Antibodies binding to clostridium difficile

Abstract

The inventors of the present disclosure have undergone in-depth studies and found that an antibody that binds to and inhibits the proliferation of intestinal bacteria, particularly clostridium difficile cells and other intestinal bacteria cells. The present disclosure exerts the effect of providing antibodies and antigen-binding fragments thereof that bind to enteral bacteria, in particular, clostridium difficile and other enteral bacteria, and uses thereof.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to antibodies and antigen-binding fragments thereof having an effect of binding to, inhibiting proliferation of, and the like, Clostridioides difficile (C. difficile), and applications of the antibodies. BACKGROUND

[0002] The intestines are equivalent to "outside the body" for an organism, and thus are in a state of being always exposed to antigens derived from a wide variety of and large number of intestinal resident bacteria, viruses, food, and the like. Such intestinal resident bacteria form an intestinal flora.

[0003] It is known that the intestinal flora exerts various functions on an organism through a mucosal surface constituted by intestinal epithelial cells and the like, and it is also known that the intestinal immune system cannot develop normally in the absence of intestinal bacteria (Non-Patent Literatures 1 to 3).

[0004] If the intestinal flora changes and the symbiotic relationship with the host is disrupted, the intestinal immune system is excessively stimulated and its homeostasis is disrupted, and further, various diseases such as inflammatory bowel disease, colon cancer, asthma, allergy, obesity, and the like are induced. As a result, it is known that the intestinal immune system not only plays an important role in the elimination of pathogens and the like, but also plays an important role in the maintenance of the homeostasis of the entire immune system (Non-Patent Literatures 4 to 7).

[0005] Many attempts have been made in the past to control the intestinal flora using antibiotics, but there are still cases where the effects are no longer exerted due to antibiotic-resistant bacteria. Therefore, despite the attempts in the past, many patients are still suffering from C. difficile enteritis or other inflammatory bowel diseases associated with the intestinal flora. In addition, although attempts have been made as a relatively novel method such as fecal transplantation, the therapeutic effects are not stable. As a result, in some attempts, the development thereof as a therapeutic method has been stopped. In addition, research has been conducted in the past to utilize an antibody against a toxin released by the intestinal flora as a therapeutic drug and commercialization has been achieved. However, this antibody drug is a drug against only the toxin, and thus the therapy using this antibody drug is a symptomatic therapy. Therefore, it is desirable to develop a drug for fundamentally treating inflammatory bowel diseases associated with the intestinal flora.

[0006] Clostridium difficile associated with inflammatory bowel disease is a bacterium that causes nosocomial infection as well as MRSA, and is a bacterium that requires attention. In fact, according to statistics in the United States, about 40 to 50 million people are infected and about 15,000 to 20,000 people die every year. Clostridium difficile infection occurs in most cases in patients who have been administered antibiotics for a long time, and is a disease with a high recurrence probability of 15 to 35% once the disease occurs. From this aspect, among the inflammatory bowel disease-associated bacteria, a particularly effective treatment is required. At present, treatment using strong antibiotics such as vancomycin is performed, but bacterial toxins remain and the production of bacteria having antibiotic resistance is promoted, and thus alternative methods are being sought.

[0007] Prior art documents

[0008] Non-patent literature

[0009] Non-patent literature 1: Cerf-Bensussan N, and Gaboriau-Routhiau V., Nat Rev Immunol 2010, 10:735

[0010] Non-patent literature 2: Hooper LV, and Macpherson AJ., Nat Rev Immunol 2010, 10:159

[0011] Non-patent literature 3: Round JL, and Mazmanian SK, Proc Natl Acad Sci USA 2010, 107:12204

[0012] Non-patent literature 4: Vijay-Kumar M, et al., Science 2010, 328:228

[0013] Non-patent literature 5: Shulzhenko N, et al., Nat Med 2011, 17:1585

[0014] Non-patent literature 6: Bry L, et al., Science 1996, 273:1380

[0015] Non-patent literature 7: Turnbaugh PJ, et al., Nature 2006, 444:1027 SUMMARY

[0016] Problems to be solved by the invention

[0017] The present disclosure provides an antibody and antigen-binding fragment thereof that binds to Clostridium difficile (C. difficile) bacterial cells and inhibits proliferation thereof, and applications thereof.

[0018] Means for solving the problem

[0019] The present inventors conducted intensive studies and as a result, found an antibody that binds to bacterial cells of Clostridium difficile (C. difficile) and other intestinal bacteria and inhibits proliferation thereof. It was also clarified that the antibody further binds to bacteria present in dysbiosis.

[0020] In one embodiment, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to Clostridium difficile (C. difficile) bacterial cells and inhibits proliferation thereof.

[0021] In one embodiment, the present disclosure provides an immortalized cell that produces an antibody that binds to Clostridium difficile (C. difficile) bacterial cells and inhibits proliferation thereof.

[0022] In one embodiment, the present disclosure provides a composition comprising an antibody or antigen-binding fragment thereof that binds to Clostridium difficile (C. difficile) bacterial cells and inhibits proliferation thereof.

[0023] In one embodiment, the present disclosure provides a method for treating a disease using an antibody or antigen-binding fragment thereof that binds to Clostridium difficile (C. difficile) bacterial cells and inhibits proliferation thereof.

[0024] In one embodiment, the present disclosure provides the following regimen.

[0025] [Item 1]

[0026] An antibody or antigen-binding fragment thereof that binds to Clostridium difficile (C. difficile) bacterial cells and inhibits proliferation thereof, the antibody comprising:

[0027] the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the heavy chain variable region represented by SEQ ID NO: 3, and the light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain variable region represented by SEQ ID NO: 4; or,

[0028] the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the heavy chain variable region represented by SEQ ID NO: 13, and the light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain variable region represented by SEQ ID NO: 14.

[0029] [Item 2]

[0030] The antibody or antigen-binding fragment thereof as described in Project 1, wherein the antibody comprises:

[0031] Heavy chain CDR1 represented by sequence number 5, heavy chain CDR2 represented by sequence number 6, and heavy chain CDR3 represented by sequence number 7; and,

[0032] Light chain CDR1 represented by sequence number 8, light chain CDR2 represented by sequence number 9, and light chain CDR3 represented by sequence number 10.

[0033] [Project 3]

[0034] The antibody or antigen-binding fragment thereof as described in Project 1, wherein the antibody comprises:

[0035] Heavy chain CDR1 represented by sequence number 15, heavy chain CDR2 represented by sequence number 16, and heavy chain CDR3 represented by sequence number 17; and,

[0036] Light chain CDR1 represented by sequence number 18, light chain CDR2 represented by sequence number 19, and light chain CDR3 represented by sequence number 20.

[0037] [Project 4]

[0038] A nucleic acid that encodes the antibody or antigen-binding fragment described in item 1.

[0039] [Project 5]

[0040] An expression vector comprising the nucleic acid described in Item 4.

[0041] [Project 6]

[0042] A cell containing the nucleic acids described in item 4.

[0043] [Project 7]

[0044] A composition comprising the antibody or antigen-binding fragment thereof described in item 1.

[0045] [Project 8]

[0046] The composition described in Project 7 is in a freeze-dried state.

[0047] The effects of the invention

[0048] This disclosure provides an antibody and its antigen-binding fragment that bind to intestinal bacteria, particularly Clostridium difficile and other intestinal bacteria, and their application. Attached Figure Description

[0049] Figure 1is a graph showing results obtained by testing the binding ability of SNK0004 antibody and SNK0005 antibody to C. difficile.

