Classifications

• • 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
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material 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
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/10—Cells modified by introduction of foreign genetic material
• • 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
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

• the present disclosure relates to an antibody or an antigen-binding fragment thereof that binds to Clostridioides difficile bacteria (C. difficile bacteria) and has the effect of inhibiting its proliferation, and uses of the antibody.
• the intestinal tract is, so to speak, "outside the body" of the living body, so it is constantly exposed to a very large variety and large number of intestinal resident bacteria, viruses, food-derived antigens, etc. These intestinal resident bacteria form the intestinal flora.
• Non-Patent Documents 4-7) When the intestinal flora changes and the symbiotic relationship with the host is disrupted, the intestinal immune system is overstimulated and its homeostasis is disrupted, which can lead to the induction of many diseases such as inflammatory bowel disease, colon cancer, asthma, allergies, and obesity. For this reason, it is known that the intestinal immune system not only eliminates pathogens, but also plays an important role in maintaining homeostasis of the immune system as a whole (Non-Patent Documents 4-7).
• C. difficile which is associated with inflammatory bowel disease
• MRSA MRSA-resistant bacterium
• C. difficile infections often occur in patients who have been taking antibiotics for a long time, and once infected, the probability of recurrence is high at 15-35%, so there is a demand for an especially effective treatment, among the bacteria associated with inflammatory bowel disease.
• treatment is carried out with powerful antibiotics such as vancomycin, but alternative methods are required because the toxins of the bacteria remain and promote the emergence of antibiotic-resistant bacteria.
• the present disclosure aims to provide antibodies that bind to C. difficile bacteria, and antigen-binding fragments thereof, as well as uses thereof.
• the inventors of the present disclosure have discovered an antibody that binds to enterobacteria, particularly C. difficile bacteria and other enterobacteria, and inhibits their proliferation. They have also demonstrated that the antibody binds to bacteria present in abnormal enteric flora (dysbiosis).
• the present disclosure provides an antibody or antigen-binding fragment thereof that binds to and inhibits the growth of C. difficile bacteria.
• the present disclosure provides an immortalized cell that produces an antibody that binds to and inhibits the growth of C. difficile bacteria.
• the present disclosure provides a composition comprising an antibody or antigen-binding fragment thereof that binds to and inhibits the growth of C. difficile bacteria.
• the present disclosure provides a method for treating disease using an antibody or antigen-binding fragment thereof that binds to and inhibits the growth of C. difficile bacteria.
• the present disclosure provides: [Item 1] Binds to C. difficile bacteria and inhibits the growth of C. difficile; heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of a heavy chain variable region represented by SEQ ID NO: 3; and light chain CDR1, light chain CDR2, and light chain CDR3 of a light chain variable region represented by SEQ ID NO: 4; or heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of a heavy chain variable region represented by SEQ ID NO: 13; and light chain CDR1, light chain CDR2, and light chain CDR3 of a light chain variable region represented by SEQ ID NO: 14;
• An antibody or antigen-binding fragment thereof comprising: [Item 2] Heavy chain CDR1 represented by SEQ ID NO:5, heavy chain CDR2 represented by SEQ ID NO:6, heavy chain CDR3 represented by SEQ ID NO:7; and light chain CDR1 represented by SEQ ID NO:8, light chain CDR2 represented by SEQ ID NO:9, light chain CDR3 represented by
• [Item 5] 5. An expression vector comprising the nucleic acid according to item 4.
• [Item 6] A cell comprising the nucleic acid according to item 4.
• [Item 7] A composition comprising the antibody or antigen-binding fragment thereof described in item 1.
• the present disclosure has the effect of providing antibodies and antigen-binding fragments thereof that bind to enterobacteria, particularly C. difficile bacteria and other enterobacteria, and uses thereof.
• FIG. 1 shows the results of testing the binding ability of SNK0004 antibody and SNK0005 antibody to C. difficile bacteria.
• FIG. 2 shows the results of testing the C. difficile proliferation inhibitory ability of SNK0004 antibody, SNK0005 antibody, W27 antibody IgG1 type antibody (rW27IgG), and negative control IgA antibody (Control rIgA).
• Figure 3 shows the results of evaluating the binding characteristics of individual bacterial species derived from stool samples of IBD patients that bind to the SNK0004 antibody, using quantitative data from the IgA Index.
• Figure 4 shows the results of evaluating the binding characteristics of individual bacterial species derived from stool samples of IBD patients that bind to the SNK0005 antibody, using quantitative data from the IgA Index.
• FIG. 1 shows the results of testing the binding ability of SNK0004 antibody and SNK0005 antibody to C. difficile bacteria.
• FIG. 2 shows the results of testing the C. difficile proliferation inhibitory ability of SNK0004 antibody, SNK0005 antibody, W27 antibody I
• antibody is used in the broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, and antibody fragments that exhibit the intended antigen-binding activity.
• a full-length antibody includes a heavy chain and a light chain that are primarily composed of polypeptides.
• the heavy chain and the light chain each include a site called a variable region that recognizes an antigen, and the site is generally called the heavy chain variable region and the light chain variable region, respectively.
• the variable region has sites called CDR1 to 3, which are further specified as sites that recognize the antigen, in order from the amino terminus.
• CDR1 to 3 are also referred to in more detail as heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, light chain CDR3, etc.
• regions other than CDR1 to 3 of the heavy chain and the light chain are referred to as heavy chain FR1 to 4 and light chain FR1 to 4, in order from the amino terminus, respectively.
