaluate the antitumor effect.
• an anti-PD-1 antibody anti-mouse PD-1 [CD279], clone: RMP1-14, Bio X Cell
• Mouse colon cancer cell line MC38 (Cat. No. ENH204-FP, Kerafast) is a C57BL/6J mouse (6 weeks old, female, Jackson Laboratory Japan Co., Ltd.), and CT26 (Cat. No. CRL-2638, ATCC) is a BALB/c mouse (6 weeks old, female, Jackson Laboratory Japan Co., Ltd.), and 100 ⁇ L of each cell suspended in physiological saline is subcutaneously transplanted into the right flank of each mouse.
• the tumor diameter of the mouse is measured, and the estimated tumor volume (long diameter x short diameter x short diameter / 2) is calculated. Based on the estimated tumor volume, the group is divided and designated as Day 0. On Days 0, 2, 4, 7, 9, 11, 14, 16, 18, 21, 23, and 25, E. The E.
• casseriflavus wild-type strain and its genome-edited strain are prepared in phosphate-buffered saline to give 10 9 CFU/100 ⁇ L, and 100 ⁇ L per individual is forcibly administered orally.
• the anti-PD-1 antibody is administered into the tail vein at 5 mg/kg per individual on days 0, 3, 7, 10, and 14. Under the above administration conditions of the bacterial solution and anti-PD-1 antibody, the E. casseriflavus wild-type strain or its genome-edited strain alone, or in combination with the anti-PD-1 antibody, is administered.
• the estimated tumor volume and body weight of the mice are measured twice a week from day 0.
• Tumor growth is expected to be suppressed in the group administered the genome-edited strain alone and in the group administered the genome-edited strain in combination with an anti-PD-1 antibody.
• Example 5 Cancer Treatment A strain with enhanced TLR5 activation ability is orally administered to a cancer patient (regardless of the type of cancer) in the form of a tablet or capsule, and the strain is allowed to exist transiently or ideally become established in the intestine of the patient, stimulating the host's immune cells and enhancing the immune response to cancer cells.
• ICI in combination, it is possible to inhibit the suppressive effect of cancer cells on immune cells, and it is expected that the reactivity of activated immune cells to cancer cells will be enhanced.
• the timing of administration of the strain can be either simultaneous administration or administration of the strain prior to ICI.
• the strain may be administered multiple times. Even in patients who have a low response to cancer immunotherapy using ICI alone, it is expected that the response of ICI will be increased by combining it with the strain.
• THP-1 cells (RCB3686, RIKEN BioResource Research Center) were cultured using 10% FBS-containing RPMI1640 medium. After seeding 2 ⁇ 10 5 cells/well in a 24-well plate, all-trans retinoic acid was added to each well at 100 ⁇ M. After 48 hours of culture in an incubator (5% CO2, 37 ° C.), E. casseriflavus wild-type strain or its genome-edited strain was added to 10 8 CFU/well for stimulation. After 2 hours of stimulation, antibiotics were added to stop the growth of the strain, and the cells were further cultured for 22 hours. The cell culture supernatant was collected, and the amount of TNF ⁇ in the culture supernatant was measured by ELISA (Enzyme-Linked Immuno Sorbent Assay).
• ELISA Enzyme-Linked Immuno Sorbent Assay
• the genome-edited strain produced increased amounts of the inflammatory cytokine TNF ⁇ compared to the wild-type strain.
• Example 7 Reactivity test using human peripheral blood mononuclear cells
• Human peripheral blood mononuclear cells (CC-2705, Lonza) were cultured using 10% FBS-containing RPMI1640 medium. After seeding in a 96-well plate at 5 x 10 5 cells/well, the cells were cultured for 2 hours in an incubator (5% CO 2 , 37°C). E. casseriflavus wild-type strain or its genome-edited strain was then added at 10 4 CFU/well for stimulation. After 2 hours of stimulation, antibiotics were added to stop the growth of the strain, and the cells were further cultured for 22 hours. The cell culture supernatant was collected, and the amount of TNF ⁇ and IFN ⁇ in the culture supernatant was measured by ELISA (Enzyme-Linked Immuno Sorbent Assay).
• the genome-edited strain had increased production of the inflammatory cytokine TNF ⁇ and the immune activating factor IFN ⁇ . This suggests that the genome-edited strain is expected to activate immunity against cancer, infectious diseases, etc. through human immune cells.