[0050] Figure 2 is a graph showing results obtained by testing the C. difficile proliferation inhibitory ability of SNK0004 antibody and SNK0005 antibody, and IgG1 type antibody of W27 antibody (rW27 IgG) and negative control IgA antibody (control rIgA).

[0051] Figure 3 is a graph showing results obtained by evaluating the binding property of each bacterial species of bacteria from IBD patient feces bound to SNK0004 antibody by quantitative data based on IgA Index.

[0052] Figure 4 is a graph showing results obtained by evaluating the binding property of each bacterial species of bacteria from IBD patient feces bound to SNK0005 antibody by quantitative data based on IgA Index.

[0053] Figure 5 is a graph showing results obtained by testing the proportion of bacteria from IBD patient feces bound to SNK0004, SNK0005 antibody and previously produced RS_H000_L001, SNK0001, SNK0002, SNK0003 antibody (WO2023 / 277142) against them. DETAILED DESCRIPTION

[0054] (Definitions)

[0055] In the present specification, in the case where two or more numerical ranges are shown, a range constituted by any combination of lower limit values and upper limit values of two or more of these ranges also has the same meaning.

[0056] In the present specification, "antibody" is used in the broadest sense to include monoclonal antibodies, polyclonal antibodies, and antibody fragments that show the intended antigen-binding activity, but is not limited to these. A full-length antibody mainly includes a heavy chain and a light chain composed of polypeptides. The heavy chain and the light chain each include a site called a variable region that recognizes an antigen, which is generally called a heavy chain variable region and a light chain variable region, respectively. The variable region has, in order from the amino terminal, sites called CDR1 to 3, which are determined to be sites that further specifically recognize an antigen. Note that these CDR1 to 3 are also called heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, light chain CDR3, and the like in more detail. In addition, the regions other than CDR1 to 3 of the heavy chain and the light chain are called heavy chain FR1 to 4 and light chain FR1 to 4, respectively, in order from the amino terminal.

[0057] The antibody can be in a form of an antibody including one heavy chain and one light chain (also called a single-chain antibody) in addition to a form including two heavy chains and two light chains. In addition, the antibody can be in a form of a dimer or a multimer having a J (joining) chain and, if necessary, an SC (secretory component) in addition to two heavy chains and two light chains.

[0058] The antibody can be of any type (for example, IgG, IgE, IgM, IgD, IgA, or IgY, and the like) or subtype (for example, IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2, and the like).

[0059] The antibody can have an amino acid sequence derived from the same species or an amino acid derived from different species. In the case of being derived from the same species, for example, a human-derived, a mouse-derived, a rat-derived, a hamster-derived, a rabbit-derived, a goat-derived, a donkey-derived, a pig-derived, a bovine-derived, a horse-derived, a chicken-derived, a monkey-derived, a chimpanzee-derived, a camel-derived, a llama-derived, and the like can be given.

[0060] In the case of being derived from different species, there is no particular limitation, and for example, an antibody derived from any two or more of a human, a mouse, a rat, a hamster, a rabbit, a goat, a sheep, a donkey, a pig, a bovine, a horse, a chicken, a monkey, a chimpanzee, a camel, a llama, and the like can be given.

[0061] In the present specification, the "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 is known that the ability of an antibody to specifically bind to an antigen can also be maintained by a fragment containing a portion thereof. As one mode, the "antigen-binding fragment" of an antibody can be a Fab fragment containing a light chain variable region (VL), a heavy chain variable region (VH), a light chain constant region (CL), and a CH1 region which is a part of a heavy chain constant region; a F(ab')2 fragment containing two Fab fragments linked by a disulfide bond at the hinge region; an Fd fragment containing a VH and a CH1 region; an Fv fragment containing a VL and a VH region of a single arm of an antibody; a dAb fragment containing a single variable region; and an isolated complementarity determining region (CDR), but is not limited thereto.

[0062] The antibody of the present disclosure can also be a CDR-grafted antibody. In one example, in a CDR-grafted antibody, part or all of the sequences of the CDR regions of an antibody derived from one animal species are replaced with CDR sequences of other animal species. For example, the CDRs of one or more mouse antibodies are replaced with CDR sequences of a human antibody.

[0063] When the heavy chain CDR1 to 3 in the heavy chain variable region and the light chain CDR1 to 3 in the light chain variable region of an antibody are identified, methods known to those skilled in the art can be used. For example, the "Kabat definition" (Kabat et al., Ann. NY Acad, Sci. 1971, Vol. 190, pp. 382-391 and Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th Ed., 1991, U.S. Department of Health and Human Services, NIH Publication, pp. 91-3242), the "Chothia definition" (Chothia et al., Nature, 1989, Vol. 342, pp. 877-883), the "contact definition" (MacCallum et al., J. Mol. Biol., 1996, Vol. 262, pp. 732-745), and the like known to those skilled in the art can be used. In order to identify the CDR sequences within an antibody, the CDRs can also be identified based on the information of public databases (for example, https: / / www.ncbi.nlm.nih.gov / igblast).

[0064] In the present specification, "identity" refers to the extent to which two or more comparable amino acid sequences or base sequences are identical to each other with respect to the amino acid sequences or base sequences. Thus, the higher the identity of two amino acid sequences or base sequences, the higher the identity or similarity of their sequences. The level of identity of amino acid sequences or base sequences is usually determined using the sequence analysis tool FASTA using the default parameters. Alternatively, it can be determined using the algorithm BLAST proposed 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.). Programs called BLASTN, BLASTX were developed based on such BLAST algorithm (e.g., Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. J Mol Biol. 215:403-10 (1990), etc.). The specific methods of these analysis methods are well known, and can be referred to the website of NCBI. For example, that a certain amino acid sequence A is a certain percentage identical to another amino acid sequence B means that the amino acid sequence A has that percentage of identity to the amino acid sequence B.

[0065] In the present specification, "monoclonal" is a modifier that characterizes an antibody or the like obtained from a substantially homogeneous antibody population. Each antibody contained in such a homogeneous antibody population is identical except for possible naturally occurring mutations that can be present in minor amounts.

[0066] In the present specification, "conservative substitution technique" refers to a technique of substituting an amino acid residue with an amino acid residue having a similar side chain.

[0067] For example, substitution of amino acid residues having basic side chains such as lysine, arginine, histidine for each other belongs to the conservative substitution technique. In addition, substitution of amino acid residues having acidic side chains such as aspartic acid, glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; amino acid residues having β-branched side chains such as threonine, valine, isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, histidine for each other also belong to the conservative substitution technique.

[0068] Antibody or antigen-binding fragment thereof binding to Clostridium difficile cell

[0069] The antibody of the present disclosure binding to Clostridium difficile cell binds to Clostridium difficile. Typically, the antibody of the present disclosure binds to Clostridium difficile that is not destroyed present in culture supernatant.

[0070] The antibody of the present disclosure binding to Clostridium difficile cell can use the antibody produced by B cell-derived antibody-producing cells such as hybridoma, and can use the nucleic acid encoding the antibody introduced into cells other than the immune system using genetic recombination technology and the produced antibody as a recombinant antibody.

[0071] The antibody of the present disclosure binding to Clostridium difficile cell is preferably an IgA antibody.

[0072] In one mode, the antibody of the present disclosure binding to Clostridium difficile cell comprises:

[0073] a heavy chain variable region comprising an amino acid sequence of a heavy chain CDR1 of a heavy chain variable region having an amino acid sequence represented by SEQ ID NO: 3, an amino acid sequence of a heavy chain CDR2, and an amino acid sequence of a heavy chain CDR3; and,

[0074] a light chain variable region comprising an amino acid sequence of a light chain CDR1 of a light chain variable region having an amino acid sequence represented by SEQ ID NO: 4, an amino acid sequence of a light chain CDR2, and an amino acid sequence of a light chain CDR3.