• Antibodies may be in the form of an antibody consisting of one heavy chain and one light chain (also called a single-chain antibody) in addition to a form consisting of two heavy chains and two light chains. Furthermore, antibodies may be in the form of a dimer or polymer having a J (joining) chain and, in some cases, a SC (secretary component) in addition to two heavy chains and two light chains.
• the antibody may be of any class, such as IgG, IgE, IgM, IgD, IgA, or IgY, or subclass, such as IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.
• Antibodies may have amino acid sequences derived from the same species or from different species. When derived from the same species, examples include antibodies derived from humans, mice, rats, hamsters, rabbits, goats, donkeys, pigs, cows, horses, chickens, monkeys, chimpanzees, camels, llamas, etc.
• antibodies derived from a different species there is no particular limitation, but examples include antibodies derived from two or more of the following: human, mouse, rat, hamster, rabbit, goat, sheep, donkey, pig, cow, horse, chicken, monkey, chimpanzee, camel, llama, etc.
• an "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been found that the ability of an antibody to specifically bind to an antigen can also be maintained by fragments consisting of a portion of the antibody.
• an "antigen-binding fragment" of an antibody can be, but is not limited to, a Fab fragment consisting of a light chain variable region (VL), a heavy chain variable region (VH), a light chain constant region (CL) and a CH1 domain that is a part of the heavy chain constant region, an F(ab')2 fragment containing two Fab fragments linked by a disulfide bridge at the hinge region, an Fd fragment consisting of a VH and a CH1 domain, an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a dAb fragment containing a single variable domain, and an isolated complementarity determining region (CDR).
• VL light chain variable region
• VH heavy chain variable region
• CL light chain constant region
• CH1 domain that is a part of the heavy chain constant region
• F(ab')2 fragment containing two Fab fragments linked by a disulfide bridge at the hinge region
• an Fd fragment consisting of
• An antibody of the present disclosure may be a CDR-grafted antibody.
• a CDR-grafted antibody has some or all of the sequences of the CDR regions of an antibody derived from one animal species replaced with the CDR sequences of another animal species.
• one or more CDRs of a mouse antibody are replaced with the CDR sequences of a human antibody.
• CDR sequences in an antibody can be identified based on information in public databases (e.g., https://www.ncbi.nlm.nih.gov/igblast).
• identity refers to the degree to which two or more comparable amino acid or nucleotide sequences are identical to each other. Thus, the higher the identity between two amino acid or nucleotide sequences, the higher the identity or similarity of those sequences.
• the level of identity between amino acid or nucleotide sequences is usually determined using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (e.g., Karlin S, Altschul SF. Proc. Natl Acad Sci USA. 87:2264-2268 (1990), Karlin S, Altschul SF. Natl Acad Sci USA. 90:5873-7 (1993), etc.).
• BLASTN and BLASTX Programs based on this BLAST algorithm, called BLASTN and BLASTX, have been developed (for example, Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. J. Mol. Biol. 215: 403-10 (1990)). Specific techniques for these analysis methods are publicly known and can be found on the NCBI website. For example, when a certain amino acid sequence A is a certain percentage identical to another amino acid sequence B, it means that amino acid sequence A and amino acid sequence B have the same percentage of identity.
• monoclonal is a modifier characterizing an antibody or the like as being obtained from a population of substantially homogeneous antibodies.
• the individual antibodies within such a population of antibodies are identical except for possible naturally occurring mutations, which may be present in minor amounts.
• conservative substitution technique refers to a technique in which an amino acid residue is replaced with an amino acid residue having a similar side chain.
• substitution between amino acid residues with basic side chains such as lysine, arginine, and histidine is a conservative substitution technique.
• Other conservative substitution techniques include substitution between amino acid residues with acidic side chains such as aspartic acid and glutamic acid; amino acid residues with non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues with non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues with ⁇ -branched side chains such as threonine, valine, and isoleucine, and amino acid residues with aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.
• Antibodies or antigen-binding fragments thereof that bind to C. difficile bacteria Antibodies that bind to C. difficile bacterial organisms of the present disclosure bind to C. difficile bacteria. Typically, antibodies of the present disclosure bind to undisrupted C. difficile bacteria present in the culture supernatant.
• the antibody that binds to the C. difficile bacteria of the present disclosure may be produced from antibody-producing cells derived from B cells, such as hybridomas, or may be produced by using recombinant gene technology to introduce nucleic acid encoding the antibody into cells outside the immune system and used as a recombinant antibody.
• the antibody that binds to the C. difficile bacteria of the present disclosure is preferably an IgA antibody.
• an antibody that binds to a C. difficile bacterial organism of the present disclosure is A heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 3; and It comprises a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of the light chain variable region comprising the amino acid sequence represented by SEQ ID NO:4.
• an antibody that binds to a C. difficile bacterial organism of the present disclosure is A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:5; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:6, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:7; and a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:8; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:9; Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 10
• the light chain variable region comprises:
• an antibody that binds to a C. difficile bacterial organism of the present disclosure is a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:1 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:2 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.
• an antibody that binds to a C. difficile bacterial organism of the present disclosure is a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO:3 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO:4 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto;
• a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:5;
• a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:6, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:7; and a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:8;
• a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:9;
• Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 10
• the IgA antibody of the present disclosure is SNK0004, which was produced by determining the nucleic acid sequence encoding an IgA antibody produced by a hybridoma obtained from mouse-derived B cells and applying recombinant technology to produce the IgA antibody.