• Mouse colon cancer cell line CT26 (Cat. No. CRL-2638, ATCC) was subcutaneously transplanted into the right flank of BALB/c mice (6 weeks old, female, Jackson Laboratory Japan Co., Ltd.) at 3 x 10 7 cells/mL of each cell suspended in physiological saline, 100 ⁇ L each.
• the tumor diameter of the mouse was measured, and the estimated tumor volume (long diameter x short diameter x short diameter/2) was calculated. Groups were divided based on the estimated tumor volume and designated as Day 0 (FIGS. 6 and 9).
• Anti-PD-1 antibody was administered into the tail vein at 5 mg/kg per individual on Days 0, 3, 7, 10, and 14.
• E. casseriflavus wild-type strains and their genome-edited strains were prepared in phosphate-buffered saline to give 10 9 CFU/100 ⁇ L, and 100 ⁇ L per individual was forcibly administered (FIG. 9).
• the bacterial liquid administration was started simultaneously with the administration of anti-PD-1 antibody, the bacterial strain administration started on Day 0 (total of 9 times, FIG. 6).
• E. casseriflavus wild-type strains or their genome-edited strains alone, or in combination with anti-PD-1 antibody were administered.
• the estimated tumor volume and body weight of the mice were measured twice a week from Day 0.
• Example 9 Urinary tract infection mouse model A BALB/c mouse (7 weeks old, female, Jackson Laboratory Japan Co., Ltd.) is anesthetized with isoflurane, and the bladder is lightly pressed to drain urine from the bladder. The urethral opening is disinfected with ethanol cotton, and 100 ⁇ L (1 x 10 9 CFU / mL) of E. coli (E. coli ATCC700928) suspension is injected transurethrally into the bladder using a 1 mL syringe with a blunted 26G needle. After infection by injection, the urethral opening is blocked with an eye clip for 4 hours.
• E. coli E. coli ATCC700928
• the efficacy of the genome-edited strain is evaluated by raising the mouse normally for 2 hours after removing the clip, and administering 10 9 CFU of the strain transurethrally under isoflurane anesthesia similar to that during infection. After raising the mouse for a certain period of time, the mouse is bled under isoflurane anesthesia, and each organ is aseptically removed, and then a homogenate is prepared to measure the number of bacteria in the organs.
• Example 10 Influenza virus-infected mouse model BALB/c mice (6 weeks old, female, Jackson Laboratory Japan Co., Ltd.) are used in the experiment after an acclimation period.
• the influenza virus H1N1 strain is prepared to 4 x 10 4 PFU/mL. Mice are anesthetized with isoflurane and infected by administering 50 ⁇ L of the virus suspension into the nasal cavity.
• E. casseriflavus wild strain or its genome-edited strain is prepared in phosphate-buffered saline to 10 9 CFU/100 ⁇ L, and 100 ⁇ L per individual is forcibly administered. Mice are observed daily, and mortality, weight loss, and visual score are recorded.
• blood, bronchoalveolar lavage fluid, and lung tissue are collected and stored. Viral RNA is extracted from the lung tissue, and the amount of virus is measured by quantitative PCR.
• the microorganism disclosed herein can improve the host's immune activity through activation of the host's Toll-Like Receptor 5 (TLR5) in cancer immunotherapy using immune checkpoint inhibitors, and can also enhance the antitumor effects of immune checkpoint inhibitors, so it is expected to have a wide range of applications in the medical field.
• TLR5 Toll-Like Receptor 5
• SEQ ID NO:1 Nucleic acid sequence of fliC(hag) of E_casseliflavus
• SEQ ID NO:2 Nucleic acid sequence of fliD of E_casseliflavus
• SEQ ID NO:3 Nucleic acid sequence of flgE of E_casseliflavus
• SEQ ID NO:4 Nucleic acid sequence of fliF of E_casseliflavus
• SEQ ID NO:5 Nucleic acid sequence of flgK of E_casseliflavus
• SEQ ID NO:6 Nucleic acid sequence of flgM of E_casseliflavus
• SEQ ID NO:7 Nucleic acid sequence of flgN of E_casseliflavus
• SEQ ID NO:8 Nucleic acid sequence of fliT of E_casseliflavus
• SEQ ID NO:9 Nucleic acid sequence of sigD of E_casseliflavus

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