[0075]

[0076] In one mode, the antibody of the present disclosure binding to Clostridium difficile cell comprises:

[0077] a heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO: 6, and a heavy chain CDR3 having an amino acid sequence represented by SEQ ID NO: 7; and,

[0078] a light chain variable region comprising a light chain CDR1 having an amino acid sequence represented by SEQ ID NO: 8, a light chain CDR2 having an amino acid sequence represented by SEQ ID NO: 9, and a light chain CDR3 having an amino acid sequence represented by SEQ ID NO: 10.

[0079]

[0080] In one embodiment, the antibody of the present disclosure that binds to a Clostridium difficile cell comprises:

[0081] a heavy chain variable region having the amino acid sequence set forth in 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,

[0082] a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2 or a sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0083] In one embodiment, the antibody of the present disclosure that binds to a Clostridium difficile cell comprises:

[0084] a heavy chain variable region having the amino acid sequence set forth in 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,

[0085] a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 4 or a sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto,

[0086] and comprises:

[0087] a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 5, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 6, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 7; and,

[0088] a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 8, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 9, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 10.

[0089] In one embodiment, the IgA antibody of the present disclosure is SNK0004 which is obtained by determining the nucleic acid sequence encoding the IgA antibody produced by a hybridoma derived from a mouse-derived B cell, and producing the IgA antibody using a recombinant technique.

[0090] The antibody SNK0004 has the following amino acid sequence.

[0091]

[0092] In one embodiment, the antibody SNK0004 is encoded by the following base sequence.

[0093]

[0094]

[0095] In one embodiment, the disclosed antibody binding to *C. difficile* cells comprises:

[0096] The heavy chain variable region includes the amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, which are the heavy chain variable regions having the amino acid sequences represented by sequence number 13; and,

[0097] The light chain variable region comprises the amino acid sequences of light chain CDR1, light chain CDR2, and light chain CDR3, which have the amino acid sequences represented by sequence number 14.

[0098]

[0099] In one embodiment, the disclosed antibody binding to *C. difficile* cells comprises:

[0100] The heavy chain variable region comprises heavy chain CDR1 having the amino acid sequence represented by sequence number 15, heavy chain CDR2 having the amino acid sequence represented by sequence number 16, and heavy chain CDR3 having the amino acid sequence represented by sequence number 17; and,

[0101] The light chain variable region comprises light chain CDR1 having the amino acid sequence represented by sequence number 18, light chain CDR2 having the amino acid sequence represented by sequence number 19, and light chain CDR3 having the amino acid sequence represented by sequence number 20.

[0102]

[0103] In one embodiment, the disclosed antibody binding to *C. difficile* cells comprises:

[0104] The heavy chain variable region having the amino acid sequence represented by sequence number 13 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it; and,

[0105] The light chain variable region has the amino acid sequence represented by sequence number 14 or a sequence that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0106] In one embodiment, the disclosed antibody binding to *C. difficile* cells comprises:

[0107] The heavy chain variable region having the amino acid sequence represented by sequence number 13 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it; and,

[0108] The light chain variable region has the amino acid sequence represented by sequence number 14 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0109] And includes:

[0110] The heavy chain variable region comprises heavy chain CDR1 having the amino acid sequence represented by sequence number 15, heavy chain CDR2 having the amino acid sequence represented by sequence number 16, and heavy chain CDR3 having the amino acid sequence represented by sequence number 17; and,

[0111] The light chain variable region comprises light chain CDR1 having the amino acid sequence represented by sequence number 18, light chain CDR2 having the amino acid sequence represented by sequence number 19, and light chain CDR3 having the amino acid sequence represented by sequence number 20.

[0112] In one embodiment, the IgA antibody disclosed herein is SNK0005, obtained by determining the nucleic acid sequence encoding an IgA antibody produced by a hybridoma derived from mouse small intestinal B cells and preparing the IgA antibody using recombinant technology.

[0113] The antibody SNK0005 has the following amino acid sequence.

[0114]

[0115] In one embodiment, antibody SNK0005 is encoded by the following base sequence.

[0116]

[0117]

[0118] In one embodiment, the antibody disclosed herein may be provided in the form of an antibody comprising one heavy chain and one light chain (also referred to as a single-chain antibody), in addition to comprising two heavy chains and two light chains. Furthermore, the antibody disclosed herein may also be provided in the form of a dimer or polymer having a J (linking) chain and, if necessary, an SC (secretory component) in addition to two heavy chains and two light chains.

[0119] In one embodiment, the antibodies disclosed herein may be provided of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY) or subtype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2).

[0120] In one embodiment, the antibodies disclosed herein may have amino acid sequences derived from the same species or from different species. Examples of amino acid sequences derived from the same species include those from humans, mice, rats, hamsters, rabbits, goats, donkeys, pigs, cattle, horses, chickens, monkeys, chimpanzees, camels, and llamas.

[0121] There are no particular restrictions on the origin of antibodies from different species. For example, antibodies can be derived from any two or more of the following: human, mouse, rat, hamster, rabbit, goat, sheep, donkey, pig, cow, horse, chicken, monkey, chimpanzee, camel, and llama.

[0122] (Method for manufacturing antibodies that bind to Clostridium difficile cells)

[0123] The antibody that binds to Clostridium difficile cells disclosed herein can be manufactured by performing the following steps: (1) preparing antibody-producing immortalized cells from B cells that produce the antibody, taken from the lamina propria of the intestinal mucosa; (2) culturing the antibody-producing immortalized cells prepared in step 1 above to identify cells that produce antibodies that bind to Clostridium difficile cells; and (3) recovering the antibody from the cells that produce antibodies that bind to Clostridium difficile cells.

[0124] In one approach, to configure the heavy chain variable region and / or the light chain variable region to be appropriately combined as needed, the process may include, for example, a step of treatment with a protease or the like; a step of introducing functional groups capable of forming chemical bonds such as disulfide bonds; and a subsequent step of forming the aforementioned chemical bonds through the aforementioned functional groups; and so on.

[0125] In the case of creating a hybridoma by fusing B cells with cells of other types, there is no particular limitation on the type of cells other than B cells (sometimes referred to as "other cells" in this specification), as long as they fuse with the B cells to form a hybridoma and the hybridoma does not lose the antibody-producing function of the B cells.

[0126] As other such cells, those that fuse with the aforementioned B cells to form a hybridoma and that are capable of immortalization are preferred. Specifically, examples include cancer cells, especially myeloma cells derived from bone marrow. Preferred other cells include myeloma cells, such as mouse NS1 cells.

[0127] Other cell sources are not specifically limited, and examples include humans, mice, rats, hamsters, rabbits, goats, sheep, donkeys, pigs, cattle, horses, chickens, monkeys, chimpanzees, camels, llamas, etc.

[0128] Fusion refers to the merging of the aforementioned B cells with other cells in an inseparable manner. Here, "inseparable" does not include the phenomenon of cell division during proliferation of the fused cells. Thus, the fused cell can be cited as an example of a hybridoma in this specification.

[0129] There are no particular limitations on the conditions for mixing and fusion; any conditions commonly used in methods for cell fusion can be appropriately adopted. For example, the conditions used when culturing B cells or other cells can be appropriately modified. Examples of such methods include: mixing B cells with other cell types in a suitable culture medium and bringing them into contact in the presence of polyethylene glycol or the like; applying electrical stimulation after the above mixing; and culturing the mixture at 37°C and 5% carbon dioxide after using a virus such as Sendai virus.