• the antibody SNK0004 has the following amino acid sequence configuration:
• the antibody SNK0004 is encoded by the following nucleic acid sequence:
• an antibody that binds to a C. difficile bacterial organism of the present disclosure is A heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 13; and It comprises a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of the light chain variable region comprising the amino acid sequence represented by SEQ ID NO:14.
• an antibody that binds to a C. difficile bacterial organism of the present disclosure is A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 15; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 16, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 17; and a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 18; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 19; Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:20
• the light chain variable region comprises:
• an antibody that binds to a C. difficile bacterial organism of the present disclosure is a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:13 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:14 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.
• an antibody that binds to a C. difficile bacterial organism of the present disclosure is a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 13 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 14 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto;
• a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 15;
• a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 16, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 17; and a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 18;
• a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 19;
• Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:20
• the IgA antibody of the present disclosure is SNK0005, which was produced by determining the nucleic acid sequence encoding an IgA antibody produced by a hybridoma obtained from mouse small intestine-derived B cells and applying recombinant technology to produce the IgA antibody.
• the antibody SNK0005 has the following amino acid sequence configuration:
• the antibody SNK0005 is encoded by the following nucleic acid sequence:
• the antibody of the present disclosure may be provided in the form of an antibody consisting of one heavy chain and one light chain (also called a single-chain antibody) in addition to a form consisting of two heavy chains and two light chains.
• the antibody of the present disclosure may also be provided in the form of a dimer or multimer having a J (joining) chain and, in some cases, a SC (secretary component) in addition to two heavy chains and two light chains.
• the antibodies disclosed herein may be provided as any class, such as IgG, IgE, IgM, IgD, IgA, or IgY, or any subclass, such as IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.
• the antibodies disclosed herein may have an amino acid sequence derived from the same species or from a different species.
• examples include human, mouse, rat, hamster, rabbit, goat, donkey, pig, cow, horse, chicken, monkey, chimpanzee, camel, llama, etc.
• antibodies derived from a different species there is no particular limitation, but examples include antibodies derived from two or more of the following: human, mouse, rat, hamster, rabbit, goat, sheep, donkey, pig, cow, horse, chicken, monkey, chimpanzee, camel, llama, etc.
• the antibody that binds to C. difficile bacteria can be produced by carrying out the steps of: (1) preparing an antibody-producing immortalized cell from an antibody-producing B cell collected from the intestinal mucosa lamina basement; (2) culturing the antibody-producing immortalized cell prepared in step 1 and determining a cell that produces an antibody that binds to C. difficile bacteria; and (3) recovering the antibody from the cell that produces the antibody that binds to C. difficile bacteria.
• the process may include, for example, a step of treating the heavy chain variable region with a protease or the like, a step of introducing a functional group that enables chemical bonding such as a disulfide bond, and a subsequent step of forming the chemical bond via the functional group.
• the type of cell other than B cells (sometimes referred to as "other cells" in this specification) is not particularly limited as long as it fuses with the B cells upon contact to form a hybridoma and the hybridoma does not lose the antibody-producing function exhibited by the B cells described above.
• Such other cells are preferably cells that can fuse with the B cells to form hybridomas, and the hybridomas can acquire immortalization functions.
• Specific examples include cancer cells, particularly cells derived from bone marrow types such as myelomas.
• a preferred other cell is a myeloma, for example, a mouse NS1 cell.
• the origin of the other cells is not particularly limited, but examples include humans, mice, rats, hamsters, rabbits, goats, sheep, donkeys, pigs, cows, horses, chickens, monkeys, chimpanzees, camels, llamas, etc.
• Fusion means that the B cell and another cell are united in an inseparable unity.
• inseparable unity does not include cell division, which is a phenomenon that occurs when the fused cells grow. Cells fused in this manner are an example of hybridomas in this specification.
• the conditions for mixing and fusion are not particularly limited, and any conditions normally used in methods for fusing cells may be appropriately adopted.
• the conditions used for culturing B cells or other cells may be appropriately modified.
• Such methods include, for example, a method in which B cells are mixed with a type of cell other than B cells in an appropriate medium and brought into contact with each other in the presence of polyethylene glycol or the like; a method in which electrical stimulation is applied after the above mixing; a method in which a virus such as Sendai virus is used, and then the mixture is incubated at 37°C under 5% carbon dioxide.
• the time required for fusion is not particularly limited, and may be set as long as the time required for the fusion itself to be completed. Completion of fusion may be confirmed by appropriately modifying a known method used in normal cell fusion. For example, such a method may be used to observe the progress of fusion under a microscope, and a known kit may be appropriately selected to fuse the cells according to the conditions of use.
• the specific recovery method in the step of recovering an antibody from cells that produce an antibody that binds to C. difficile bacterial bodies is not particularly limited, but examples include a method of recovering the supernatant of a culture medium of the antibody-producing cells and a method of recovering a lysate of the cells.
• the cell lysate is prepared by appropriately combining known mechanical means such as ultrasonic disruption and French press, and/or known chemical treatment methods using surfactants, cell wall digestive enzymes, and cell membrane digestive enzymes, and then subjecting the cells to a solid-liquid separation step, and recovering the liquid phase fraction to produce the antibody that binds to the C. difficile bacterial bodies of the present disclosure.
• the antibody that binds to the C. difficile bacteria of the present disclosure is produced by a method in which a nucleic acid encoding the antibody is introduced into a cell capable of producing the monoclonal antibody, the cell is cultured, and the monoclonal antibody is recovered from the cell extract or culture supernatant, and purified as necessary.
• Methods well known to those skilled in the art can be used to confirm that an antibody binds to C. difficile bacteria, for example, by identifying the antibody molecule or the cells that produce the antibody using ELISA, EIA, RIA, FLISA, FIA, FACS, or other methods.
