Antibody binding to clostridium difficile

Abstract

To provide an antibody binding to a Clostridium difficile bacterial body, or an antigen-binding fragment thereof.SOLUTION: In one aspect, the present invention provides an antibody binding to a Clostridium difficile bacterial body, or an antigen-binding fragment thereof, and including a heavy chain variable region comprising an amino acid sequence of a heavy chain CDR1, an amino acid sequence of a heavy chain CDR2, and an amino acid sequence of a heavy chain CDR3, the heavy chain variable region comprising a specific amino acid sequence, and a light chain variable region comprising an amino acid sequence of a light chain CDR1, an amino acid sequence of a light chain CDR2, and an amino acid sequence of a light chain CDR3, the light chain variable region comprising a specific amino acid sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to antibodies and antigen-binding fragments thereof that bind to Clostridium difficile (C. difficile), and uses thereof. [Background technology]

[0002] The intestinal tract is, so to speak, "outside the body" of the living body, and is therefore constantly exposed to a large number of different types of intestinal resident bacteria, viruses, food-derived antigens, etc. These intestinal resident bacteria form the intestinal flora.

[0003] It has become clear that this intestinal flora performs various functions for the body via the mucosal surface composed of intestinal epithelial cells, etc., and it is also known that without the presence of intestinal bacteria, the intestinal immune system cannot develop normally (Non-Patent Documents 1 to 3).

[0004] When the intestinal flora changes and the symbiotic relationship with the host is broken, the intestinal immune system is overstimulated and its homeostasis is disrupted, which ultimately induces many diseases such as inflammatory bowel disease, colon cancer, asthma, allergies, obesity, etc. For these reasons, it is known that the intestinal immune system not only eliminates pathogens, but also plays an important role in maintaining the homeostasis of the entire immune system (Non-Patent Documents 4 to 7).

[0005] Although many attempts have been made to control the intestinal flora using antibiotics, the problem of antibiotic resistance has been cited as a problem of ineffectiveness. Therefore, despite the previous attempts, there are still many patients suffering from difficile colitis and other inflammatory bowel diseases associated with the intestinal flora. In addition, fecal transplants have been attempted as a relatively new approach, but their therapeutic effects are not stable. As a result, development as a treatment has already been discontinued in some attempts. In addition, the use of antibodies against toxins released by the intestinal flora as a therapeutic agent has been researched and commercialized. However, since the antibody drug is only against the toxins, the treatment using this is only a symptomatic treatment. Therefore, there has been a need for the development of a drug to fundamentally treat inflammatory bowel diseases associated with the intestinal flora.

[0006] C. difficile, which is related to inflammatory bowel disease, is a bacterium that, along with MRSA, requires attention as a bacterium that can cause hospital-acquired infections. In fact, according to statistics in the United States, about 400,000 to 500,000 people are infected with it every year, and about 15,000 to 20,000 people die from it. C. difficile infection often occurs 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 need for an effective treatment, especially among the bacteria related to inflammatory bowel disease. Currently, treatment is carried out with strong antibiotics such as vancomycin, but alternative methods are required because the toxins of the bacteria remain and promote the emergence of antibiotic-resistant bacteria. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Cerf-Bensussan N, and Gaboriau-Routhiau V., Nat Rev Immunol 2010;10:735 [Non-Patent Document 2] Hooper LV, and Macpherson AJ., Nat Rev Immunol 2010;10:159 [Non-Patent Document 3] Round JL,and Mazmanian SK,Proc Natl Acad Sci USA 2010;107:12204 [Non-Patent Document 4] Vijay-Kumar M,et al.,Science 2010;328:228 [Non-Patent Document 5] Shulzhenko N, et al., Nat Med 2011;17:1585 [Non-Patent Document 6] Bry L,et al.,Science 1996;273:1380 [Non-Patent Document 7] Turnbaugh PJ,et al.,Nature 2006;444:1027 Summary of the Invention [Problem to be solved by the invention]

[0008] An objective of the present invention is to provide an antibody that binds to intestinal bacteria including C. difficile bacteria, with the aim of regulating the intestinal bacterial flora. [Means for solving the problem]

[0009] As a result of extensive research, the present inventors have discovered antibodies that bind to enterobacteria, particularly C. difficile bacteria and other enterobacteria.

[0010] In one aspect, the present invention comprises: Binds to Clostridium difficile bacteria 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:7; and The present invention provides an antibody or antigen-binding fragment thereof, comprising 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, the light chain variable region comprising the amino acid sequence represented by SEQ ID NO:8.

[0011] In one aspect, the present invention comprises: Binds to Clostridium difficile bacteria 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: 17; and The present invention provides an antibody or antigen-binding fragment thereof, comprising 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, the light chain variable region comprising the amino acid sequence represented by SEQ ID NO:18.

[0012] In one aspect, the present invention comprises: Binds to Clostridium difficile bacteria The present invention provides an antibody or antigen-binding fragment thereof, comprising 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, the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:27, and 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, the light chain variable region comprising the amino acid sequence represented by SEQ ID NO:28.

[0013] In one aspect, the present invention comprises: Binds to Clostridium difficile bacteria A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:1; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:2, and Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:3 and a heavy chain variable region comprising A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:4; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:5, and Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:6 The present invention provides an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising:

[0014] In one aspect, the present invention comprises: Binds to Clostridium difficile bacteria A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 11; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 12, and Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:13 and a heavy chain variable region comprising A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 14; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 15, and Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:16 The present invention provides an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising:

[0015] In one aspect, the present invention comprises: Binds to Clostridium difficile bacteria A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22, and Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:23 and a heavy chain variable region comprising A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 24; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 25, and Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:26 The present invention provides an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising:

[0016] In one aspect, the present invention comprises: A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 37, and For a reference antibody comprising a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 38, having at least one amino acid mutation in at least one region selected from heavy chain CDR1 to 3, light chain CDR1 to 3, and light chain FR1; The present invention provides an antibody or antigen-binding fragment thereof that binds to the amino acid sequence RQEEHIELIAS in the E. coli SHMT protein and the amino acid sequence VLDMMKLEKPE in the iPGM protein of C. difficile.

[0017] In one aspect, the present invention comprises: Binds to Clostridium difficile bacteria a heavy chain CDR1 comprising the amino acid sequence of X1YYIH; a heavy chain CDR2 comprising the amino acid sequence RIDPENX2X3TTYAPKFQ; a heavy chain CDR3 comprising the amino acid sequence of YCARSTVL; and a heavy chain variable region comprising a light chain CDR1 comprising the amino acid sequence RX4SQSIVHTNG; a light chain CDR2 comprising the amino acid sequence of KLLIYKV; a light chain CDR3 comprising the amino acid sequence GVYYFQGS; and a light chain variable region comprising Light chain FR1 is The amino acid sequence of TPLSLPVSLGDQA or Contains the amino acid sequence of SPASX5SVSLGDRX6 X1, X2, and X3 are each independently a neutral polar amino acid or an acidic polar amino acid, X4 is a nonpolar amino acid or a neutral polar amino acid, and X5 and X6 are each independently a nonpolar amino acid; The antibodies or antigen-binding fragments thereof are provided except for those wherein X1 is aspartic acid, X2 is aspartic acid, X3 is glutamic acid, X4 is alanine, and the light chain FR1 comprises the sequence TPLSLPVSLGDQA.

[0018] In one aspect, the present invention provides a nucleic acid encoding an antibody or antigen-binding fragment thereof that binds to Clostridium difficile bacteria and comprises a combination of the heavy chain variable region and the light chain variable region described above.

[0019] In one aspect, the present invention provides an expression vector comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof that binds to Clostridium difficile bacteria and comprises a combination of the heavy chain variable region and the light chain variable region.

[0020] In one aspect, the present invention provides a nucleic acid encoding an antibody or antigen-binding fragment thereof that binds to Clostridium difficile bacteria and comprises a combination of the heavy chain variable region and the light chain variable region described above, or a cell comprising an expression vector containing the nucleic acid.

[0021] In one aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to Clostridium difficile bacteria and comprises a combination of the heavy chain variable region and the light chain variable region, and a pharma- ceutically acceptable carrier or excipient.

[0022] In one aspect, the present invention provides a method for inhibiting the growth of Clostridium difficile bacteria associated with inflammatory bowel disease in an individual, the method comprising administering to the individual an effective amount of the pharmaceutical composition.

[0023] In one aspect, the present invention comprises: 1) mixing and fusing B cells collected from the intestinal mucosa lamina propria or the spleen with other types of cells to produce hybridomas; 2) culturing the hybridomas and determining which cells produce antibodies that bind to Clostridium difficile bacteria; and 3) recovering antibodies from cells that produce antibodies that bind to the Clostridium difficile bacteria. The present invention provides a method for producing an antibody that binds to Clostridium difficile bacteria, comprising:

[0024] More specifically, the present invention provides the following: [Item 1] Binds to Clostridium difficile bacteria a) The amino acid sequence of heavy chain CDR1 of the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:7; a heavy chain variable region comprising the amino acid sequence of a heavy chain CDR2 and the amino acid sequence of a heavy chain CDR3; And The amino acid sequence of light chain CDR1 of the light chain variable region comprising the amino acid sequence represented by SEQ ID NO:8; an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of a light chain CDR2 and the amino acid sequence of a light chain CDR3; b) 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: 17; and 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:18 an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising: c) 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:27; and an antibody or antigen-binding fragment thereof comprising 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:28; or d) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 37, and has at least one amino acid mutation in at least one region selected from heavy chain CDR1 to 3, light chain CDR1 to 3, and light chain FR1, compared to a reference antibody comprising a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 38; An antibody or an antigen-binding fragment thereof that binds to the amino acid sequence RQEEHIELIAS in the Escherichia coli SHMT protein and the amino acid sequence VLDMMKLEKPE in the iPGM protein of C. difficile. [Item 2] A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:1; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:2, and Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:3 and a heavy chain variable region comprising A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:4; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:5, and Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:6 a light chain variable region comprising: A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 11; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 12, and Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:13 and a heavy chain variable region comprising A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 14; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 15, and Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:16 a light chain variable region comprising: A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22, and Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:23 and a heavy chain variable region comprising A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 24; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 25, and Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:26 A light chain variable region comprising 2. The antibody or antigen-binding fragment thereof according to item 1, [Item 3] A heavy chain variable region comprising a sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO:7; and 2. The antibody or antigen-binding fragment thereof according to item 1, comprising a light chain variable region comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO:8. [Item 4] A heavy chain variable region comprising a sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 17; and A light chain variable region comprising a sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO:18. The antibody or antigen-binding fragment thereof according to item 1, [Item 5] A heavy chain variable region comprising a sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO:27; and A light chain variable region comprising a sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO:28. The antibody or antigen-binding fragment thereof according to item 1, [Item 6] a heavy chain CDR1 comprising the amino acid sequence of X1YYIH; a heavy chain CDR2 comprising the amino acid sequence RIDPENX2X3TTYAPKFQ; a heavy chain CDR3 comprising the amino acid sequence of YCARSTVL; and a heavy chain variable region comprising a light chain CDR1 comprising the amino acid sequence RX4SQSIVHTNG; a light chain CDR2 comprising the amino acid sequence of KLLIYKV; a light chain CDR3 comprising the amino acid sequence GVYYFQGS; and a light chain variable region comprising Light chain FR1 is The amino acid sequence of TPLSLPVSLGDQA or comprising the amino acid sequence of SPASX5SVSLGDRX6, X1, X2, and X3 are each independently a neutral polar amino acid or an acidic polar amino acid, X4 is a nonpolar amino acid or a neutral polar amino acid, and X5 and X6 are each independently a nonpolar amino acid; 2. The antibody or antigen-binding fragment thereof according to item 1, except that X1 is aspartic acid, X2 is aspartic acid, X3 is glutamic acid, X4 is alanine, and the light chain FR1 comprises the sequence TPLSLPVSLGDQA. [Item 7] a heavy chain CDR1 comprising the amino acid sequence of X1YYIH; A heavy chain CDR2 comprising the amino acid sequence of RIDPENX2X3TTYAPKFQ, and a heavy chain CDR3 comprising the amino acid sequence of YCARSTVL; and a heavy chain variable region comprising a light chain CDR1 comprising the amino acid sequence RX4SQSIVHTNG; A light chain CDR2 comprising the amino acid sequence of KLLIYKV, and a light chain CDR3 comprising the amino acid sequence GVYYFQGS; and a light chain variable region comprising Light chain FR1 The amino acid sequence of TPLSLPVSLGDQA or comprising the amino acid sequence of SPASX5SVSLGDRX6, X1 and X2 are each independently asparagine or aspartic acid, X3 is glutamine or glutamic acid, X4 is alanine or serine, X5 is leucine or methionine, and X6 is alanine or valine; 2. The antibody or antigen-binding fragment thereof according to item 1, except that X1 is aspartic acid, X2 is aspartic acid, X3 is glutamic acid, and the light chain FR1 comprises the sequence TPLSLPVSLGDQASISCRA. [Item 8] A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31 or 41; A heavy chain CDR2 comprising an amino acid sequence represented by any one of SEQ ID NOs: 32, 42 to 44, and A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 34 or 52; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and A light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; and A light chain FR1 comprising an amino acid sequence represented by any one of SEQ ID NOs: 53 to 56; 1. An antibody comprising a light chain variable region comprising: A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 37, and 2. The antibody or antigen-binding fragment thereof according to item 1, excluding the antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 38. [Item 9] A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of items 1 to 8. [Item 10] 10. An expression vector comprising the nucleic acid according to item 9. [Item 11] A cell comprising the nucleic acid according to item 9 or the expression vector according to item 10. [Item 12] 9. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 8, and a pharma- ceutically acceptable carrier or excipient. [Item 13] 13. The pharmaceutical composition according to item 12, which is in a lyophilized state. [Item 14] 1) preparing antibody-producing immortalized cells from B cells collected from the intestinal lamina propria or the spleen; 2) culturing the antibody-producing immortalized cells and determining which cells produce antibodies that bind to Clostridium difficile bacteria; and 3) recovering antibodies from cells that produce antibodies that bind to the Clostridium difficile bacteria. A method for producing an antibody that binds to Clostridium difficile bacteria, comprising: [Item 15] Item 15. The method for producing an antibody that binds to Clostridium difficile bacterial bodies according to Item 14, wherein the step 2 is carried out by determining cells that produce an antibody that binds to the bacterial bodies immobilized on a carrier. Effect of the Invention

[0025] The present invention advantageously provides antibodies and antigen-binding fragments thereof that bind to Enterobacteriaceae, particularly C. difficile bacterial organisms and other Enterobacteriaceae, and uses thereof. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 shows the inhibitory effect of antibodies that bind to C. difficile bacteria on the proliferation of C. difficile. [Diagram 2] 2 is a graph showing the inhibitory effect of antibodies that bind to C. difficile bacteria on C. difficile infection, showing the results when SNK0002M and W27G2 were used as antibodies that bind to C. difficile bacteria. [Diagram 3] 3 is a graph showing the inhibitory effect of antibodies that bind to C. difficile bacteria on C. difficile infection. The numbers of viable C. difficile bacteria in feces are shown 4 days (left panel) and 10 days (right panel) after C. difficile infection. The results are shown when SNK0002M and W27G2 were used as antibodies that bind to C. difficile bacteria. [Figure 4] Figure 4 shows the growth inhibitory effect of antibodies that bind to C. difficile bacteria on Fusobacterium nucleatum, a bacterium thought to be the causative bacterium of colon cancer. The results shown are for antibodies SNK0001M, SNK0003A, SNK0001MR, SNK0002MR, SNK0001AR, SNK0002AR, and RS_H000_L001 that bind to C. difficile bacteria. [Diagram 5]Figure 5 shows the two-dimensional electrophoresis data obtained when identifying the antigen molecule of C. difficile. The red arrows indicate the same spots in the three SDS-PAGE images. Mass spectrometry was performed on the samples obtained from the spots. [Figure 6] Figure 6 shows the results of Western blot and CBB staining of various synthetic peptides using W27G2 antibody. Gray letters indicate amino acid sequences common to each bacterial species. The underlined synthetic peptides recognized by W27G2 antibody. [Figure 7] Figure 7 shows the results of Western blot and CBB staining of various synthetic peptides using W27G2 antibody. Gray letters indicate amino acid sequences common to each bacterial species. The underlined synthetic peptides recognized by W27G2 antibody. [Figure 8] FIG. 8 shows some of the results of database analysis of bacteria sharing the epitope of the SHMT of E. coli recognized by the W27G2 antibody. [Figure 9] FIG. 9 shows the crystal of RS_H000_L000GR Fab-E. coli SHMT (25-45). [Figure 10] FIG. 10 shows crystals of the RS_H000_L000GR Fab-C. difficile iPGM (486-509) complex. [Figure 11] Figure 11 is a 3D reconstruction image showing the binding mode between the W27G2 antibody and the E. coli SHMT antigen (left) and the binding mode between the W27G2 antibody and the C. difficile bacterial iPGM antigen. [Figure 12] FIG. 12 shows 3D reconstruction images showing the binding mode between the W27G2 antibody and the E. coli SHMT antigen (left) and the binding mode between the W27G2 antibody and the C. difficile bacterial iPGM antigen. [Figure 13] FIG. 13 shows an alignment of amino acid sequences of the heavy chain variable regions of mutants of the W27G2 antibody, an antibody of the present invention that binds to C. difficile bacteria. [Figure 14]FIG. 14 shows an alignment of amino acid sequences of the light chain variable regions of mutants of the W27G2 antibody, an antibody of the present invention that binds to C. difficile bacteria. [Figure 15] Figure 15 shows the results of subjecting RS mutant recombinant purified antibodies to non-reducing SDS-PAGE and staining with Coomassie Blue. Lane 1 shows the results of an antibody sample crudely purified from W27G2 hybridoma culture fluid using a hydroxyapatite column. Lanes 2 to 14 show the results of various RS mutant recombinant purified antibody samples produced by CHO cells (antibodies produced in CHO cells by introducing a mutation into the heavy or light chain of the W27G2 antibody so as not to substantially change the antigen specificity, called RS mutant recombinant purified antibodies; details are omitted in Figure 15). [Figure 16] FIG. 16 shows the binding of the W27G2 antibody and various RS mutant recombinant purified antibodies obtained from hybridomas to E. coli SHMT and SHMT mutants. [Figure 17] 17 is a diagram showing the specific binding properties of the W27G2 antibody and various RS mutant recombinant purified antibodies to total proteins of multiple bacteria. As the W27G2 antibody, W27G2-CHT (a crude purification of the W27 hybridoma culture supernatant using a hydroxyapatite column) and W27G2-GF (a multimer fraction of W27CHT further purified using a gel filtration column) were used, and as the RS mutant recombinant purified antibodies, RS_H000_L001, RS_H000_L005, and RS_H007_L005 were used. [Figure 18] FIG. 18 shows the inhibitory effect of various RS mutant recombinant purified antibodies on the proliferation of E. coli. [Figure 19] FIG. 19 shows that two recombinant IgA antibodies, antibodies RS_H000_L001 (rW27) and SNK0002AR, each suppressed the weight loss and the decrease in survival rate in C. difficile-infected mice. [Figure 20]FIG. 20 shows the C. difficile bacteria content in stool after administration of two recombinant IgA antibodies, antibodies RS_H000_L001 (rW27 and SNK0002AR, and Vancomycin. [Figure 21] FIG. 21 shows the results of analyzing the relative abundance ratios of order levels of bacteria by 16S rRNA analysis in feces collected 14 days after administration of two recombinant IgA antibodies, antibodies RS_H000_L001 (rW27) and SNK0002AR, rW27 IgG antibody, and vancomycin (at the time of death for mice that died). [Figure 22] FIG. 22 shows the results of β-diversity analysis using two recombinant IgA antibodies, antibodies RS_H000_L001 (rW27) and SNK0002AR, as well as rW27 IgG antibody, and the results of 16S rRNA analysis of stool collected 14 days after administration of vancomycin (at the time of death for mice that died). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] (definition) In the present specification, when a plurality of ranges of numerical values ​​are indicated, the same applies to any combination of the lower limit values ​​and upper limit values ​​of those plurality of ranges.

[0028] In the present specification, the term "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 mainly 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 a heavy chain variable region and a light chain variable region, respectively. The variable region has sites called CDR1 to 3, which are further specified as sites that recognize antigens, in order from the amino terminus. These 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. Furthermore, 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, in order from the amino terminus, respectively. The antibody may be in the form of an antibody consisting of two heavy chains and two light chains, or in the form of an antibody consisting of one heavy chain and one light chain (also called a single-chain antibody).

[0029] 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.

[0030] Antibodies may have amino acid sequences derived from the same species or from different species, such as human, mouse, rat, hamster, rabbit, goat, donkey, pig, cow, horse, chicken, monkey, chimpanzee, camel, and llama.