[0130] There is no particular time limit for fusion; simply set an appropriate time until fusion is complete. As for confirming that fusion is complete, any known methods used for normal cell fusion can be modified as needed. For example, observing the progress of fusion under a microscope can be used, or a known reagent kit can be selected and the cells fused according to its operating conditions.

[0131] In one embodiment, the specific recovery method in the step of recovering antibodies from cells that produce antibodies binding to *C. difficile* cells is not particularly limited. Examples include methods for recovering the culture supernatant of the antibody-producing cells and methods for recovering the cell lysates. Regarding the cell lysates, known mechanical means such as ultrasonic disruption or cell disruptors, and / or known chemical treatments based on the use of surfactants, cell wall digestive enzymes, or cell membrane digestive enzymes, can be appropriately combined to lyse the cells. The liquid phase component is then recovered after a solid-liquid separation step to produce the disclosed antibody binding to *C. difficile* cells.

[0132] (Methods for manufacturing recombinant antibodies)

[0133] In one embodiment, the antibody of the present disclosure that binds to Clostridium difficile cells is manufactured by introducing a nucleic acid encoding the antibody into a cell capable of producing monoclonal antibodies, culturing the cell, recovering the monoclonal antibody from the cell extract or culture supernatant, and purifying it as needed.

[0134] To confirm that the antibody binds to Clostridium difficile cells, methods known to those skilled in the art can be used, such as identifying the antibody molecules or the cells that produce the antibody using methods based on ELISA, EIA, RIA, FLISA, FIA, etc., or FACS-based methods.

[0135] The cells that can produce monoclonal antibodies are not particularly limited as long as they are cells commonly derived from mammals or other organisms that produce various proteins that function by forming higher-order structures. For example, appropriately selected cells include mammalian-derived cells such as COS cells, HEK cells (HEK293, HEK293T, etc.), HELA cells, CHO cells, and insect-derived cells such as Sf9. In another approach, plant cells such as yeast cells, filamentous fungal cells, soybean cells, and Arabidopsis thaliana cells can also be used to produce monoclonal antibodies.

[0136] The specific methods for nucleic acid introduction, the culture conditions of cells containing the nucleic acid, the recovery method, and the purification method vary depending on the type of cells used and are not particularly limited. The antibodies that bind to Clostridium difficile cells disclosed herein can be manufactured by appropriately modifying and combining known methods.

[0137] In one approach, after monoclonal antibodies are generated within these cells, the monoclonal antibodies secreted from these cells into the culture medium can be dried or freeze-dried together with the culture medium, or the monoclonal antibodies accumulated in these cells can be pulverized, dried, or freeze-dried together with these cells, thereby producing monoclonal antibodies in the form of an antibody-containing composition suitable for enteral uptake (Virdi et al., Nat. Biotechnol., 2019, Vol.37, pp.527-530). In one approach, the cells that produce the antibody-containing composition suitable for enteral uptake are preferably plant cells such as yeast cells, filamentous fungal cells, soybean, or Arabidopsis thaliana, and the monoclonal antibodies produced are preferably in the form of a VHH fragment fused with IgAFc.

[0138] In the case of the disclosed antibody binding to Clostridium difficile cells being a chimeric antibody, regarding the aforementioned nucleic acid, nucleic acids having base sequences encoding heavy chain variable regions and light chain variable regions, as well as base sequences encoding heavy chain constant regions and light chain constant regions derived from different species, are prepared. The prepared nucleic acids having base sequences encoding heavy chain variable regions and heavy chain constant regions are then bound to the antibody, and the prepared nucleic acids having base sequences encoding light chain variable regions and light chain constant regions are also bound to the antibody. These bound nucleic acids are then introduced into antibody-producing cells as described above.

[0139] Furthermore, in the case of the disclosed antibody binding to *C. difficile* cells, where the heavy chain and / or light chain CDR1-3 have amino acid sequences derived from mice, and the remaining regions have amino acid sequences derived from humans (sometimes referred to as humanized antibodies), the manufacturing method is the same as for the chimeric antibodies described above: nucleic acids encoding the heavy chain and / or light chain CDR1-3, and the remaining regions, are prepared. The nucleic acids are then recombined in a manner that constitutes the heavy and light chains, and introduced into cells capable of producing monoclonal IgA antibodies as described above. To maintain the antibody's binding ability, it is sometimes necessary to replace some bases with other bases.

[0140] (Nucleic acid encoding an antibody or its antigen-binding fragment that binds to Clostridium difficile cells)

[0141] In one embodiment, this disclosure provides a nucleic acid encoding an antibody or antigen-binding fragment thereof that binds to Clostridium difficile cells. The nucleic acid encodes the heavy and / or light chains of the antibody. The morphology of the nucleic acid is not particularly limited and can be either ribonucleotides or deoxyribonucleotides. Furthermore, the morphology of the nucleic acid is not particularly limited and can be either single-stranded or double-stranded. The codons used in the nucleic acid sequence are not particularly limited, and various codons can be appropriately selected according to the purpose. For example, they can be appropriately selected based on the type of host cell used in manufacturing and considering codon frequencies. In one embodiment, the nucleic acid encoding the antibody or antigen-binding fragment thereof of this disclosure is used to express or manufacture the antibody or antigen-binding fragment thereof of this disclosure.

[0142] In cases where the nucleic acid encoding the antigen-binding fragment of the disclosed antibody or its antigen-binding fragment encodes the antigen-binding fragment, for example, each nucleic acid molecule can be used to encode the two domains VL and VH of the Fv fragment, or a single protein chain (single-chain Fv (scFv)) can be obtained by using recombination technology to encode the VL and VH regions with a single nucleic acid to form a monovalent molecule.

[0143] In one embodiment, the nucleic acid encoding the antibody or its antigen-binding fragment disclosed herein may be in the form of a vector. For example, the vector may be an expression vector, or alternatively, a vector for use in gene recombination. The composition of the vector is not particularly limited and may include various constituent elements used in known vectors.

[0144] (Cells containing nucleic acids encoding antibodies or antigen-binding fragments that bind to Clostridium difficile cells)

[0145] In one embodiment, this disclosure provides a cell comprising nucleic acid encoding an antibody or antigen-binding fragment thereof that binds to Clostridium difficile cells. The cell may contain one or more heavy and / or light chains of nucleic acid encoding the antibody or antigen-binding fragment thereof of this disclosure. In one embodiment, the cell of this disclosure produces an antibody or antigen-binding fragment thereof that binds to Clostridium difficile cells. In one embodiment, the cell of this disclosure replicates nucleic acid encoding an antibody or antigen-binding fragment thereof that binds to Clostridium difficile cells. The type of cell disclosed is not particularly limited; cells used for protein production, nucleic acid amplification, gene recombination, etc., may be used. In one embodiment, the cell of this disclosure may be the cell illustrated in the above-described method for manufacturing antibodies that bind to Clostridium difficile cells, or the cell illustrated in the following description (antibody-producing immortalized cells).

[0146] (Antibody-producing immortalized cells)

[0147] In one embodiment, this disclosure provides immortalized cells that generate the antibodies described above that bind to Clostridium difficile cells.

[0148] Specifically, antibody-producing immortalized cells are cells created by mixing and fusing B cells that produce the antibody with other types of cells, i.e., cells other than B cells, or by creating immortalized B cells through infection with Epstein-Barr virus (EBV).