• the cells capable of producing the monoclonal antibodies are not particularly limited as long as they are capable of producing various proteins that function by forming higher-order structures derived from mammals, etc., and may be appropriately selected from known cells such as mammalian cells such as COS cells, HEK cells (HEK293, HEK293T, etc.), HELA cells, CHO cells, and insect cells such as Sf9.
• monoclonal antibodies can also be produced using yeast cells, filamentous fungal cells, and plant cells such as soybean and Arabidopsis thaliana.
• the specific method of introducing nucleic acid, the culture conditions for cells into which the nucleic acid has been introduced, the recovery method, and the purification method vary depending on the type of cells used, and are not particularly limited. By appropriately modifying and combining known methods, it is possible to produce an antibody that binds to the C. difficile bacterial cells of the present disclosure.
• the monoclonal antibody secreted from these cells into the culture medium can be dried or lyophilized together with the culture medium, or the monoclonal antibody accumulated in these cells can be crushed, dried or lyophilized together with these cells to produce a monoclonal antibody as an antibody-containing composition suitable for enteral intake (Virdi et al., Nat. Biotechnol., 2019, Vol. 37, pp. 527-530).
• the cells producing the antibody-containing composition suitable for enteral intake are preferably yeast cells, filamentous fungal cells, or plant cells such as soybean and Arabidopsis, and the produced monoclonal antibody is preferably in a form in which a VHH fragment is fused to IgA Fc.
• the above-mentioned nucleic acid may be prepared by preparing a nucleic acid having a base sequence encoding a heavy chain variable region and a light chain variable region, as well as a base sequence encoding a heavy chain constant region and a light chain constant region of a different species, combining the prepared base sequence encoding the heavy chain variable region with a nucleic acid having a base sequence encoding the heavy chain constant region, combining the prepared base sequence encoding the light chain variable region with a nucleic acid having a base sequence encoding the light chain constant region, and introducing the combined nucleic acids into a cell capable of producing the antibody as described above.
• the antibody that binds to C. difficile bacteria according to the present disclosure is an antibody in which heavy and/or light chain CDR1-3 have amino acid sequences derived from mouse and other regions have amino acid sequences derived from human (sometimes referred to as a humanized antibody)
• the method of production is similar to that of the chimeric antibody described above, in that nucleic acid having a base sequence encoding heavy and/or light chain CDR1-3 and a base sequence encoding other regions is prepared, the nucleic acid is recombined to form heavy and light chains, and these are introduced into cells capable of producing monoclonal IgA antibodies as described above. In order to maintain the binding ability of the antibody, it may be necessary to replace some bases with other bases.
• the present disclosure provides a nucleic acid encoding an antibody or an antigen-binding fragment thereof that binds to C. difficile bacteria.
• the nucleic acid encodes the heavy and/or light chains of the antibody.
• the form of the nucleic acid is not particularly limited, and may be ribonucleotides or deoxynucleotides.
• the form of the nucleic acid is also not particularly limited, and may be a single-stranded form or a double-stranded form.
• the codons used in the nucleic acid sequence are not particularly limited, and various codons can be appropriately selected and used depending on the purpose.
• the nucleic acid encoding the antibody or antigen-binding fragment thereof of the present disclosure is used to express and produce the antibody or antigen-binding fragment thereof according to the present disclosure.
• the nucleic acid encoding the antibody or antigen-binding fragment thereof of the present disclosure encodes an antigen-binding fragment
• the two domains of the Fv fragment, VL and VH may be encoded by separate nucleic acid molecules, or, using recombinant techniques, a single protein chain in which the pair of VL and VH domains form a monovalent molecule (single-chain Fv (scFv)) may be encoded by a single nucleic acid.
• scFv single-chain Fv
• the nucleic acid encoding the antibody or antigen-binding fragment thereof of the present disclosure may be in the form of a vector.
• the vector may be an expression vector or a vector for use in genetic recombination.
• the configuration of the vector is not particularly limited, and may include various components used in known vectors.
• the present disclosure provides a cell comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof that binds to C. difficile bacteria.
• the cell may comprise one or more nucleic acids encoding the heavy and/or light chains of the antibody or antigen-binding fragment thereof of the present disclosure.
• the cell of the present disclosure produces an antibody or antigen-binding fragment thereof that binds to C. difficile bacteria.
• the cell of the present disclosure replicates a nucleic acid encoding an antibody or antigen-binding fragment thereof that binds to C. difficile bacteria.
• the type of cell of the present disclosure is not particularly limited, and cells used for protein production, nucleic acid amplification, genetic recombination, etc. may be used.
• the cell of the present disclosure may be the cell exemplified in the above (Method for producing an antibody that binds to C. difficile bacteria) or the cell exemplified in the below (Antibody-producing immortalized cell).
• the present disclosure provides an immortalized cell that produces an antibody that binds to a C. difficile bacterial organism according to the present disclosure described above.
• Specific antibody-producing immortalized cells are cells that are made by mixing and fusing the antibody-producing B cells with other types of cells, i.e., cells of a type other than B cells, or cells that have been made into immortal B cells by infecting them with EB virus or the like.
• the hybridomas may be hybridomas derived from the same species or from different species.
• Hybridomas derived from the same species may be those in which the B cells and the types of cells other than the B cells are derived from the same species in the above-mentioned [Method for producing monoclonal IgA antibodies], while hybridomas derived from different species may be those in which the B cells and the types of cells other than the B cells are derived from different species.