[0031] In the case of antibodies derived from a different species, there is no particular limitation, and examples include antibodies derived from two or more of human, mouse, rat, hamster, rabbit, goat, sheep, donkey, pig, cow, horse, chicken, monkey, chimpanzee, camel, llama, etc.

[0032] As used herein, the term "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 a fragment consisting of a part of the antibody. In one embodiment, the "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).

[0033] The antibody of the present invention may be a CDR-grafted antibody.In one example, in a CDR-grafted antibody, a part or whole sequence of the CDR region of an antibody derived from one animal species is replaced with the CDR sequence of another animal species.For example, one or more CDRs of a mouse antibody are replaced with the CDR sequence of a human antibody.

[0034] A method well known to those skilled in the art can be used to identify the heavy chain CDR1-3 in the heavy chain variable region and the light chain CDR1-3 in the light chain variable region of an antibody. For example, the "Kabat definition" (Kabat et al., Ann. NY Acad. Sci. 1971, Vol. 190, pp. 382-391 and Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Edition, 1991, USDepartment of Health and Human Services, NIH Publication, pp. 91-3242), "Chothia definition" (Chothia et al., Nature, 1989, Vol. 342, pp. 877-883), "contact definition" (MacCallum et al., J. Mol. Biol., 1996, Vol. 262, pp. 732-745), etc., all of which are well known to those skilled in the art, can be used. To identify the CDR sequence in an antibody, the CDR may be identified based on information in a public database.

[0035] As used herein, "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 between 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.). Programs called BLASTN and BLASTX based on such BLAST algorithms have been developed (e.g., Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. J Mol Biol. 215:403-10 (1990) etc.). Specific techniques for these analysis methods are known and can be found on the NCBI website. For example, when a certain amino acid sequence A is identical to another amino acid sequence B to a certain percentage, it means that amino acid sequence A and amino acid sequence B have the said percentage of identity.

[0036] As used herein, "monoclonal" is a modifier indicating the character of an antibody or the like obtained from a population of substantially homogeneous antibodies, the individual antibodies within such a population being identical except for possible naturally occurring mutations which may be present in minor amounts.

[0037] As used herein, the term "conservative substitution techniques" refers to techniques in which an amino acid residue is replaced with an amino acid residue having a similar side chain.

[0038] For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine is a conservative substitution technique. Other conservative substitution techniques include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid, amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine, amino acid residues having non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan, amino acid residues having β-branched side chains such as threonine, valine, and isoleucine, and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0039] In the present specification, the term "intestinal bacteria" is not particularly limited as long as it is a bacterium that normally resides in the intestine of a living body. Examples of such intestinal bacteria include bacteria that form intestinal flora in a living body and are involved in maintaining homeostasis of the intestinal immune system.

[0040] Specific examples of intestinal bacteria include, but are not limited to, Bacteria belonging to the genus Prevotella Bacteria belonging to the genus Bacteroides Bacteria belonging to the genus Megamonas Bacteria belonging to the genus Bifidobacterium Bacteria belonging to the genus Faecalibacterium Bacteria belonging to the genus Coprococcus Bacteria belonging to the genus Ruminococcus Bacteria belonging to the genus Blautia Bacteria belonging to the genus Eubacterium Bacteria belonging to the genus Roseburia Bacteria belonging to the genus Lactobacillus Bacteria belonging to the genus Clostridium Bacteria belonging to the genus Escherichia Staphylococcus bacteria, Bacteria belonging to the genus Enterococcus Bacteria belonging to the genus Pseudomonas Bacteria belonging to the genus Enterorhabdus Bacteria belonging to the genus Fusobacterium etc.

[0041] (Antibody or antigen-binding fragment thereof that binds to C. difficile bacteria) Antibodies that bind to C. difficile bacterial bodies of the invention bind to C. difficile bacteria. Typically, antibodies of the invention bind to undisrupted C. difficile bacteria present in the culture supernatant.

[0042] The antibodies that bind to C. difficile bacteria of the present invention may be produced from antibody-producing cells derived from B cells, such as hybridomas, or may be produced using genetic recombination technology to introduce nucleic acid encoding the antibody into cells other than those of the immune system, and used as recombinant antibodies.

[0043] In one embodiment, the antibody that binds to C. difficile bacteria of the present invention 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:7; 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:8. TIFF2024041901000001.tif49167

[0044] In one embodiment, the antibody that binds to C. difficile bacteria of the present invention is A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:1; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:2, and A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:3; and a heavy chain variable region comprising A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:4; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:5; Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:6 The light chain variable region comprises: TIFF2024041901000002.tif53168

[0045] In one embodiment, the antibody that binds to C. difficile bacteria of the present invention is A heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:7 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and It comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0046] In one embodiment, the antibody of the present invention that binds to C. difficile bacteria is an IgM antibody SNK0001M produced by a hybridoma obtained from spleen-derived B cells. The nucleic acid sequence encoding the antibody SNK0001M was determined, and the recombinant purified antibody SNK0001MR and the recombinant purified antibody SNK0001AR, which are IgA antibodies, were produced by applying recombinant technology. The antibody SNK0001AR has the following amino acid sequence configuration: TIFF2024041901000003.tif195169

[0047] In one embodiment, the antibody that binds to C. difficile bacteria of the present invention 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: 17; 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:18. TIFF2024041901000004.tif46164

[0048] In one embodiment, the antibody that binds to C. difficile bacteria of the present invention is A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 11; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 12, and A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 13; and a heavy chain variable region comprising A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 14; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 15; Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:16 The light chain variable region comprises: TIFF2024041901000005.tif50160

[0049] In one embodiment, the antibody that binds to C. difficile bacteria of the present invention is A heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 17 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and It comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:18 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0050] In one embodiment, the antibody of the present invention that binds to C. difficile bacteria is the IgM antibody SNK0002M produced by a hybridoma obtained from spleen-derived B cells. The nucleic acid sequence encoding the antibody SNK0002M was determined, and the recombinant purified antibody SNK0002MR and the recombinant purified antibody SNK0002AR, which is an IgA antibody, were produced by applying recombinant technology. The antibody SNK0002AR has the following amino acid sequence configuration: TIFF2024041901000006.tif178164

[0051] In one embodiment, the antibody that binds to C. difficile bacteria of the present invention 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: 27; 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:28. TIFF2024041901000007.tif48158

[0052] In one embodiment, the antibody that binds to C. difficile bacteria of the present invention is A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22, and A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23; and a heavy chain variable region comprising A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 24; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 25; Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:26 The light chain variable region comprises: TIFF2024041901000008.tif50158

[0053] In one embodiment, the antibody that binds to C. difficile bacteria of the present invention is A heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:27 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and It comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:28 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0054] In one embodiment, the antibody of the present invention that binds to C. difficile bacteria is the IgA antibody SNK0003A produced by a hybridoma obtained from B cells derived from the intestinal mucosa lamina propria. The nucleic acid sequence encoding the antibody SNK0003M was determined, and the recombinant purified antibody SNK0003MR and the recombinant purified antibody SNK0003AR, which is an IgA antibody, were produced by applying recombinant technology. The antibody SNK0003A has the following amino acid sequence configuration: TIFF2024041901000009.tif183164

[0055] In one embodiment, the antibody that binds to C. difficile bacteria of the present invention is A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 37, and For a reference antibody comprising a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 38, having at least one amino acid mutation in at least one region selected from heavy chain CDR1 to 3, light chain CDR1 to 3, and light chain FR1; This is an antibody that binds to the amino acid sequence RQEEHIELIAS (SEQ ID NO: 72) in the E. coli SHMT protein and the amino acid sequence VLDMMKLEKPE (SEQ ID NO: 73) in the iPGM protein of C. difficile. TIFF2024041901000010.tif54165

[0056] The at least one amino acid mutation in the antibody can be identified by applying techniques available to those skilled in the art using the results of structural analysis of the conjugates of an antibody having the amino acid sequence of the reference antibody and E. coli SHMT protein, and an antibody having the amino acid sequence of the reference antibody and C. difficile iPGM protein. The number of amino acid mutations relative to the reference antibody may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.

[0057] Such a reference antibody is not particularly limited as long as it has W27G2_HV (SEQ ID NO: 37) as a heavy chain variable region and W27G2_LV (SEQ ID NO: 38) as a light chain variable region. For example, an IgG antibody (W27RS_H000_L000GR) can be used. The antibody W27RS_H000_L000GR has the same heavy chain variable region and light chain variable region as the antibody W27G2 produced by a hybridoma obtained from B cells derived from the intestinal mucosa lamina propria. TIFF2024041901000011.tif98167

[0058] Whether an antibody having the specified mutation actually binds to the amino acid sequence RQEEHIELIAS in the E. coli SHMT protein and the amino acid sequence VLDMMKLEKPE in the iPGM protein of C. difficile can be confirmed by methods well known to those skilled in the art, such as ELISA or Western blotting.

[0059] In one embodiment, the antibody that binds to C. difficile bacteria of the present invention is a heavy chain CDR1 comprising the amino acid sequence of X1YYIH; a heavy chain CDR2 comprising the amino acid sequence RIDPENX2X3TTYAPKFQ; a heavy chain CDR3 comprising the amino acid sequence of YCARSTVL; and a heavy chain variable region comprising a light chain CDR1 comprising the amino acid sequence RX4SQSIVHTNG; a light chain CDR2 comprising the amino acid sequence of KLLIYKV; a light chain CDR3 comprising the amino acid sequence GVYYFQGS; and a light chain variable region comprising Light chain FR1 The amino acid sequence of TPLSLPVSLGDQA or comprising the amino acid sequence of SPASX5SVSLGDRX6, X1, X2, and X3 are each independently a neutral polar amino acid or an acidic polar amino acid, X4 is a nonpolar amino acid or a neutral polar amino acid, and X5 and X6 are each independently a nonpolar amino acid; This antibody excludes those in which X1 is aspartic acid, X2 is aspartic acid, X3 is glutamic acid, X4 is alanine, and the light chain FR1 contains the sequence TPLSLPVSLGDQA.

[0060] The above antibody was designed, based on three-dimensional structural analysis, to have a binding mode similar to that of the W27G2 antibody with respect to the amino acid sequence RQEEHIELIAS in the E. coli SHMT protein and the amino acid sequence VLDMMKLEKPE in the iPGM protein of C. difficile, and actually exhibits a binding mode and bacterial growth inhibitory effect similar to that of the W27G2 antibody with respect to these bacteria.