[0149] Hybridomas can be from the same species or from different species. Regarding hybridomas from the same species, in the above-described [method for manufacturing monoclonal IgA antibodies], the B cells and other cell types should be of the same species of origin; regarding hybridomas from different species, the B cells and other cell types should be of different species of origin.

[0150] Hybridomas of the same species origin, such as those described in step 1 of the above-mentioned [method for manufacturing monoclonal IgA antibodies], can be appropriately selected from overlapping sources including B cells and cell types other than B cells. Examples of hybridomas of the same species origin include those from humans, mice, rats, hamsters, rabbits, goats, donkeys, pigs, cattle, horses, chickens, monkeys, chimpanzees, camels, and llamas.

[0151] Hybridomas from different species can be created by appropriately selecting and combining sources from those described in step 1 of the above-mentioned [method for manufacturing monoclonal IgA antibodies], including B cells and cell types other than B cells. Examples of hybridomas from different species include those created by appropriately combining sources such as humans, mice, rats, hamsters, rabbits, goats, sheep, donkeys, pigs, cattle, horses, chickens, monkeys, and chimpanzees.

[0152] (Composition)

[0153] In one embodiment, this disclosure provides a composition comprising the antibody or antigen-binding fragment thereof that binds to Clostridium difficile cells as described above. The compositions of this disclosure are preferably provided as pharmaceutical compositions, oral compositions, or enteric compositions, but are not limited thereto.

[0154] (Pharmaceutical Composition)

[0155] In one embodiment, the pharmaceutical compositions disclosed herein are not particularly limited, for example, they may be suitable for the treatment of diseases associated with Clostridium difficile.

[0156] Diseases associated with Clostridium difficile are not specifically limited, but can include inflammatory bowel disease, ulcerative colitis, Crohn's disease, allergies, asthma, obesity, autoimmune diseases, and necrotizing enterocolitis of the newborn, with inflammatory bowel disease being the preferred option.

[0157] In one embodiment, the pharmaceutical compositions disclosed herein are suitable for improving the intestinal flora.

[0158] Such a pharmaceutical composition of the present disclosure only needs to contain an effective amount of the antibody of the present disclosure that binds to Clostridium difficile cells or its antigen-binding fragment. For example, it can be appropriately set according to the proportion of the antibody of the present disclosure in 100% by weight in the pharmaceutical composition, in the range of 0.001 to 99.99% by weight, taking into account the type of intestinal disease of the target, dosage form, method of administration, target of administration, severity of symptoms of the target of administration, and degree of effect exerted by administration.

[0159] As used in this specification, the term "effective amount" refers to the amount of antibody or antigen-binding fragment thereof that binds to Clostridium difficile cells to exert a therapeutic effect on intestinal diseases, etc.

[0160] Pharmaceutically acceptable carriers or additives may be co-formulated with the disclosed antibody that binds to Clostridium difficile cells in the pharmaceutical composition thereof. Pharmaceutically acceptable carriers or additives refer to any known carrier, diluent, excipient, suspension, lubricant, adjuvant, medium, delivery system, emulsifier, tablet disintegrant, absorbent, preservative, surfactant, colorant, flavoring, or sweetener.

[0161] The pharmaceutical composition disclosed herein is available for use in the treatment of enteric diseases, including the administration of medication to individuals suffering from the aforementioned enteric diseases associated with Clostridium difficile. It is also available for use in the prevention of enteric diseases, including the administration of medication to individuals who, while not exhibiting the aforementioned symptoms or condition of enteric disease, may have predisposing factors for enteric disease. These individuals can be used as recipients of the pharmaceutical composition disclosed herein.

[0162] There are no particular limitations on the individuals that can be used as subjects for such drug administration. Examples include humans, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cattle, pigs, and other mammals, as well as birds such as chickens.

[0163] The dosage and method of administration of this pharmaceutical composition shall be appropriately adjusted according to the type of intestinal disease suffered by the individual, sex, species, age, general condition, severity of disease, and desired effect. The dosage is generally set appropriately within the range of 0.001 to 100 mg / kg / day.

[0164] In addition, there are no particular restrictions on the method of administration, but direct administration through the digestive tract is preferred. Examples of such methods include oral administration, nasal administration, mucosal administration, and intestinal administration.

[0165] It should be noted that enteral administration is not limited to administration via the anus, but also includes administration via methods such as gastrostomy, where tubes or other devices are inserted into the digestive tract from outside the individual. The location of insertion into the digestive tract is not limited to the intestine, but can include the esophagus, stomach, small intestine (including the duodenum, jejunum, ileum, etc.), and large intestine (including the cecum, colon, rectum, etc.).

[0166] It should be noted that the pharmaceutical composition disclosed herein can be administered in the above-mentioned amount once a day or in several divided doses. Furthermore, within the range of therapeutic effects on the above-mentioned diseases, the dosing interval can be daily, every other day, weekly, every other week, every 2-3 weeks, monthly, every other month, or every 2-3 months.

[0167] (Oral or intestinal combination)

[0168] The oral or enteral compositions of this disclosure comprise the disclosed antibody or antigen-binding fragment thereof that binds to Clostridium difficile cells. By using such oral or enteral compositions, it is possible to treat and / or prevent diseases associated with Clostridium difficile and to improve the intestinal flora.

[0169] There is no particular limitation on the mixing ratio of the antibodies that bind to Clostridium difficile in such oral or intestinal compositions. The ratio can be adjusted appropriately according to the morphology and purpose of the oral or intestinal composition. Generally, it is about 0.001 to 99% by weight relative to the total amount of the oral or intestinal composition.

[0170] There are no particular limitations on the individuals to whom the oral or intestinal compositions of this disclosure are intended to be used, but examples include humans, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cattle, pigs and other mammals, and birds such as chickens.

[0171] The antibodies that bind to Clostridium difficile bacteria contained in the oral or intestinal compositions disclosed herein are particularly useful as intestinal regulation compositions, intestinal environment improvement compositions, intestinal environment optimization compositions, or intestinal putrefaction prevention compositions.

[0172] The dosage of the oral or enteral composition disclosed herein is not particularly limited as long as it can exert the effects of the oral or enteral composition. It can be further set according to the type of individual ingesting the oral or enteral composition, the target effect, the degree of the target effect, and other various conditions. Specifically, the amount of antibody that binds to Clostridium difficile cells disclosed herein is usually about 0.001 to 100 mg / kg / day, which can be taken once or several times a day.

[0173] It should be noted that enteric administration is not limited to administration via the anus. For example, the enteric composition of this disclosure can also be mixed with known ingredients for use as an intestinal cleansing solution.

[0174] The oral or enteral compositions of this disclosure contain the disclosed antibody that binds to *C. difficile* cells, which exerts an effect of inhibiting abnormal proliferation of intestinal bacteria and / or causing intestinal flora disorders, and can be suitably used in the food or feed industries where such effects are desired. Therefore, the oral or enteral compositions of this disclosure can be formulated into food compositions or feed compositions.

[0175] The above-described food compositions are compositions specifically adapted for the food industry from the oral or enteral compositions of this disclosure. Such food compositions can be provided in the form of food compositions labeled as for intestinal regulation, intestinal environment improvement, intestinal environment optimization, intestinal putrefaction prevention, etc.

[0176] In addition to ordinary foods, other examples of the aforementioned food compositions include foods for specific health purposes that include conditionally specified health foods, nutritional supplements, functional foods, and foods for patients.