• Hybridomas derived from the same species may be appropriately selected from those origins described in step 1 of the above-mentioned [Method for producing monoclonal IgA antibodies] that overlap in the origin of B cells and types of cells other than B cells.
• Examples include hybridomas derived from the same species, such as human, mouse, rat, hamster, rabbit, goat, donkey, pig, cow, horse, chicken, monkey, chimpanzee, camel, and llama.
• Hybridomas of different species may be, for example, a combination of cells appropriately selected from the origins described in step 1 of the above-mentioned [Method for producing monoclonal IgA antibodies] for the origins of B cells and types of cells other than B cells.
• Examples include hybridomas of different species that are an appropriate combination of origins such as human, mouse, rat, hamster, rabbit, goat, sheep, donkey, pig, cow, horse, chicken, monkey, chimpanzee, etc.
• composition provides a composition comprising an antibody or antigen-binding fragment thereof that binds to the C. difficile bacterial organism of the present disclosure.
• the composition of the present disclosure is preferably provided as, but is not limited to, a pharmaceutical composition, an oral composition, or an enteral composition.
• compositions in one aspect, can be suitably used for the treatment of, but is not limited to, a disease associated with, for example, C. difficile bacteria.
• C. difficile bacteria Diseases associated with C. difficile bacteria are not particularly limited, but examples include inflammatory bowel disease, ulcerative colitis, Crohn's disease, allergies, asthma, obesity, autoimmune diseases, neonatal necrotizing enterocolitis, etc., with inflammatory bowel disease being preferred.
• the pharmaceutical composition disclosed herein can be suitably used to improve the intestinal flora.
• Such pharmaceutical compositions according to the present disclosure may contain an effective amount of an antibody or antigen-binding fragment thereof that binds to the C. difficile bacteria according to the present disclosure, and can be appropriately set, for example, so that the content of the antibody according to the present disclosure in 100% by weight of the pharmaceutical composition is in the range of 0.001 to 99.99% by weight, taking into consideration the type of intestinal disease to be treated, the dosage form, the method of administration, the subject to be treated, the severity of the subject's symptoms, and the degree of effect exerted by administration.
• the term "effective amount” refers to an amount in which the antibody or antigen-binding fragment thereof that binds to C. difficile bacteria according to the present disclosure can exert a therapeutic effect against intestinal diseases, etc.
• the pharmaceutical composition of the present disclosure may contain a pharma- ceutically acceptable carrier or additive together with the antibody that binds to the C. difficile bacteria of the present disclosure.
• the pharma- ceutically acceptable carrier or additive means any carrier, diluent, excipient, suspending agent, lubricant, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, flavoring, or sweetener, and any known carrier or additive may be used.
• the pharmaceutical composition disclosed herein may be used in a method for treating intestinal disease, which includes administering the composition to an individual suffering from an intestinal disease associated with the above-mentioned C. difficile bacteria.
• the pharmaceutical composition may also be used in a method for preventing intestinal disease, which includes administering the composition to an individual who may have a predisposition to intestinal disease, but has not developed the pathology or symptoms of the above-mentioned intestinal disease. These individuals may be subjects for administration of the pharmaceutical composition disclosed herein.
• Individuals to which the drug is administered are not particularly limited, but examples include mammals such as humans, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cows, and pigs, and birds such as chickens.
• the dosage and method of administration of the pharmaceutical composition are adjusted appropriately depending on the type of intestinal disease suffered by the individual to be administered, the sex, species, age, general condition, severity of the disease, the degree of the desired effect, etc.
• the dosage is usually set appropriately within the range of 0.001 to 100 mg/kg/day.
• the administration method is not particularly limited, but it is preferable to administer directly to the digestive tract.
• Examples of such administration methods include oral administration, nasal administration, transmucosal administration, and enteral administration.
• Enteral administration is not limited to administration via the anus, but also includes administration via a tube or the like inserted into the digestive tract from outside the individual, such as a gastrostomy.
• the location into which the digestive tract is inserted is not limited to the intestine, but may include the esophagus, stomach, small intestine (including the duodenum, jejunum, ileum, etc.), large intestine (including the cecum, colon, rectum, etc.), etc.
• the pharmaceutical composition according to the present disclosure may be administered in the above amount once a day, or in several divided doses. Furthermore, the administration interval may be daily, every other day, weekly, biweekly, every 2-3 weeks, monthly, bimonthly, or every 2-3 months, as long as it has a therapeutic effect against the above diseases.
• the oral or enteral composition according to the present disclosure includes an antibody or an antigen-binding fragment thereof that binds to the C. difficile bacteria according to the present disclosure.
• diseases associated with C. difficile bacteria can be treated and/or prevented, and the intestinal flora can be improved.
• the proportion of the antibody that binds to the C. difficile bacteria in such an oral or enteral composition is not particularly limited and may be adjusted as appropriate depending on the form and use of the oral or enteral composition, but is usually about 0.001 to 99% by weight of the total amount of the oral or enteral composition.
• oral or enteral composition of the present disclosure are not particularly limited, but examples include mammals such as humans, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cows, and pigs, and birds such as chickens.
• the antibodies that bind to C. difficile bacteria contained in the oral or enteral composition of the present disclosure are particularly useful as a composition for regulating the intestines, improving the intestinal environment, optimizing the intestinal environment, or preventing intestinal putrefaction.