[0061] In one embodiment, the antibody that binds to C. difficile bacteria of the present invention is a heavy chain CDR1 comprising the amino acid sequence of X1YYIH; a heavy chain CDR2 comprising the amino acid sequence RIDPENX2X3TTYAPKFQ; a heavy chain CDR3 comprising the amino acid sequence of YCARSTVL; and a heavy chain variable region comprising a light chain CDR1 comprising the amino acid sequence RX4SQSIVHTNG; a light chain CDR2 comprising the amino acid sequence of KLLIYKV; a light chain CDR3 comprising the amino acid sequence GVYYFQGS; and a light chain variable region comprising Light chain FR1 The amino acid sequence of TPLSLPVSLGDQA or comprising the amino acid sequence of SPASX5SVSLGDRX6, X1 and X2 are each independently asparagine or aspartic acid, X3 is glutamine or glutamic acid, X4 is alanine or serine, X5 is leucine or methionine, and X6 is alanine or valine; This antibody excludes those in which X1 is aspartic acid, X2 is aspartic acid, X3 is glutamic acid, X4 is alanine, and the light chain FR1 contains the sequence TPLSLPVSLGDQA.

[0062] In one embodiment, the heavy chain of an antibody that binds to C. difficile bacteria of the invention comprises: A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31 or 41; A heavy chain CDR2 comprising an amino acid sequence represented by any one of SEQ ID NOs: 32, 42 to 44; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; Includes. TIFF2024041901000012.tif57154

[0063] In one embodiment, the heavy chain of an antibody that binds to C. difficile bacteria of the invention comprises: A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:31; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; Includes.

[0064] In one embodiment, the heavy chain of an antibody that binds to C. difficile bacteria of the invention comprises: A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:31; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 42; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; Includes.

[0065] In one embodiment, the heavy chain of an antibody that binds to C. difficile bacteria of the invention comprises: A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:31; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 43; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; Includes.

[0066] In one embodiment, the heavy chain of an antibody that binds to C. difficile bacteria of the invention comprises: A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:31; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:44; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; Includes.

[0067] In one embodiment, the heavy chain of an antibody that binds to C. difficile bacteria of the invention comprises: A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:41; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; Includes.

[0068] In one embodiment, the heavy chain of an antibody that binds to C. difficile bacteria of the invention comprises: A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:41; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 42; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; Includes.

[0069] In one embodiment, the heavy chain of an antibody that binds to C. difficile bacteria of the invention comprises: A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:41; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 43; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; Includes.

[0070] In one embodiment, the heavy chain of an antibody that binds to C. difficile bacteria of the invention comprises: A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:41; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:44; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; Includes.

[0071] In one embodiment, the light chain of an antibody that binds to C. difficile bacteria of the invention is A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34 or 52; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35; A light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; Includes. TIFF2024041901000013.tif36158

[0072] In one embodiment, the light chain of an antibody that binds to C. difficile bacteria of the invention is A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35; A light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; Includes.

[0073] In one embodiment, the light chain of an antibody that binds to C. difficile bacteria of the invention is A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:52; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35; A light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; Includes.

[0074] In one embodiment, the antibody or antigen-binding fragment thereof that binds to C. difficile bacteria of the present invention contains in its light chain variable region a sequence that binds to Protein L. The antibody molecule or antigen-binding fragment thereof that contains the sequence that binds to Protein L can be purified using a Protein L column.

[0075] In one aspect, the antibody or antigen-binding fragment of the present invention that binds to C. difficile bacteria and comprises a sequence that binds to Protein L comprises a light chain variable region comprising the amino acid sequence SPASX5SVSLGDRX6, wherein X5 and X6 are each independently a non-polar amino acid.

[0076] In one aspect, the antibody or antigen-binding fragment thereof that binds to C. difficile bacteria of the present invention and comprises a sequence that binds to Protein L comprises a light chain variable region comprising the amino acid sequence SPASX5SVSLGDRX6, wherein X5 is leucine or methionine and X6 is alanine or valine.

[0077] In one aspect, the antibody or antigen-binding fragment thereof of the present invention that binds to C. difficile bacteria and comprises a sequence that binds to Protein L comprises a light chain variable region comprising an amino acid sequence represented by one selected from the group consisting of SEQ ID NOs: 53 to 55. TIFF2024041901000014.tif37160

[0078] In one embodiment, the antibody or antigen-binding fragment thereof that binds to C. difficile bacteria of the present invention and comprises a sequence that binds to Protein L comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:54. This sequence was obtained by introducing a mutation into the sequence TPLSLPVSLGDQA (SEQ ID NO: 56) of the light chain FR1 region of the W27G2 antibody so that Protein L binds to the antibody molecule.

[0079] In one embodiment, the antibody or antigen-binding fragment thereof that binds to C. difficile bacteria of the present invention and comprises a sequence that binds to Protein L comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:55. This sequence further includes a mutation introduced into the light chain FR1 region of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO:54, so that Protein L binds to the antibody molecule more tightly.

[0080] In one embodiment, the antibody or antigen-binding fragment thereof that binds to C. difficile bacteria of the present invention and comprises a sequence that binds to Protein L comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:58. This sequence further includes a mutation introduced into the light chain FR1 region of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO:54, so that Protein L binds to the antibody molecule more tightly.

[0081] The antibodies of the present invention that bind to C. difficile bacteria may have any combination of the above-mentioned heavy and light chains, and may or may not contain a sequence that binds to Protein L.

[0082] In one embodiment, the antibody or antigen-binding fragment thereof that binds to C. difficile bacteria of the present invention is A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31 or 41; A heavy chain CDR2 comprising an amino acid sequence represented by any one of SEQ ID NOs: 32, 42 to 44, and A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; a heavy chain variable region comprising: A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34 or 52; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and A light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; and A light chain FR1 comprising an amino acid sequence represented by any one of SEQ ID NOs: 53 to 56; 1. An antibody comprising a light chain variable region comprising: A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 37, and This antibody does not include an antibody having a light chain variable region containing the amino acid sequence represented by SEQ ID NO:38.

[0083] For example, an antibody of the present invention that binds to C. difficile bacteria comprises any of the heavy chain variable regions described below in combination with any of the light chain variable regions. TIFF2024041901000015.tif165167TIFF2024041901000016.tif118162

[0084] Preferably, the antibodies of the present invention that bind to C. difficile bacteria comprise the combination of heavy and light chain variable regions described below. TIFF2024041901000017.tif103157

[0085] Preferably, the antibodies that bind to C. difficile bacteria of the present invention include: TIFF2024041901000018.tif213156TIFF2024041901000019.tif233157TIFF2024041901000020.tif232156TIFF2024041901000021.tif245156Antibodies produced in CHO cells by introducing mutations into the heavy or light chain of the W27G2 antibody so as not to substantially alter the antigen specificity are called RS mutant recombinant purified antibodies. These RS mutant recombinant purified antibodies further have mutations for binding to protein L.

[0086] The antibodies of the present invention that bind to C. difficile bacteria can have multiple specificities that bind to other disease-related bacteria in addition to binding to C. difficile bacteria. For example, the antibodies of the present invention that bind to C. difficile bacteria can also bind to Fusobacterium nucleatum, which is thought to be a causative bacterium of colon cancer, and preferably inhibit its proliferation.

[0087] The antibody that binds to C. difficile bacteria of the present invention can have a mutation in its constituent amino acid sequence, for example, its heavy chain variable region, light chain variable region, heavy chain CDR1-3, or light chain CDR1-3, to the extent that it does not lose its antigen-binding property. The mutation may be, but is not limited to, substitution, deletion, insertion, etc. For example, in the case of substitution, a conservative substitution technique can be adopted. Furthermore, by analyzing in detail the binding mode between the antibody and the antigen using three-dimensional structural analysis, etc., various mutations can be introduced into the antibody that binds to C. difficile bacteria of the present invention to the extent that it does not lose its antigen-binding property.

[0088] (nucleic acid) The nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention may be ribonucleotides or deoxynucleotides. The form of the nucleic acid is not particularly limited, and may be single-stranded or double-stranded. 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. For example, appropriate codons can be selected in consideration of codon frequency and the like depending on the type of host cell used during production. In one aspect, the nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention is used to express and produce the antibody or antigen-binding fragment thereof according to the present invention.

[0089] When a nucleic acid encoding an antibody or antigen-binding fragment thereof of the present invention encodes an antigen-binding fragment, for example, 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.

[0090] Exemplary sequence information regarding nucleic acids encoding the antibodies or antigen-binding fragments thereof of the present invention is provided below. TIFF2024041901000022.tif225160TIFF2024041901000023.tif229159TIFF2024041901000024.tif223156TIFF20240419010 00025.tif222156TIFF2024041901000026.tif230155TIFF2024041901000027.tif226155TIFF2024041901000028.tif229154 TIFF2024041901000029.tif228153TIFF2024041901000030.tif229154TIFF2024041901000031.tif229153TIFF20240419010 00032.tif230154TIFF2024041901000033.tif229153TIFF2024041901000034.tif229153TIFF2024041901000035.tif230153

[0091] (Method of producing an antibody that binds to C. difficile bacteria) The method for producing an antibody that binds to C. difficile bacteria according to the present invention is characterized by comprising the following steps 1 to 3: (Process 1) A process for producing antibody-producing immortalized cells from B cells collected from the intestinal lamina propria or the spleen. (Process 2) A step of culturing the antibody-producing immortalized cells prepared in step 1 above and determining which cells produce antibodies that bind to C. difficile bacteria. (Step 3) A step of recovering antibodies from the cells that produce the antibodies that bind to the C. difficile bacteria.

[0092] The method of the present invention for producing an antibody that binds to C. difficile bacteria may include, after step 3, a step of treating the heavy chain variable region and / or the light chain variable region with, for example, a protease or the like, as necessary, in order to form an appropriate combination of these as described below, 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.

[0093] The above steps 1 to 3 in the method for producing an antibody that binds to C. difficile bacteria of the present invention are described in detail below.

[0094] (Regarding step 1) Step 1 in the method of the present invention for producing an antibody that binds to C. difficile bacteria is a step of preparing antibody-producing immortalized cells from B cells collected from the intestinal mucosa lamina propria or the spleen.

[0095] The intestinal lamina propria is not particularly limited, but may be one of the layers constituting the mucosa present in, for example, the esophagus, stomach, small intestine (including the duodenum, jejunum, ileum, etc.), large intestine (including the cecum, colon, rectum, etc.), etc., and may be a layer located between a layer containing epithelial cells and a mucosal substrate (Muscularis mucosae). Among these, the intestinal lamina propria present in the small intestine, which contains lymphatic tissue, capillaries, lymphatic vessels, etc., is preferred.

[0096] The spleen cells that can be used are not particularly limited, but include cells containing B cells.