[0177] The specific form of the aforementioned food compositions is not particularly limited. Examples include: beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, and milk drinks; ice cream, shaved ice, and other frozen treats; snacks such as maltose, candy, chewing gum, chocolate, compressed candy, snacks, biscuits, jelly, jam, cream, and baked goods; noodles such as buckwheat noodles, udon noodles, vermicelli, Chinese noodles, and instant noodles; processed aquatic and livestock products such as fish cakes, ham, and sausages; dairy products such as processed milk and fermented milk; oils and fats such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and salad dressing, as well as oil-based processed foods; seasonings such as sauces and condiments; soups, stews, salads, home-style dishes, rice seasonings, pickles, bread, and cereal products. Additionally, in the case of specific health foods, nutritional supplements, and functional foods, forms such as powders, granules, capsules, lozenges, tablets, and syrups can be cited.

[0178] The above-described feed composition is a composition specifically designed for use in the feed industry. Such feed compositions can be provided in the form of food compositions labeled as for intestinal regulation, intestinal environment improvement, intestinal environment optimization, intestinal putrefaction prevention, etc.

[0179] The specific form of the above-mentioned feed composition is not particularly limited. For example, as long as it does not impair the effect of the feed composition disclosed herein, it can be mixed in ordinary feed or mixed with ingredients that can be mixed in ordinary feed as needed to form a feed composition. Alternatively, the feed composition itself can be used as feed.

[0180] (Methods for the prevention or treatment of disease, or for the maintenance or improvement of health)

[0181] In one embodiment, this disclosure provides a method for the prevention or treatment of a disease, or a method for maintaining health, which is a method for the prevention or treatment of intestinal diseases associated with Clostridium difficile, or a method for maintaining or improving health related to the improvement of intestinal flora. This includes administering an effective amount of the aforementioned antibody or antigen-binding fragment of the present disclosure that binds to Clostridium difficile cells, the pharmaceutical composition of the present disclosure, or the oral composition of the present disclosure to a subject. The subject may be a subject requiring prevention or treatment of intestinal diseases associated with Clostridium difficile, or a subject requiring health maintenance or improvement related to the improvement of intestinal flora.

[0182] For those who require prevention or treatment of intestinal diseases related to Clostridium difficile, the recipients described in the [Pharmaceutical Composition] of this disclosure may be selected. Furthermore, there is no particular limitation on the recipients who require health maintenance or improvement related to the improvement of intestinal flora, including those who wish to maintain or improve their intestinal flora.

[0183] There are no particular limitations on the individuals that can be used as subjects for such drug administration. Examples include humans, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cattle, pigs, and other mammals, as well as birds such as chickens.

[0184] For specific administration methods and dosages, the administration methods and dosages described in the above-disclosed [Pharmaceutical Compositions] or [Oral or Enteral Compositions] can be used.

[0185] For example, when administered in the form of a pharmaceutical composition, the dosage and method of administration are appropriately adjusted according to the type of intestinal disease suffered by the individual, their sex, species, age, general condition, severity of the disease, and the desired effect. The dosage is typically set appropriately within the range of 0.001 to 100 mg / kg / day.

[0186] There are no particular restrictions on the method of administration, but direct administration to the digestive tract is preferred. Examples of such methods include oral administration, nasal administration, administration through the mucosa, and administration through the intestines.

[0187] Intestinal drug administration is not limited to administration via the anus, but also includes administration via inserting a tube or similar device into the digestive tract from outside the individual, such as through a gastrostomy fistula. The location of insertion into the digestive tract is not limited to the intestine, but can include the esophagus, stomach, small intestine (including the duodenum, jejunum, ileum, etc.), and large intestine (including the cecum, colon, rectum, etc.).

[0188] The pharmaceutical composition can be administered in the above-mentioned amount once a day or in several divided doses. Furthermore, within the range of therapeutic effects on the above-mentioned diseases, the dosing interval can be daily, every other day, weekly, every other week, every 2-3 weeks, monthly, every other month, or every 2-3 months.

[0189] The pharmaceutical composition may contain an effective amount of the antibody or antigen-binding fragment thereof that binds to Clostridium difficile cells as disclosed herein. For example, the composition may be appropriately set in a manner that the content of the antibody disclosed herein in 100% by weight ranges from 0.001% to 99.99% by weight, taking into account the type of intestinal disease of the target, dosage form, method of administration, target patient, severity of symptoms of the target patient, and degree of effect exerted by administration.

[0190] Pharmaceutically acceptable carriers or additives may be co-formulated with the antibodies or antigen-binding fragments thereof that bind to Clostridium difficile cells disclosed herein in a pharmaceutical composition. Pharmaceutically acceptable carriers or additives refer to any known carrier, diluent, excipient, suspension, lubricant, adjuvant, medium, delivery system, emulsifier, tablet disintegrant, absorbent, preservative, surfactant, colorant, flavoring, or sweetener.

[0191] Pharmaceutically acceptable carriers or additives may be co-formulated with the antibodies or antigen-binding fragments thereof that bind to Clostridium difficile cells disclosed herein in a pharmaceutical composition. Pharmaceutically acceptable carriers or additives refer to any known carrier, diluent, excipient, suspension, lubricant, adjuvant, medium, delivery system, emulsifier, tablet disintegrant, absorbent, preservative, surfactant, colorant, flavoring, or sweetener.

[0192] When the composition is administered orally or enterally, the dosage is not particularly limited as long as it is within the range that allows the composition to exert its effect. It can be further set according to the type of individual ingesting the oral or enteral composition, the target effect, the degree of the target effect, and other various conditions. Specifically, the amount of antibody that binds to Clostridium difficile cells, as disclosed in this literature, is typically around 0.001 to 100 mg / kg / day, and can be administered once or several times a day.

[0193] It should be noted that enteric administration is not limited to administration via the anus. For example, the enteric composition of this disclosure can also be mixed with known ingredients for use as an intestinal cleansing solution.

[0194] Oral or enteral compositions can be administered in the form of food compositions or feed compositions.

[0195] Food compositions can be administered in the form of food compositions labeled as for intestinal regulation, intestinal environment improvement, intestinal environment optimization, or intestinal putrefaction prevention.

[0196] In addition to ordinary food, food compositions can also be administered in the form of specific health foods containing conditions for use, nutritional supplements, functional foods, patient foods, etc.

[0197] The specific form of the food composition is not particularly limited. Examples include: beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, and milk drinks; ice cream, shaved ice, and other frozen treats; snacks such as maltose, candy, chewing gum, chocolate, compressed candy, snacks, biscuits, jelly, jam, cream, and baked goods; noodles such as buckwheat noodles, udon noodles, vermicelli, Chinese noodles, and instant noodles; processed aquatic and livestock products such as fish cakes, ham, and sausages; dairy products such as processed milk and fermented milk; oils and fats such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and salad dressing, as well as oil-based processed products; seasonings such as sauces and condiments; soups, stews, salads, home-style dishes, rice seasonings, pickles, bread, and cereal products. Additionally, in the case of specific health foods, nutritional supplements, and functional foods, administration may be in the form of powders, granules, capsules, lozenges, tablets, or syrups.

[0198] Feed compositions can be administered in the form of food compositions labeled as for intestinal regulation, intestinal environment improvement, intestinal environment optimization, or intestinal putrefaction prevention.

[0199] The specific form of the feed composition is not particularly limited. For example, as long as it does not impair the effect of the feed composition disclosed above, it can be mixed in ordinary feed or mixed with ingredients that can be mixed in ordinary feed as needed to prepare the feed composition. The feed composition itself can also be administered as feed.