• the amount of the oral or enteral composition according to the present disclosure used is not particularly limited as long as it is within a range in which the oral or enteral composition exhibits the effects described above, and may be set according to the type of individual taking the oral or enteral composition, the desired effect, the desired degree of the effect, and other conditions. Specifically, converted into the amount of antibodies that bind to the C. difficile bacteria according to the present disclosure, the amount is usually about 0.001 to 100 mg/kg/day, which may be taken once or in divided doses a day.
• enteral administration is not limited to administration via the anus.
• the enteral composition disclosed herein can be mixed with known ingredients and used as an intestinal cleansing liquid.
• the oral or enteral composition according to the present disclosure contains an antibody that binds to the C. difficile bacteria according to the present disclosure, which exerts the effect of suppressing abnormal proliferation of intestinal bacteria and/or pathological changes in the intestinal flora, and can be suitably used in the fields of food and feed, expecting that such an effect will be exerted. Therefore, the oral or enteral composition according to the present disclosure can be a food composition or a feed composition.
• the above-mentioned food composition is a composition that is suitable for exclusively using the oral or enteral composition of the present disclosure in the field of food.
• a food composition can be provided as a food composition labeled for intestinal regulation, for improving the intestinal environment, for optimizing the intestinal environment, for preventing intestinal putrefaction, etc.
• the above-mentioned food compositions include general foods, as well as foods for specified health uses, including conditional foods for specified health uses, nutritional supplements, functional foods, and foods for the sick.
• the specific form of the above-mentioned food composition is not particularly limited, but examples include beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, and milk drinks; cold desserts such as ice cream, ice sherbet, and shaved ice; sweets such as candy, candy, gum, chocolate, tablet candy, snacks, biscuits, jellies, jams, creams, and baked goods; noodles such as soba, udon, harusame, Chinese noodles, and instant noodles; processed seafood and livestock foods such as kamaboko, ham, and sausages; dairy products such as processed milk and fermented milk; oils and fats and oil-and-fat processed foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressings; seasonings such as sauces and sauces; soups, stews, salads, side dishes, furikake, pickles, bread, and cereals.
• beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks,
• the above-mentioned feed composition is a composition in which the feed composition of the present disclosure is used exclusively in the field of feed.
• a feed composition can be provided as a food composition labeled for intestinal regulation, improving the intestinal environment, optimizing the intestinal environment, preventing intestinal putrefaction, etc.
• the specific form of the above-mentioned feed composition is not particularly limited, but for example, as long as the effect of the feed composition according to the present disclosure described above is not impaired, the feed composition may be prepared by mixing it with normal feed or, if necessary, mixing it with ingredients that can be incorporated into normal feed, or the feed composition itself may be used as feed.
• the present disclosure provides a method for preventing or treating an intestinal disease associated with C. difficile bacteria, or a method for maintaining or improving health associated with the improvement and maintenance of intestinal flora, comprising administering to a subject an effective amount of an antibody or antigen-binding fragment thereof that binds to the C. difficile bacterial organism according to the present disclosure, a pharmaceutical composition according to the present disclosure, or an oral composition according to the present disclosure.
• the subject may be a subject in need of prevention or treatment of an intestinal disease associated with C. difficile bacteria, or a subject in need of health maintenance or improvement associated with the improvement and maintenance of intestinal flora.
• Those who require prevention or treatment of intestinal diseases associated with the above-mentioned C. difficile bacteria can be selected from those described in the [pharmaceutical composition] of the present disclosure.
• those who require health maintenance or improvement related to the improvement and maintenance of the intestinal flora are not particularly limited, and include those for whom it is desirable to maintain or improve the intestinal flora.
• the subjects of such administration are not particularly limited, but examples include mammals such as humans, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cows, and pigs, and birds such as chickens.
• the dosage and method of administration of the composition are appropriately adjusted depending on the type of intestinal disease suffered by the individual to be administered, the sex, species, age, general condition, severity of the disease, the degree of the desired effect, etc.
• the dosage is usually set appropriately within the range of 0.001 to 100 mg/kg/day.
• the administration method is not particularly limited, but it is preferable to administer directly to the digestive tract.
• Examples of such administration methods include oral administration, nasal administration, transmucosal administration, and enteral administration.
• Enteral administration is not limited to administration via the anus, but also includes administration via a tube or the like inserted into the digestive tract from outside the individual, such as a gastrostomy.
• the location into which the digestive tract is inserted is not limited to the intestine, but can include the esophagus, stomach, small intestine (including the duodenum, jejunum, ileum, etc.), large intestine (including the cecum, colon, rectum, etc.), etc.
• the pharmaceutical composition may be administered in the above amount once a day, or in several divided doses. Furthermore, as long as it has a therapeutic effect against the above diseases, the administration interval may be daily, every other day, weekly, every other week, every 2-3 weeks, monthly, every other month, or every 2-3 months.
• the pharmaceutical composition may contain an effective amount of an antibody or antigen-binding fragment thereof that binds to the C. difficile bacteria of the present disclosure, and can be appropriately set, for example, so that the content of the antibody of the present disclosure in 100% by weight of the composition is in the range of 0.001 to 99.99% by weight, taking into consideration the type of intestinal disease to be treated, the dosage form, the method of administration, the subject to be treated, the severity of the subject's symptoms, and the degree of effect exerted by administration.
• the pharmaceutical composition may contain a pharma- ceutically acceptable carrier or additive in combination with the antibody or antigen-binding fragment thereof that binds to C. difficile bacteria of the present disclosure.
• a pharma- ceutically acceptable carrier or additive means any carrier, diluent, excipient, suspending agent, lubricant, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, flavoring, or sweetener, and any known carrier or additive may be used.