[0097] The method for collecting B cells from the intestinal mucosa lamina propria or spleen is not particularly limited, but for example, the intestine is collected, washed, and then cut to expose the mucosa. Then, the epithelial cells are released and removed by shaking in physiological saline containing an appropriate concentration of EDTA. Then, the B cells are collected by treating with a digestive enzyme such as collagenase at an appropriate concentration. Other known methods may be used, and B cells derived from the intestinal mucosa lamina propria or spleen may be purchased and obtained commercially.

[0098] The origin of the intestinal mucosa lamina propria or spleen, i.e., the origin of the above-mentioned B 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.

[0099] Any method known to those skilled in the art can be used for the step of preparing antibody-producing immortalized cells from B cells collected from the intestinal lamina propria or spleen. For example, as the antibody-producing immortalized cells, hybridomas can be prepared by fusing the B cells with a type of cell other than B cells, and immortalized B cells can be prepared by infecting B cells with EB virus or the like, but are not limited thereto.

[0100] When producing a hybridoma by fusing a B cell with a type of cell other than a B cell, the type of cell other than the B cell (sometimes referred to as "other cell" in this specification) is not particularly limited as long as it fuses with the B cell upon contact to form a hybridoma and the hybridoma does not lose the antibody-producing function exhibited by the B cell described above.

[0101] Such other cells are preferably cells that can be fused with the B cells to form hybridomas, and the hybridomas can acquire immortalization functions, and specific examples thereof 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.

[0102] 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, and llamas.

[0103] Fusion means that the B cell and another cell are inseparably united. In this case, inseparably united does not include cell division, which is a phenomenon that occurs when the fused cells grow. The fused cells are an example of a hybridoma in this specification.

[0104] The conditions for mixing and fusion are not particularly limited, and may be appropriately selected from conditions commonly used in methods for fusing cells. For example, the conditions used for culturing B cells or other cells may be appropriately modified. Examples of such methods include a method of mixing B cells with cells of a type other than B cells in an appropriate medium and contacting them in the presence of polyethylene glycol or the like; a method of applying an electric stimulus after the above mixing; a method of using a virus such as Sendai virus, and then incubating the mixture under conditions of 37°C and 5% carbon dioxide.

[0105] 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. The completion of fusion may be confirmed by appropriately modifying a known method used in normal cell fusion. For example, such a method may include a method of observing the progress of fusion under a microscope, and a known kit may be appropriately selected to fuse cells according to the conditions of use.

[0106] (Regarding step 2) Step 2 in the method of the present invention for producing an antibody that binds to C. difficile bacteria is a step of culturing the antibody-producing immortalized cells prepared in step 1 above and determining which cells produce antibodies that bind to C. difficile bacteria.

[0107] Preferably, step 2 is performed by determining cells that produce antibodies that bind to whole C. difficile bacterial bodies immobilized on a carrier. Preferably, the immobilized C. difficile bacterial bodies have not been subjected to a lysis treatment. For example, the C. difficile bacterial bodies are immobilized on a carrier such as an ELISA plate in a state of being suspended in a Na2CO3 buffer.

[0108] Prior to step 2 of the present invention, the antibody-producing immortalized cells obtained in step 1 may be subjected to a subcloning step such as limiting dilution, which is commonly used in producing monoclonal antibodies.

[0109] The antibody-producing immortalized cells that produce antibodies that bind to C. difficile bacteria can be determined by means of ELISA, EIA, RIA, FLISA, FIA, or the like, or by means of FACS, or the like.

[0110] Furthermore, after determining antibody-producing immortalized cells that bind to C. difficile bacteria determined by the above-mentioned method, the method may further include a step of determining antibody-producing immortalized cells that produce antibodies that also bind to another enterobacteria.

[0111] (Regarding step 3) Step 3 in the method of the present invention for producing an antibody that binds to C. difficile bacteria is a step of recovering the antibody from the cells that produce the antibody that binds to C. difficile bacteria, as determined in step 2 above. The specific recovery method in step 3 is not particularly limited, and examples thereof include a method of recovering the supernatant of a culture solution of cells producing IgA antibodies, 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 treatments using surfactants, cell wall digestive enzymes, and cell membrane digestive enzymes to lyse the cells, and then subjecting the cell to a solid-liquid separation step, and recovering the liquid fraction, thereby producing the antibody of the present invention that binds to C. difficile bacteria.

[0112] Before recovering antibody molecules from the above-mentioned antibody-producing immortalized cells, the antibody-producing immortalized cells may be cultured for a certain period of time in an appropriate medium, and then subjected to the above-mentioned method of recovering the supernatant or the method of recovering the antibody from a cell lysate. Alternatively, the above-mentioned antibody-producing immortalized cells may be administered intraperitoneally to an individual animal such as an immunodeficient mouse, and the antibody may be recovered from the ascites of the individual.

[0113] The antibody that binds to C. difficile bacteria collected by the above-mentioned method may be subjected to a known purification step as appropriate. The specific purification means is not particularly limited, but may be a suitable combination of known protein purification means, such as a purification means based on precipitation using acetone, ammonium sulfate, or the like, or a purification means based on column chromatography such as affinity, anion exchange, cation exchange, size exclusion, or reverse phase.

[0114] (Method of Producing Recombinant Antibodies) In another aspect, an antibody that binds to C. difficile bacteria of the present invention can be produced by introducing a nucleic acid encoding an antibody confirmed to bind to C. difficile bacteria into cells capable of producing a monoclonal antibody, culturing the cells, and recovering the monoclonal antibody from the cell extract or culture supernatant, and purifying it as necessary.

[0115] To confirm that an antibody binds to C. difficile bacteria, means well known to those skilled in the art can be used, for example, by identifying the antibody molecule or cells producing the antibody using means such as ELISA, EIA, RIA, FLISA, FIA, or FACS.

[0116] The cells capable of producing the monoclonal antibodies are not particularly limited as long as they are capable of producing various proteins derived from mammals or the like that exert their functions by forming higher-order structures, 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. In one embodiment, monoclonal antibodies can also be produced using yeast cells, filamentous fungal cells, and plant cells such as soybean and Arabidopsis thaliana.

[0117] 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 body of the present invention.

[0118] In one embodiment, after producing a monoclonal antibody in these cells, 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). In one embodiment, the cells producing the antibody-containing composition suitable for enteral intake are preferably yeast cells, filamentous fungal cells, soybeans, Arabidopsis, or other plant cells, and the produced monoclonal antibody is preferably in the form in which a VHH fragment is fused to IgAFc.

[0119] When the antibody of the present invention that binds to C. difficile bacteria is a chimeric antibody, with regard to the above-mentioned nucleic acid, nucleic acids having base sequences encoding the heavy chain variable region and the light chain variable region, and base sequences encoding the heavy chain constant region and the light chain constant region of a different species are prepared, the prepared base sequence encoding the heavy chain variable region is bound to the nucleic acid having the base sequence encoding the heavy chain constant region, and the prepared base sequence encoding the light chain variable region is bound to the nucleic acid having the base sequence encoding the light chain constant region, and these bound nucleic acids are introduced into cells capable of producing the antibody as described above.

[0120] Furthermore, when the antibody according to the present invention that binds to C. difficile bacteria is an antibody in which the heavy and / or light chain CDR1-3 have amino acid sequences derived from mouse and the other regions have amino acid sequences derived from human (this may be referred to as a humanized antibody), the method of production may be the same as that of the chimeric antibody described above, in which nucleic acids having base sequences encoding the heavy and / or light chain CDR1-3 and base sequences encoding the other regions are prepared, recombined to form heavy and light chains, and introduced into cells capable of producing monoclonal IgA antibodies as described above. In order to maintain the binding ability of the antibody, some bases may need to be replaced with other bases.

[0121] (Antibody-producing immortalized cells) The antibody-producing immortalized cells according to the present invention are immortalized cells which produce antibodies that bind to the above-mentioned C. difficile bacteria according to the present invention.

[0122] A specific antibody-producing immortalized cell is one obtained in step 1 of the above-mentioned [Method for producing an antibody that binds to C. difficile bacteria]. That is, the antibody-producing immortalized cell is a cell obtained by mixing and fusing the B cell described in step 1 of the above-mentioned method for producing an antibody that binds to C. difficile bacteria according to the present invention with a cell of another type, i.e., a cell of a type other than B cells, or a cell that has been made into an immortalized B cell by infection with EB virus or the like.

[0123] The hybridoma may be a hybridoma derived from the same species or a hybridoma derived from a different species. A hybridoma derived from the same species may be obtained by using the above-mentioned [Method for producing a monoclonal IgA antibody] in which the B cells and the cells other than the B cells are derived from the same species, whereas a hybridoma derived from a different species may be obtained by using the B cells and the cells other than the B cells from different species.

[0124] 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 origins of B cells and types of cells other than B cells, for example. 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.

[0125] The hybridomas of different species may be appropriately selected and combined from the origins described in the section on the origins of B cells and types of cells other than B cells among those described in step 1 of the above-mentioned [Method for producing monoclonal IgA antibodies]. Examples of the hybridomas of different species include an appropriate combination of origins of human, mouse, rat, hamster, rabbit, goat, sheep, donkey, pig, cow, horse, chicken, monkey, chimpanzee, etc.

[0126] Pharmaceutical Composition The pharmaceutical compositions of the present invention include an antibody or antigen-binding fragment thereof that binds to the C. difficile bacterial organism of the present invention.

[0127] The pharmaceutical composition according to the present invention can be suitably used for the treatment of, but is not particularly limited to, diseases associated with C. difficile bacteria.

[0128] Diseases associated with C. difficile bacteria include, but are not limited to, inflammatory bowel disease, ulcerative colitis, Crohn's disease, allergies, asthma, obesity, autoimmune diseases, neonatal necrotizing enterocolitis, etc., with inflammatory bowel disease being preferred.

[0129] Such a pharmaceutical composition of the present invention may contain an effective amount of an antibody or an antigen-binding fragment thereof that binds to C. difficile bacteria of the present invention, and can be appropriately set, for example, so that the content of the antibody of the present invention 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 subjects to be treated, the severity of symptoms in the subjects to be treated, and the degree of effect exerted by administration.

[0130] The term "effective amount" as used herein refers to an amount in which the antibody or antigen-binding fragment thereof that binds to C. difficile bacteria according to the present invention can exert a therapeutic effect against intestinal diseases and the like.

[0131] The pharmaceutical composition of the present invention may contain a pharma- ceutically acceptable carrier or additive together with the antibody that binds to C. difficile bacteria of the present invention. The pharma- ceutically acceptable carrier or additive means any carrier, diluent, excipient, suspending agent, lubricant, adjuvant, vehicle, delivery system, emulsifier, tablet disintegrant, absorbent, preservative, surfactant, colorant, flavor, or sweetener, and any known carrier or additive may be used.