[0200] Example

[0201] The present disclosure is further illustrated in detail below by way of examples, but these are merely illustrative and do not limit the present disclosure.

[0202] Example 1 : Acquisition of antibodies binding to Clostridium difficile (C. difficile)

[0203] Antibodies that bind to Clostridium difficile were obtained using the following method.

[0204] (Materials and methods)

[0205] (1-1) Isolation of lamina propria cells of small and large intestinal mucosa

[0206] After euthanasia of mice, the entire small intestine was removed via laparotomy. Connective tissue and Peyer's lymph nodes were removed from the removed small intestine. The small intestine was then longitudinally cut, and its contents were washed with PBS. Next, the washed small intestine was cut into approximately 1 cm lengths and added to a 100 ml beaker containing 50 ml of PBS containing 1 mM EDTA. After shaking at 37°C for 20 minutes, the small intestine fragments were collected through a sieve, and the PBS containing EDTA was discarded. The small intestine fragments were then added to a 50 ml tube, followed by 20 ml of PBS. After vigorous shaking for 10 seconds, the small intestine fragments were again collected through a sieve, and the PBS was discarded. This procedure was repeated twice to remove only the small intestinal epithelial cells from the small intestinal tissue. Next, add 50 ml of warmed (37°C) digestive enzyme solution (adjusted using 100 ml RPMI 1640, 5 ml FCS (final concentration 5%), 50 μl 2-mercaptoethanol (final concentration 55 μM), 0.15 g collagenase, and 1 ml dispersant (50 U / ml) (final concentration 0.5 U / ml)) to a 100 ml beaker. Further finely chop the small intestinal tissue and add it to the solution. Shake at 37°C for 30 minutes. Afterward, allow the 100 ml beaker to stand to allow the small intestinal tissue to precipitate. Transfer the supernatant to a new 50 ml tube. Repeat the digestion step using this enzyme solution for 20 minutes once. Centrifuge the digest at 1,500 rpm for 5 minutes at room temperature and discard the supernatant. Wash the resulting lamina propria cells once with RPMI 1640 containing 2% FCS, then resuspend them in 2 ml of RPMI 1640 containing 2% FCS and store on ice. The same procedure was performed on the supernatant after the second digestion step. The cell suspensions from the first and second digestion steps were combined, and the tissue slices were removed through a filter to prepare small intestinal (large intestine) lamina propria cells.

[0207] (1-2) Preparation and cloning of antibody-generating hybridomas

[0208] Hybridomas were created by fusing intrinsic cells of the small (large) intestinal mucosa or spleen cells with NS1 cells (mouse myeloma cells). Cell fusion was performed according to the reagents and procedures of the ClonaCell-HY Hybridoma Cloning Kit (STEMCELL Technologies). The resulting hybridomas were proliferated in methylcellulose medium according to the ClonaCell-HY Hybridoma Cloning Kit (STEMCELL Technologies). Clones were visually selected and further proliferated in 96-well plates. Frozen cell stock was then prepared from the cloned hybridomas, and the culture supernatant was simultaneously obtained. Antibodies contained in the supernatant were confirmed using a standard sandwich ELISA method to identify antibody isotypes. The antibody titers in the supernatant were then measured, and hybridomas were generated from each isotype antibody for isolation. Regarding the antibodies used in the ELISA, the plate coating consisted of 2 μg / ml of anti-goat mouse IgA (Southern Biotech), anti-goat mouse IgG (Southern Biotech), or anti-goat mouse IgM (Southern Biotech). For assays, 0.5 μg / ml of alkaline phosphatase (ALP) conjugated to anti-goat mouse IgA (Southern Biotech), alkaline phosphatase (ALP) conjugated to anti-goat mouse IgG (Southern Biotech), or alkaline phosphatase (ALP) conjugated to anti-goat mouse IgM (Southern Biotech) was used. As control antibodies, mouse IgAκ (Immunology Consultants Laboratory), purified mouse IgG1κ isotype control (BD Pharmingen), and PE-CF594 mouse IgMκ isotype control (BD Horizon) were used. The assay was performed using TriStar. 2 LB942(BERTHOLD TECHNOLOGIES), to OD 405 nm was measured. Through the above operations, a total of more than 500 hybridoma clones were obtained.

[0209] (1-3) Analysis of the binding affinity of hybridoma IgA antibody to Clostridium difficile.

[0210] Clostridium difficile was anaerobically cultured at 37°C (brain and heart infusion medium, 80% N2, 10% H2, 10% CO2) and collected by centrifugation. The bacteria were resuspended in 0.05M Na2CO3 buffer and coated onto ELISA plates. After blocking with PBS supplemented with 1% BSA, the supernatant of each antibody culture from the 96-well plates was added, and the reaction was carried out at room temperature for approximately 1 hour. The plates were then washed with PBS supplemented with 0.05% Tween 20, and the corresponding secondary detection antibodies (as described above) were added for reaction. Colorimetric reaction was then performed using alkaline phosphatase tablets (Sigma). To ensure sufficient reaction, the ELISA plates were incubated overnight at 4°C using TriStar. 2 LB942 (BERTHOLD TECHNOLOGIES) determination of OD 405 nm. OD 405 Clones with a nm value greater than 2.0 were identified as antibodies binding to Clostridium difficile.

[0211] Selected clones were cultured in approximately 100 mL of medium. 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). Antibody elution was performed using 10 mM citrate buffer (pH 2.5), and the eluent was immediately neutralized with 1 M citrate buffer (pH 9.0). The eluent was then concentrated using Amicon (100 kDa) and dialyzed through a dialysis membrane (100 kDa pore size) to replace the PBS. After dialysis, the antibody solution was recovered in a clean bench and sterilized using a syringe filter (0.22 μm) and stored at 4°C. Antibody concentration was determined using a sandwich ELISA method, as described above. RNA was extracted from clones selected by ELISA, and the full-length antibody gene was cloned for the production of recombinant antibodies.

[0212] (1-4) Cloning of the full-length antibody gene from antibodies derived from hybridomas

[0213] RNA was extracted from cells of each clone using IsogenII (Nippon Gene Co., Ltd.). cDNA was synthesized using the extracted RNA as a template, and a specific antibody-targeted V-type RNA was used. H RT-PCR was performed using seven primers (MH1-7) for the Cα, Cμ, or Cγ regions, as well as Cα-, Cμ-, or Cγ-specific primers. For the light chain, PCR was performed using Vκ and Cκ primers. The primer sequences are shown in Table 1. The amplified Vκ... HThe PCR products of the Vκ region or Vκ region were directly sequenced to obtain the variable region gene sequences of each clone. Ig blast was then used to search for specific Vκ regions. H Alternatively, the upstream sequence of the Vκ gene (including the signal sequence) can be used. PCR primers are prepared based on this upstream sequence, and PCR is performed together with the reverse primers at the 3' end of each C-region secretory sequence to obtain the full-length gene base sequences of the H and L chains. Based on this, the H, L, and J chains (based on base sequences retrieved from databases, and the full-length gene sequences obtained by PCR using hybridoma cDNA) are cloned into the pcDNA 3.1(+) vector (Invitrogen).

[0214]

[0215] Table 1. Base sequences of primers used for amplifying the variable region of antibody genes derived from hybridomas.

[0216] In the base 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.

[0217] The above expression vector was transfected into Expi CHOS cells (Thermo Fischer Scientific) using the kit's operating procedures at a ratio of H:L:J = 2:1:1 (Thermo Fischer Scientific, ExpiCHO expression system). On days 10-12 post-transfection, the culture supernatant was collected, and the antibody was purified and concentrated using a protein L column, as described above. After dialysis to replace the supernatant with PBS, the filter was sterilized for use in antibody titer and activity assays.