• the pharmaceutical composition may contain a pharma- ceutically acceptable carrier or additive in combination with the antibody or antigen-binding fragment thereof that binds to C. difficile bacteria of the present disclosure.
• a pharma- ceutically acceptable carrier or additive means any carrier, diluent, excipient, suspending agent, lubricant, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, flavoring, or sweetener, and any known carrier or additive may be used.
• the dosage is not particularly limited as long as it is within the range in which the effect of the composition is exerted, and may be set according to the type of individual taking the oral or enteral composition, the desired effect, the desired degree of the effect, and other conditions. Specifically, converted into the amount of antibodies that bind to the C. difficile bacteria according to the present disclosure, the dosage is usually about 0.001 to 100 mg/kg/day, which may be taken once or in divided doses a day.
• enteral administration is not limited to administration via the anus.
• the enteral composition disclosed herein can be mixed with known ingredients and used as an intestinal cleansing liquid.
• Oral or enteral compositions can be administered as food or feed compositions.
• the food composition can be administered as a food composition labeled for intestinal regulation, improving the intestinal environment, optimizing the intestinal environment, preventing intestinal putrefaction, etc.
• the food composition can be administered as general foods, as well as foods for specified health uses, including conditional foods for specified health uses, dietary supplements, functional foods, foods for the sick, etc.
• the specific form of the food composition is not particularly limited, but examples include beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, and milk drinks; frozen desserts such as ice cream, ice sherbet, and shaved ice; sweets such as candy, candy, gum, chocolate, tablet candy, snacks, biscuits, jellies, jams, creams, and baked goods; noodles such as soba, udon, harusame, Chinese noodles, and instant noodles; processed seafood and livestock foods such as kamaboko, ham, and sausages; dairy products such as processed milk and fermented milk; oils and fats and oil-processed foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressings; seasonings such as sauces and sauces; soups, stews, salads, side dishes, furikake, pickles, bread, and cereals.
• beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, and milk
• the feed composition can be administered as a food composition labeled for intestinal regulation, improving the intestinal environment, optimizing the intestinal environment, preventing intestinal putrefaction, etc.
• the specific form of the feed composition is not particularly limited, but for example, as long as the effect of the feed composition according to the present disclosure described above is not impaired, the feed composition may be prepared by mixing it with normal feed or, if necessary, mixing it with ingredients that can be incorporated into normal feed, or the feed composition itself may be administered as feed.
• Example 1 Obtaining an antibody that binds to C. difficile An antibody that binds to C. difficile was obtained by the following method. Materials and Methods (1-1) Separation of small intestinal and large intestinal mucosal lamina intestinal cells After euthanasia, mice were opened and the entire length of the small intestine was extracted. After removing connective tissue and Peyer's patches from the extracted small intestine, the small intestine was incised longitudinally and the small intestinal contents were washed with PBS. Next, the washed small intestine was cut into approximately 1 cm lengths and added to a 100 ml beaker filled with 50 ml of PBS containing 1 mM EDTA.
• the small intestinal fragments were collected in a strainer and the PBS containing EDTA was discarded.
• the small intestinal fragments were added to a 50 ml tube, 20 ml of PBS was added, and the tube was shaken vigorously for 10 seconds, after which the small intestinal fragments were collected again in a strainer and the PBS was discarded. This procedure was repeated twice to remove only the small intestinal epithelial cells from the small intestinal tissue.
• the digestive solution was centrifuged at 1,500 rpm at room temperature for 5 minutes, and the supernatant was discarded.
• the lamina propria cells obtained as a precipitate were washed once with RPMI 1640 containing 2% FCS, suspended in 2 ml of RPMI 1640 containing 2% FCS, and stored on ice.
• the same procedure was performed for the supernatant from the second digestion step, and the first and second cell suspensions were combined and passed through a filter to remove tissue fragments, yielding small intestinal (large intestinal) lamina intestinal cells.
• Hybridomas were prepared by fusing small intestinal (large intestine) mucosal specific cells or spleen cells with mouse myeloma cells, NS1 cells. Cell fusion was performed according to the ClonaCell-HY Hybridoma Cloning Kit (STEMCELL Technologies) using the kit's reagents and procedures. The obtained hybridomas were grown in a methylcellulose-containing medium according to the ClonaCell-HY Hybridoma Cloning Kit (STEMCELL Technologies), clones were picked up with the naked eye, and each clone was further grown in a 96-well plate.
• frozen cell stocks were prepared from the cloned hybridomas, and the culture supernatant was obtained at the same time.
• the isotype of the antibody contained in the supernatant was confirmed by a conventional sandwich ELISA method, and then the antibody titer of the antibody contained in the supernatant was measured and separated as hybridomas producing antibodies of each isotype.
• the antibodies used in the ELISA were anti-goat-mouse IgA (Southern Biotech), anti-goat-mouse IgG (Southern Biotech), or anti-goat-mouse IgM (Southern Biotech) at 2 ⁇ g/ml for plate coating, and alkaline phosphatase (ALP)-conjugated anti-goat mouse IgA (Southern Biotech) or alkaline phosphatase (ALP)-conjugated anti-goat mouse IgG (Southern Biotech) for detection.
• ALP alkaline phosphatase
• Anti-goat mouse IgM (Southern Biotech) or alkaline phosphatase (ALP)-conjugated anti-goat mouse IgM (Southern Biotech) were used at 0.5 ⁇ g/ml.
• Mouse IgA ⁇ (Immunology Consultants Laboratory), Purified mouse IgG1 ⁇ Isotype Control (BD Pharmingen), and PE-CF594 mouse IgM ⁇ Isotype Control (BD Horizon) were used as control antibodies.