[0132] The pharmaceutical composition according to the present invention can be used in a method for treating an intestinal disease, which includes a step of administering the composition to an individual suffering from an intestinal disease associated with the above-mentioned C. difficile bacteria. It can also be used in a method for preventing an intestinal disease, which includes a step of administering the composition to an individual who does not develop the pathology or symptoms of the above-mentioned intestinal disease but may have a predisposition to the intestinal disease. These individuals may be the subjects to which the pharmaceutical composition according to the present invention is administered.

[0133] Such subjects of administration include, but are not limited to, mammals such as humans, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cows, pigs, and birds such as chickens.

[0134] The dosage and administration method of the pharmaceutical composition vary 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.

[0135] The administration method is not particularly limited, but is preferably administered directly into the digestive tract, and examples of such administration methods include oral administration, nasal administration, transmucosal administration, and enteral administration.

[0136] 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.

[0137] The pharmaceutical composition of the present invention may be administered in the above amount once a day or in several divided doses. 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.

[0138] (Oral or enteral compositions) The oral or enteral compositions of the present invention include an antibody or antigen-binding fragment thereof that binds to the C. difficile bacteria of the present invention, and can be used to treat diseases associated with C. difficile bacteria.

[0139] The proportion of the antibody that binds to the above-mentioned C. difficile bacteria in such an oral or enteral composition is not particularly limited and may be appropriately adjusted depending on the form, use, etc. 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.

[0140] The individual to which the oral or enteral composition of the present invention is applied is not particularly limited, and examples thereof include mammals such as humans, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cows, and pigs, and birds such as chickens.

[0141] The antibody that binds to C. difficile bacteria contained in the oral or enteral composition of the present invention is particularly useful as a composition for regulating the intestines, a composition for improving the intestinal environment, a composition for optimizing the intestinal environment, or a composition for preventing intestinal putrefaction.

[0142] The amount of the oral or enteral composition according to the present invention to be 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, etc. Specifically, the amount may be about 0.001 to 100 mg / kg / day in terms of the amount of the antibody that binds to the C. difficile bacterial body according to the present invention, and this may be taken once or in divided doses per day.

[0143] In addition, enteral administration is not limited to administration via the anus. For example, the enteral composition of the present invention can be used as an intestinal cleansing liquid by mixing it with known ingredients.

[0144] The oral or enteral composition of the present invention contains an antibody that binds to the C. difficile bacteria of the present invention, which exerts the effect of suppressing abnormal proliferation of intestinal bacteria and / or pathological changes in the intestinal bacterial flora, and can be suitably used in the fields of food and feed, expecting to exert such an effect. Therefore, the oral or enteral composition of the present invention can be a food composition or a feed composition.

[0145] The above-mentioned food composition is a composition that is suitable for exclusively using the oral or enteral composition of the present invention in the field of food. Such a food composition can be provided as a food composition labeled for intestinal regulation, intestinal environment improvement, intestinal environment optimization, intestinal putrefaction prevention, etc.

[0146] Examples of 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, foods for the sick, and the like.

[0147] The specific form of the above-mentioned food composition is not particularly limited, but examples thereof 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, cereals, etc. In addition, in the case of foods for specified health uses, nutritional supplements, and functional foods, the forms include powders, granules, capsules, lozenges, tablets, and syrups.

[0148] The above-mentioned feed composition is a composition in which the feed composition of the present invention is used exclusively in the field of feed. Such a feed composition can be provided as a food composition indicated for intestinal regulation, intestinal environment improvement, intestinal environment optimization, intestinal putrefaction prevention, etc.

[0149] The specific form of the above-mentioned feed composition is not particularly limited. For example, as long as the effect of the above-mentioned feed composition according to the present invention is not impaired, the feed composition may be prepared by mixing it with normal feed or, if necessary, by mixing it with other components that can be incorporated into normal feed. Alternatively, the feed composition itself may be used as feed.

[0150] (Method of Treating Disease) The method for treating a disease according to the present invention is a method for treating an intestinal disease associated with C. difficile bacteria, and comprises the step of administering an effective amount of an antibody or antigen-binding fragment thereof that binds to the C. difficile bacterial body according to the present invention, a pharmaceutical composition according to the present invention, or an oral composition according to the present invention to a human suffering from an intestinal disease associated with C. difficile bacteria.

[0151] The human suffering from the intestinal disease associated with the above-mentioned C. difficile bacteria may be the administration target described above in [Pharmaceutical composition according to the present invention]. The specific administration method and dosage may also be the same as those described above in [Pharmaceutical composition according to the present invention]. EXAMPLES

[0152] The present invention will be described in more detail below with reference to examples, but these are merely illustrative and do not limit the present disclosure.

[0153] Methods and Materials 1. Mouse We used 8- to 25-week-old C57BL / 6 or BALB / c mice, which were raised under specific pathogen-free conditions, transplanted with human intestinal flora, or infected with specific pathogens.

[0154] Example 1: Screening for antibodies that bind to bacteria (1-1) Isolation of small intestinal and large intestinal lamina propria cells After euthanasia, the 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. After shaking for 20 minutes at 37°C, 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 again collected in a strainer and the PBS was discarded. This process was repeated twice to remove only the small intestinal epithelial cells from the small intestinal tissue. Next, 50 ml of digestive enzyme solution (adjusted with 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 dispase (50 U / ml) (final concentration 0.5 U / ml)) warmed to 37 ° C. was added to a 100 ml beaker, and the small intestinal tissue was further cut into small pieces and added, and shaken at 37 ° C. for 30 minutes. After that, the 100 ml beaker was left to stand to allow the small intestinal tissue to sink, and the supernatant was transferred to a new 50 ml tube. This digestion process using the digestive enzyme solution was repeated once for 20 minutes. 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 on the supernatant from the second digestion step, and the first and second cell suspensions were combined and passed through a filter to remove tissue debris, yielding small intestinal (large intestinal) mucosal lamina propria cells.

[0155] (1-2) Creation and cloning of antibody-producing hybridomas Hybridomas were produced by fusing small intestinal (large intestinal) 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 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. Then, frozen cell stocks were prepared from the cloned hybridomas, and the culture supernatant was obtained at the same time. The antibody isotypes of the antibodies contained in the supernatant were confirmed by the conventional sandwich ELISA method, and the antibody titers of the antibodies contained in the supernatant were measured and separated as hybridomas producing antibodies of each isotype. 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 μg / ml for plate coating, alkaline phosphatase (ALP)-conjugated anti-goat mouse IgA (Southern Biotech), alkaline phosphatase (ALP)-conjugated anti-goat mouse IgG (Southern Biotech), or alkaline phosphatase (ALP)-conjugated anti-goat mouse IgM (Southern Biotech) at 0.5 μg / ml for detection, and mouse IgAκ (Immunology Consultants Laboratory), purified mouse IgG1κ isotype control (BD Pharmingen), and PE-CF594 mouse IgMκ isotype control (BD Horizon) as control antibodies. Measurements were performed using TriStar2 Using LB942 (BERTHOLD TECHNOLOGIES), OD 405 By the above procedures, a total of 500 or more hybridoma clones were obtained.

[0156] (1-3) Analysis of the binding strength of hybridoma IgA antibodies to each enterobacteria To screen for antibodies that bind to each enterobacteria, ELISA was performed for each bacterium. In particular, the screening method performed for Clostridium difficile (C. difficile) bacteria is described below. C. difficile was cultured anaerobically at 37°C (Brain Heart Infusion media, 80% N2, 10% H2, 10% CO2) and collected by centrifugation. The bacteria were suspended in 0.05M Na2CO3 buffer and coated onto an ELISA plate. After blocking with 1% BSA-added PBS, each antibody culture supernatant in a 96-well plate was added and reacted at room temperature for about 1 hour. The plate was then washed with 0.05% Tween 20-added PBS, and the secondary detection antibody (mentioned above) corresponding to each antibody isotype was added and reacted, after which a color reaction was performed using Alkali Phosphatase tablets (Sigma). To ensure sufficient reaction, the ELISA plate after color development was reacted at 4°C overnight and then incubated with TriStar 2 Using LB942 (BERTHOLD TECHNOLOGIES), OD 405 nm was measured. OD 405 Clones showing nm values ​​of 2.0 or higher were determined to be antibodies that bind to C. difficile bacteria.

[0157] The selected clones were cultured in an expanded manner (about 100 mL culture), 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, and the antibody was sterilized using a syringe filter (0.22 μm) 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.

[0158] (1-4) Cloning of full-length antibody genes from hybridomas 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 the V H RT-PCR was performed using seven primers (MH1-7) specific to the Cα region, Cγ region, or Cγ region-specific primers. For the light chain, PCR was performed using Vκ primers and Cκ primers. The base sequences of the primers are shown in Table 1. H The Vkappa region PCR products or Vkappa region PCR products were directly sequenced to obtain the variable region gene sequences of each clone. H Alternatively, a sequence further upstream of the Vκ gene (including the signal sequence) was obtained, and a PCR primer was prepared based on the most upstream sequence, and PCR was performed with a reverse primer at the 3' end of each C region secretory sequence to obtain the full-length gene base sequences of the H chain and L chain. Based on this, the H chain, L chain, and J chain were cloned into the pcDNA 3.1(+) vector (Invitrogen) (the base sequence was searched from a database and the full-length gene sequence was obtained by PCR using the cDNA of the hybridoma). TIFF2024041901000036.tif75152Table 1. Nucleotide sequences of primers for amplifying the variable region of the antibody gene of hybridoma-derived antibodies 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, and V represents G, C or A. The above expression vector was transfected into Expi CHOS cells (Thermo Fischer Scientific) at a ratio of H:L:J = 2:2:1 according to the kit protocol (Thermo Fischer Scientific, ExpiCHO Expression System). The culture supernatant was collected 10-12 days after transfection, and the antibody was purified and concentrated using a Protein L column as described above, dialyzed against PBS, sterilized by filter, and used for antibody titer measurement and activity testing.