[0218] Example 2: Bacterial binding assay

[0219] The binding affinity of the two antibodies, SNK0004 and SNK0005, obtained in Example 1, to Clostridium difficile was tested. The test method used was the same as that in Example 1 (1-3) "Analysis of the binding affinity of hybridoma IgA antibody to Clostridium difficile".

[0220] according to Figure 1 The results shown are understandable; both antibodies exhibited binding to Clostridium difficile.

[0221] Example 3: Bacterial proliferation inhibition assay

[0222] The study investigated the inhibitory effects of SNK0004 antibody, SNK0005 antibody, and IgG1 antibody of W27 antibody (rW27IgG) and negative control IgA (control rIgA) on bacterial proliferation.

[0223] (Materials and methods)

[0224] Clostridium difficile was anaerobically cultured overnight at 37°C under the same conditions as in Example 1. The culture was centrifuged, and the bacteria were collected and washed twice with the culture medium. The bacterial culture was diluted 10-fold with the culture medium, and counting microspheres and thiazole orange from a Becton Dickinson cell viability kit were added. The number of SYTO24-positive viable bacteria was calculated by flow cytometry by comparing the count with the counting microspheres. The culture medium was diluted to a concentration of 10,000 cells / 5 μl. 5 μl of bacterial dilution was added to a microcentrifuge tube, followed by 25 μl of anaerobic PBS or purified test antibody (anaerobic) (antibody concentration 1 mg / ml), and incubated at 37°C for 1 hour. Then, 30 μl of culture medium was added, and the culture was incubated anaerobically at 37°C for 6 hours (final antibody concentration 0.42 mg / ml).

[0225] Six hours later, the reaction solution was continuously diluted, inoculated onto BHI agar plates, and anaerobically cultured at 37 degrees Celsius to determine the viable count.

[0226] (result)

[0227] The results are shown in Figure 2 Compared with the IgG1 antibody (rW27IgG) of W27 antibody and the negative control IgA antibody (control rIgA), both the tested SNK0004 and SNK0005 antibodies showed significant inhibitory ability against the proliferation of Clostridium difficile.

[0228] Example 4: Binding properties to human intestinal bacteria

[0229] It has been reported that gut microbiota abnormalities (dysbiosis) are associated with the pathogenesis of various diseases, including inflammatory bowel disease (IBD), and improving gut microbiota is considered important for maintaining health. The gut microbiota of patients with inflammatory bowel disease (IBD) differs from that of healthy individuals, and an increase in bacteria associated with the worsening of IBD (IBD-associated bacteria) has been found (PCT / JP2023 / 018019).

[0230] For bacteria from the feces of IBD patients that bind to SNK0004 and SNK0005 antibodies, the binding characteristics of each bacterial species were evaluated using quantitative data based on the IgA Index (Andrew L. Kau, et al. Sci Transl Med 7, 2015; Hirosuke Sugahara, et al., Frontiers in Microbiology 8, 1757, 2017).

[0231] (Materials and methods)

[0232] • Analysis of the ratio of IgA antibody-conjugating bacteria to bacteria in human fecal samples

[0233] As a human fecal sample, stool from one patient (P1) was used. The procedure was performed in an anaerobic chamber. After determining the bacterial count, PBS was added to prepare a bacterial culture of 6 × 10⁻⁶ bacteria from the human fecal sample. 7 Cells / ml. Then centrifuged at 8,000g for 5 minutes at 4°C to remove the supernatant. Add 100 μl of PBS containing 20% ​​normal rat serum (Wako) to the supernatant-free bacterial cells and react on ice for 30 minutes. This inhibits non-specific antibody binding. For the Fluorescence Activated Cell Sorter (FACS) buffer used for antibody staining, add 10% fetal bovine serum (FBS, Nichirei) and EDTA (final concentration 5 μM, Nacalai Tesque) to PBS and sterilize by passing through a 0.22 μm filter. Add FACS buffer to the bacterial culture and centrifuge at 8,000g for 5 minutes at 4°C to remove the supernatant. (Relative to 6 × 10⁻⁶) 6 Add 15 μg of each biotinylated antibody to each bacterial cell and react on ice for 20 minutes. Add FACS buffer to the antibody reaction solution, centrifuge at 8,000 g for 5 minutes at 4°C, and remove the supernatant. Add 20 μl of PE / cyanin 7-streptavidin (final concentration 10 μg / ml, BioLegend) to the bacterial cells and react on ice in the dark for 20 minutes. Add FACS buffer to the reaction solution, centrifuge at 8,000 g for 5 minutes at 4°C, and remove the supernatant. Add FACS buffer and thiazolyl orange (final concentration 42 nM, BD) to the bacteria and react on ice for 10 minutes. The proportion of antibody-bound bacteria was then analyzed using a SONY SH800 cell sorter (SONY).

[0234] result:

[0235] It can be seen that Enterobacteriaceae showed specific binding to SNK0004 and SNK0005, while other bacteria also bound to these antibodies with different binding characteristics.Figure 3 and Figure 4 ).

[0236] For SNK0004, SNK0005 antibodies and previously prepared RS_H000_L001, SNK0001, SNK0002, and SNK0003 antibodies (WO2023 / 277142), the proportion of bacteria from IBD patient feces (feces of one patient (P1)) that bound to them was compared. Figure 5 As shown, these antibodies all bind to Enterobacteriaceae, suggesting they have an effect on improving intestinal flora.

[0237] Based on the above results, it can be understood that SNK0004 and SNK0005 antibodies bind to bacteria present in dysbiosis-prone gut microbiota and have an effect on improving gut microbiota.

[0238] Industrial applicability

[0239] This disclosure has high industrial value by providing antibodies that specifically bind to Clostridium difficile (C. difficile) and their antigen-binding fragments, as well as their applications.

[0240] Sequence information

[0241]

[0242]

[0243]

[0244]

[0245] .

Claims

1. An antibody or its antigen-binding fragment thereof, which binds to Clostridium difficile cells and inhibits the proliferation of Clostridium difficile. The antibody comprises: The heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region represented by sequence number 3, and the light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region represented by sequence number 4; or, The heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 of the heavy chain variable region represented by sequence number 13, and the light chain CDR1, light chain CDR2 and light chain CDR3 of the light chain variable region represented by sequence number 14.

2. The antibody or its antigen-binding fragment as described in claim 1, wherein, The antibody comprises: Heavy chain CDR1 represented by sequence number 5, heavy chain CDR2 represented by sequence number 6, and heavy chain CDR3 represented by sequence number 7; and, Light chain CDR1 represented by sequence number 8, light chain CDR2 represented by sequence number 9, and light chain CDR3 represented by sequence number 10.

3. The antibody or its antigen-binding fragment as described in claim 1, wherein, The antibody comprises: Heavy chain CDR1 represented by sequence number 15, heavy chain CDR2 represented by sequence number 16, and heavy chain CDR3 represented by sequence number 17; and, Light chain CDR1 represented by sequence number 18, light chain CDR2 represented by sequence number 19, and light chain CDR3 represented by sequence number 20.

4. A nucleic acid encoding the antibody or antigen-binding fragment thereof as described in claim 1.

5. An expression vector comprising the nucleic acid of claim 4.

6. A cell comprising the nucleic acid of claim 4.

7. A composition comprising the antibody of claim 1 or an antigen-binding fragment thereof.

8. The composition of claim 7, wherein it is in a freeze-dried state.

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