• the measurement was performed using TriStar 2 LB942 (BERTHOLD TECHNOLOGIES) to measure OD 405 nm. By the above operations, a total of 500 or more hybridoma clones were obtained.
• the plate was washed with 0.05% Tween 20-added PBS, and the secondary detection antibody (described above) corresponding to each antibody isotype was added and reacted, and then a color reaction was performed using an Alkali Phosphatase tablet (Sigma).
• the ELISA plate after color development was incubated overnight at 4°C, and OD405nm was measured using TriStar 2 LB942 (BERTHOLD TECHNOLOGIES). Clones showing OD405nm of 2.0 or more were determined to be antibodies that bind to C. difficile bacteria.
• the selected clones were cultured in an expanded volume (about 100 mL), and the antibodies were purified from the culture medium using a Protein L column (Cytiva) for IgM and IgA antibodies, and a Protein A column (Cytiva) for IgG antibodies.
• the antibodies were eluted using 10 mM citrate buffer (pH 2.5), and the eluate was immediately neutralized with 1 M citrate buffer (pH 9.0), then concentrated using Amicon (100 kDa), dialyzed in a dialysis membrane (100 kDa pore size), and replaced with PBS. After dialysis, the antibody solution was collected in a clean bench, sterilized using a syringe filter (0.22 ⁇ m), and stored at 4°C. The antibody concentration was measured by sandwich ELISA as described above. RNA was extracted from the clones selected by ELISA, and the full-length antibody gene was cloned and used to produce recombinant antibodies.
• RNA was extracted from the cells of each clone using Isogen II (Nippon Gene Co., Ltd.). cDNA was synthesized using the extracted RNA as a template, and RT-PCR was performed using seven types of primers (MH1-7) for the VH region of the antibody and primers specific to the C ⁇ region, C ⁇ region, or C ⁇ region. PCR was performed with V ⁇ primers and C ⁇ primers for the light chain. The base sequences of the primers are shown in Table 1. The amplified VH region PCR product or V ⁇ region PCR product was directly sequenced to obtain the variable region gene sequence of each clone.
• Nucleotide sequences of primers for amplifying the variable region of the antibody gene of hybridoma-derived antibodies In the nucleotide sequences in the table, S represents G or C, R represents A or G, N represents A, C, G or T, W represents A or T, V represents G, C or A, and M represents A or C.
• S represents G or C
• R represents A or G
• N represents A, C
• G or T W
• W represents A or T
• V G, C or A
• M represents A or C.
• the culture supernatant was collected 10-12 days after transfection, and the antibody was purified and concentrated using a Protein L column in the same manner as above, replaced by PBS by dialysis, then filter sterilized, and used for antibody titer measurement and activity test.
• Example 2 Bacterial binding test Among the antibodies obtained in Example 1, the binding ability to C. difficile was tested for two antibodies, SNK0004 and SNK0005. The test method used was the same as that in (1-3) "Analysis of binding ability of hybridoma IgA antibodies to C. difficile" in Example 1. As can be seen from the results shown in Figure 1, all of the antibodies demonstrated binding to C. difficile bacteria.
• Example 3 Bacterial growth inhibition test The bacterial growth inhibition effects of SNK0004 antibody, SNK0005 antibody, IgG1 type antibody of W27 antibody (rW27IgG), and negative control IgA (Control rIgA) were tested.
• C. difficile bacteria were cultured overnight at 37°C under anaerobically.
• the culture solution was centrifuged, the bacteria were collected, and then washed twice with culture solution.
• the bacterial solution was diluted 10-fold with culture solution, and Counting beads and Thiazol orange contained in Cell Viability Kit (Becton Dickinson) were added, and the number of live bacteria positive for SYTO24 was calculated by comparison with the Counting beads by flow cytometry.
• the bacteria were diluted with culture solution to 10,000 cells/5 ⁇ l.
• Example 4 Binding properties to human intestinal bacteria It has been reported that dysbiosis of the intestinal flora is associated with the onset of many diseases, including inflammatory bowel disease (IBD), and improving the intestinal flora is considered important for maintaining health. It has been found that the state of the intestinal flora of patients with inflammatory bowel disease (IBD) is different from that of healthy individuals, and that bacteria associated with the exacerbation of IBD (IBD-associated bacteria) are increased (PCT/JP2023/018019).
• IBD inflammatory bowel disease
• Fluorescence activated cell sorter (FACS) buffer for antibody staining was prepared by adding 10% fetal bovine serum (FBS, Nichirei) and EDTA (final concentration 5 ⁇ M, Nacalai Tesque) to PBS and sterilizing through a 0.22 ⁇ m filter. FACS buffer was added to the bacterial solution, centrifuged at 8,000 g for 5 minutes at 4 ° C, and the supernatant was removed. 15 ⁇ g of each biotinylated antibody was added to 6 ⁇ 10 6 bacteria and reacted on ice for 20 minutes. FACS buffer was added to the antibody reaction solution, centrifuged at 8,000 g for 5 minutes at 4 ° C, and the supernatant was removed.
• FACS buffer was added to the antibody reaction solution, centrifuged at 8,000 g for 5 minutes at 4 ° C, and the supernatant was removed.
• the SNK0004 and SNK0005 antibodies bind to bacteria present in dysbiotic intestinal flora and have the effect of improving the intestinal flora.
• the present disclosure has extremely high industrial value by providing an antibody that specifically binds to Clostridioides difficile (C. difficile), an antigen-binding fragment thereof, and uses thereof.

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