[0159] Example 2: Bacterial growth inhibition test Materials and Methods Under the same conditions as in Example 1, C. difficile bacteria were cultured overnight at 37°C anaerobically. The culture solution was centrifuged to collect the bacteria, which were then washed twice with culture solution. The bacterial solution was diluted 10-fold with culture solution, and SYTO24 (Invitrogen) and Counting beads included in the Cell Viability Kit (Becton Dickinson) were added, and the number of live bacteria positive for SYTO24 was calculated by comparing with the Counting beads by flow cytometry. The bacteria were diluted to 10,000 cells / 5μl with culture solution. 5μl of the bacterial dilution solution was added to an Eppendorf tube, and 25μl (antibody concentration 1mg / ml) of PBS (anaerobic) or purified test antibody (anaerobic) was added, and the mixture was cultured at 37°C for 1 hour. Then, 30μl of culture solution was added, and the mixture was cultured at 37°C for 20 hours (final antibody concentration 0.42mg / ml). After 20 hours of incubation, SYTO24 and counting beads were added and the number of viable bacteria was measured by flow cytometry in the same manner as above. Antibodies that reduced the number of viable bacteria by 30% or less compared to the PBS sample (no antibody added) were determined to have an inhibitory effect on proliferation and were used in the next mouse infection experiment.

[0160] Seven antibodies, SNK0001MR, SNK0002MR, SNK0001AR, SMK0002AR, RS_H007_L004, RS_H000_L001, and SNK0003A, were used in the proliferation inhibition test. Antibodies SNK0001MR and SNK0002R are recombinant IgM antibodies produced by introducing expression vectors (H chain and L chain) obtained based on antibody gene sequence information extracted from IgM-producing hybridomas (SNK0001M and SNK0002M) into CHO cells. SNK0003A is an IgA antibody derived from an IgA-producing hybridoma. SNK0001AR and SNK0002AR are SNK000 An expression vector was prepared by combining the gene sequence of the antibody gene variable region derived from 1M and SNK0002M hybridomas with the gene sequence of the IgA antibody constant region, and a J chain expression vector was also constructed, and the three types of expression vectors (H chain, L chain, J chain) were introduced into CHO cells to produce recombinant multimeric IgA antibodies. RS_H007_L004 and RS_H00_L001 are recombinant multimeric IgA antibodies obtained by synthesizing genes based on the gene sequence of the W27 antibody produced by a W27-producing hybridoma obtained from intestinal mucosal lamina propria cells, as reported by the inventor in WO2014 / 142084 and the like, and applying recombinant technology. All of the recombinant antibodies were purified using a Protein L column by the above-mentioned method. These antibodies have been confirmed to bind to C. difficile by a test similar to that of Example 1.

[0161] (result) The results are shown in Figure 1. All of the antibodies tested showed a significant ability to inhibit the proliferation of C. difficile compared to the negative control (PBS).

[0162] Example 3: C. difficile mouse infection experiment Materials and Methods A spore suspension of C. difficile (VPI10483) was prepared and stored at -80°C in small aliquots. The spore suspension was thawed, cultured, and then inoculated onto a C. difficile selective medium plate (TCCFA plate) to determine the number of viable bacteria. The method for producing spores is as follows. C. difficile was plated on SMC medium and cultured anaerobically at 37°C for 7 days. 3 ml of ice-cold sterilized water was added to the plate, and colonies were scraped off with a cell scraper and transferred to a 50 ml tube. The plate was washed with 3 ml of ice-cold sterilized water, and the washing solution was transferred to the same tube. After centrifugation at 8,000 g and 4°C for 10 minutes, the supernatant was discarded and the cells were suspended in 20 ml of ice-cold sterilized water. After centrifugation at 8,000 g and 4°C for 10 minutes again, the supernatant was discarded and the cells were suspended in 10 ml of ice-cold sterilized water. After suspension, 10 ml of ethanol was added, the cells were stirred with a vortex, and the cells were left to stand at room temperature for 1 hour. After centrifugation at 8,000 g and 4°C for 10 minutes, the supernatant was discarded and the cells were suspended in 20 ml of ice-cold sterilized water. This operation was repeated twice, the supernatant was discarded, and the cells were suspended in 5 ml of ice-cold sterilized water. 100 μl of the medium was dispensed into 1.5 ml tubes and stored at −80° C. The composition of the SMC medium is shown below. SMC medium composition The above reagents were dissolved in 200 ml of sterile distilled water and sterilized by autoclaving. After cooling to about 60°C, 600 μl of 10% (w / v) L-cysteine ​​was added and the mixture was dispensed into 10 cm plates.

[0163] C. difficile selective medium plates were prepared by dissolving the reagents in Table 1 below in 400 ml of sterile distilled water and sterilizing them in an autoclave. The mixture was allowed to cool naturally to 60°C, and 2 ml of Cycloserine (Sigma-Aldrich) (50 mg / ml) and 2 ml of Cefoxitin (Sigma-Aldrich) (1.6 mg / ml) were added, and appropriate amounts were dispensed into 10 cm plates.

[0164] (Table 1) Composition of C. difficile selective medium TIFF2024041901000038.tif90153

[0165] C57BL / 6 mice (8 weeks old) were purchased from CLEA Japan and after acclimation for one week in a sterile isolator for infection experiments, a triple antibiotic mixture (gentamicin: 500 mg / kg, kanamycin: 150 mg / kg, metronidazole: 50 mg / kg) was orally administered for four days using a probe. After four days, the weight of each mouse was measured, and mice that had lost weight due to antibiotic administration were excluded, and the remaining mice were randomly divided into groups. On the day after the end of antibiotic administration, C. difficile spores (1X10 3 Mice were orally infected with C. difficile (cfu) using a probe. Six hours later, 100 μg of the test antibody was orally administered using a probe. Thereafter, 100 μg of the antibody was administered once a day for a total of 7 days. From the 8th day onwards, the mice were observed without antibody administration. During this period, the mice were weighed, the characteristics of the stool were observed, and stool samples were collected. The stool samples collected on the 4th and 10th days after infection were suspended in an amount of PBS according to the weight, and then further serially diluted with PBS. These diluted bacterial solutions were plated on C. difficile selective medium plates and cultured anaerobically to determine the number of viable bacteria per gram of stool.

[0166] (result) In mice administered W27G2 or SNK0002M antibody, weight loss after administration of C. difficile spore liquid was suppressed compared to the negative control (PBS-administered group) (Figure 2). Furthermore, a sufficient number of live C. difficile bacteria was observed in the feces on day 4, confirming that infection had been established in each mouse. The number of live bacteria in the feces on day 10 was dramatically reduced in the groups of mice administered the W27G2 or SNK0002M antibody, confirming that these antibodies significantly suppressed bacterial proliferation in vivo (Fig. 3).

[0167] Example 4: Effect on bacteria other than C. difficile The antibodies that bind to C. difficile bacteria were examined for their binding characteristics to other bacteria and their effects on the bacteria, using the same method as the ELISA used in Example 1. As an example, the results of examining the binding characteristics and effects on Fusobacterium nucleatum, which is thought to be a causative bacterium of colon cancer, are presented. After anaerobically culturing the bacteria, the number of bacteria was adjusted to 1,000 cells / tube using flow cytometry, and the bacteria were reacted with each test antibody to verify the growth inhibitory effect of the antibodies. The final concentration of the antibodies added and the incubation time were the same as in Example 2. After the antibody reacted with the bacteria, a culture medium was added and the number of live bacteria was measured after further incubation for 20 hours.

[0168] (result) The antibodies SNK0001M, SNK0001MR, SNK0001AR, SNK0002MR, SNK0002AR, RS_H000_L001, and SNK0003A exhibited a significant growth inhibitory effect on Fusobacterium nucleatum (Fig. 4), whereas SNK0004A and SNK0005A did not exhibit a growth inhibitory effect (negative control).

[0169] Example 5: Identification of C. difficile antigen molecules of W27 antibody It has been confirmed that the W27 antibody binds to serine hydroxymethyl transferase (SHMT) of Escherichia coli (WO2014 / 142084, etc.), and the following method was used to confirm that the W27 antibody binds to SHMT of Escherichia coli and to identify the C. difficile antigen molecule of the W27 antibody. The W27G2 antibody has the same heavy chain variable region and light chain variable region as the W27 antibody. Materials and Methods E. coli was cultured in 25 ml of LB medium for 16 hours. The bacteria were collected by centrifugation at 2,150 g for 5 minutes. After removing the supernatant, the bacteria were suspended in 1 ml of lysis buffer (sterile 1x PBS, 10% NP-40, ×100 Protease Inhibitor Cocktail (Nacalai)), sonicated, and left to stand on ice for 30 minutes. The bacterial lysate was dispensed in 200 μl portions into new tubes, and denatured (95°C, 10 min) with 50 μl of 2-ME and 200 μl of 10% SDS. 800 μl of diluent (sterile 1x PBS, 0.1% NP-40, ×100 Protease Inhibitor Cocktail) was added to dilute 2-ME and SDS. The protein solution was centrifuged at 15,000 rpm for 5 minutes to separate the soluble and insoluble fractions. The soluble fraction was used to perform 2D-PAGE to identify the target protein spots. A bacterial lysate was prepared for C. difficile in a similar manner, and the soluble fraction was used to perform 2D-PAGE.

[0170] The 2D-PAGE method is described below. First, to prepare a sample for 2D-PAGE, twice the amount of cooled acetone was added to the immunoprecipitation product, and the product was left to stand at -20°C for 20 minutes. The supernatant was removed by centrifugation (16,630g, 10 min) and the product was dried. The product was dissolved in a sample buffer for first-dimension electrophoresis (60 mM Tris-HCl (pH 8.8), 5 M urea, 1 M thiourea, 5 mM EDTA, 1% CHAPS, 1% NP-40), and 1 / 10 the amount of 1 M iodoacetamide was added and the product was left to stand at room temperature for 10 minutes to prepare a sample for the first dimension of 2D-PAGE. For the second-dimension electrophoresis, two SDS-PAGE gels were used for Western blotting and silver staining. Immobiline buffer (pH 3-8) was used. TM After the first-dimension electrophoresis using DryStrip (GE Healthcare), the second-dimension SDS-PAGE was performed, followed by Western blotting and silver staining for each gel. To make it easier to identify the spots recognized by the W27 antibody, the membrane after blotting was transferred to Pierce TMThe membrane was stained with Reversible Protein Stain to confirm the location of all proteins on the membrane. After removing the stain with Stain Eraser, the spots were confirmed by Western blot analysis using W27 antibody. Silver staining was performed using Silvest Stain One (Nacalai) according to the protocol. TM The results of staining with reversible protein stain (silver stain) were compared, and the spots specifically recognized by the W27 antibody were excised and subjected to in-gel enzymatic digestion.

[0171] The method of in-gel enzyme digestion is described below. 400 μl of MilliQ water was added to the excised gel pieces and shaken for 10 minutes, after which the supernatant was removed. This procedure was repeated twice, after which 200 μl of 100% acetonitrile was added and shaken for 10 minutes. After removing the supernatant, the gel pieces were dried under reduced pressure for 20 minutes, and 20 μl of protease solution (50 mM ammonium hydrogen carbonate, 10 μl / ml trypsin) was added and allowed to swell on ice for 30 minutes to allow the gel pieces to absorb trypsin. Excess protease solution was removed, and 100 μl of reaction solution (50 mM ammonium hydrogen carbonate) was added, and enzyme digestion was carried out overnight at 37°C. An additional 50 μl of extraction solution (5% formic acid, 50% acetonitrile) was added to the reaction solution and shaken for 30 minutes. 200μl of 0.1% formic acid was added to the collected solution, and the sample was dried under reduced pressure until the sample volume was reduced to about half. The sample was transferred to a spin filter (UltraFree 0.1μm PVDF) and centrifuged (2,460g, 5min, 4℃). The solution that fell to the bottom was transferred to a vial and analyzed by LCMS-IT-TOF (Shimadzu). Mascot search was used to identify the amino acid sequence.

[0172] (result) Using the above method, we identified SHMT of E. coli and 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGM) of C. difficile as the antigen molecules of the W27 antibody. As an example, the 2D electrophoresis data when identifying the antigen molecule of C. difficile is shown in Figure 5.

[0173] Example 6: Epitope determination of the antigen molecule of W27 antibody The epitope of the W27 antibody has been identified in the N-terminal 25-30 amino acids of SHMT of E. coli (published paper). We attempted to produce a truncated protein of iPGM of C. difficile, but the efficiency of protein expression was significantly reduced, and we were unable to confirm the reaction of the W27 antibody by Western blot analysis. Therefore, we confirmed by Western blot that the W27 antibody does not recognize the iPGM of E. coli, and created a plasmid expressing the chimeric protein of the iPGM of C. difficile and the iPGM of E. coli, which are recognized by the W27 antibody, using the In-Fusion (registered trademark) HD ​​Cloning Kit (TaKaRa). Primers were created from the iPGM gene sequence information of each bacterium in the database (Table). The chimeric protein was overexpressed in DH5α, and recognition by the W27 antibody was confirmed by Western blot to narrow down the location of the epitope sequence.

[0174] Table 2: Primer sequences used for In-Fusion cloning TIFF2024041901000039.tif113153

[0175] As a result of the analysis, amino acids 490 to 510 at the C-terminus were found to be important epitopes in iPGM of C. difficile. To further confirm that they were epitopes, peptides (BSA conjugates) containing each epitope candidate were outsourced to SIGMA. Because the molecular weight of the peptides alone is small, each peptide was conjugated with BSA so that it could be confirmed by Western blotting. 4xSDS buffer was added to the peptides, and Western blotting was performed using W27 antibody to confirm which synthetic peptides they reacted with. The results are shown in Figures 6 and 7. As a result of the above test, it was found that the epitope sequence of E. coli SHMT was RQEEHIELIASEN, and the epitope sequence of C. difficile iPGM was APTVLDMMKLEKPEEMTGHSLISK.

[0176] A database analysis of bacteria that share the epitope of the SHMT of E. coli recognized by the W27 antibody revealed that most of the bacteria with the EEHI sequence belong to Proteabacteria, including many pathogenic bacteria (Figure 8). The amino acid sequences of the same part of SHMT in lactic acid bacteria and humans are different, and it is thought that the W27 antibody recognizes this difference. (Okai et al., Nature microbiology 1, 16103, 2016)

[0177] Example 7: Crystal structure analysis of W27 antibody and antigen molecule (7-1) Preparation and purification of IgG W27 Fab To investigate the antigen recognition of the W27 antibody, we produced a W27 recombinant IgG antibody expressed in CHO (Chinese Hamster Ovary) cells. This was because it was necessary to secure a large amount of antigen-binding sites (Fab) for crystal structure analysis. Crudely purified IgG W27 was adjusted to 1 mg / ml with 10 mM Tris-HCl (pH 8.0), and then digested with 0.05 mg / ml papain (Nacalai Tesque) at 20°C for 24 hours to digest the Fc and Fab regions. It was then purified by cation exchange column chromatography using a HiTrap SP HP column (Cytiva). Buffers used were 10 mM Bis-Tris buffer (pH 6.0) (buffer A) and 10 mM Bis-Tris buffer (pH 6.0), 300 mM NaCl (buffer B), and Fab and Fc were separated and purified by linear gradient elution with gradually increasing buffer B concentration. The fraction containing a large amount of Fab was further purified by gel filtration chromatography using a HiLoad 26 / 600 Superdex 2000 pg gel filtration column (Cytiva). The gel filtration buffer was 5 mM BisTris (pH 6.5), 100 mM NaCl (gel filtration buffer). After purification by gel filtration chromatography, trace amounts of Fc impurities were adsorbed and removed using a column packed with rProtein A Sepharose Fast Flow resin (Cytiva), and Fab in the flow-through fraction was recovered. The purified Fab was concentrated to 48.2 mg / ml using an Amicon Ultra 10000 MWCO (Millipore) under the conditions of gel filtration buffer, then dispensed into 20 μl aliquots in Eppendorf tubes, flash frozen in liquid nitrogen, and stored at -80°C until use. The yield was 17.4 mg of Fab from 50 mg of IgG W27.

[0178] (7-2) Expression and purification of E. coli SHMT(25-45) for crystal structure analysis The cDNA region encoding E. coli-derived SHMT(25-45) was amplified using PrimeSTAR Max DNA Polymerase (TaKaRa), and then the expression plasmid was constructed by incorporating it into the Sma I and NotI multicloning sites of the plasmid pGEXM (Kim SY, et al. (2021) Sci Rep 11(1):2120), an improved version of the plasmid pGEX6P-3 (Cytiva), using the In-Fusion HD Cloning Kit (Clontech). The expression plasmid was transformed into E. coli Rosetta2 strain (Merck). SHMT(25-45) was expressed as a Gultathione-S-transferase (GST) fusion protein (hereinafter referred to as GST-SHMT(25-45)). In addition, the cleavage sequence LEVLFQGP (the cleavage site is between Q and G) by human rhinovirus 3C protease (HRV3C protease) is inserted between GST and the target protein, so that the expressed GST fusion protein can be cleaved between GST and the target protein by HRV3C protease. The amino acid sequence of the final purified product, E. coli SHMT(25-45), is GPRQEEHIELIASENYTSPRVMQ.

[0179] E. coli was cultured at 37°C in LB medium containing 50 μg / ml ampicillin, 25 μg / ml chloramphenicol, 0.5% (W / V) glycerol, 0.05% (W / V) glucose, 0.2% (W / V) lactose, and 1 mM magnesium sulfate. When the turbidity of the medium reached 0.6 (measured at a wavelength of 600 nm), the medium was cooled and 1 M isopropyl-β-D-thio-galactopyranoside (IPTG) was added to a final concentration of 0.1 mM to induce expression of the target protein. After addition of IPTG, the medium was cultured for another 24 hours at 18°C. The cells in the culture were collected by centrifugation at 4,000 rpm (Beckman J2-M1 JA10 rotor) at 4°C for 20 minutes. The collected cells were stored at -30°C until use in purification.

[0180] The purification of SHMT was carried out as follows. The cells were suspended in 10 mM Tris-HCl (pH 8.0), 300 mM NaCl, and then ultrasonically disrupted at 4°C. After centrifugation at 20,000 rpm at 4°C for 40 minutes, the insoluble fraction was removed, and the soluble fraction of the supernatant was used for the next purification step. The purification of GST-SHMT(25-45) from the soluble fraction was carried out using an affinity column containing Glutathione-Sepharose 4B resin (hereafter, GS4B) (Cytiva). The soluble fraction after disruption was applied to an affinity column filled with GS4B to adsorb GST-SHMT(25-45), and the GS4B was thoroughly washed with 10 mM Tris-HCl (pH 8.0), 300 mM NaCl (hereafter, washing buffer). GST-SHMT(25-45) was then eluted from the resin with 10 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 20 mM Glutathione. The eluate was digested with HRV3C protease (Merck) at 4°C for 18 hours to cleave it into GST and SHMT(25-45), and GST was removed. This SHMT(25-45) has an extra GP sequence that contains a part of the recognition sequence of HRV 3C protease at the N-terminus. SHMT(25-45) after GST cleavage was purified by gel filtration using a HiLoad 26 / 600 Superdex 75pg gel filtration column (Cytiva) under conditions of 5 mM BisTris (pH 6.5), 100 mM NaCl. The purified SHMT(25-45) was concentrated to 4.72mg / ml using a PALL Corporation centrifugal Device with 1K under the conditions of 5mM BisTris (pH6.5), 100mM NaCl (gel filtration buffer), then dispensed into 20μl aliquots in Eppendorf tubes, flash frozen in liquid nitrogen, and stored at -80℃ until use. The yield of SHMT(25-45) peptide used for crystallization was 5.9mg from the 2L culture.

[0181] (7-3) Expression and purification of C. difficile iPGM(486-509) for crystal structure analysis Cloning of the cDNA of Clostridium difficile-derived iPGM(486-509) (C. difficile iPGM(486-509)) into a pGEX vector was performed in a manner similar to that used for SHMT. In this case, since C. difficile iPGM(486-509) does not contain tyrosine or tryptophan and therefore no absorbance at 280 nm could be observed during the purification process, one tyrosine residue was added to the N-terminus and cloned. This iPGM(486-509) has an extra GPY sequence, which contains a portion of the recognition sequence for HRV 3C protease, added to the N-terminus. The amino acid sequence of the final purified product, C. difficile iPGM(486-509), is GPYAPTVLDMMKLEKPEEMTG HSLISK. The procedures for transformation, cultivation, and purification were carried out in the same manner as for SHMT(25-45), and 48.52 mg of iPGM(486-509) for crystallization was obtained from 4 L of cultured cells.

[0182] (7-4)RS_H000_L000GR Crystallization screening of Fab-E. coli SHMT(25-45) complex A mixture was prepared using the purified RS_H000_L000GR Fab and E. coli SHMT(25-45) at a molar ratio of 1:5 (0.2 mM:1.0 mM) and crystallization screening was performed. Screening of crystallization conditions was performed using commercially available crystallization screening kits, JCSG core suite I-IV and PACT suite (Qiagen), and the crystallization robot mosquito (TTP Labtech). A crystallization plate (VIOLAMO) was filled with precipitant for equilibration, and droplets were created by mixing the precipitant and protein solution at a volume ratio of 1:1 (0.2 μl:0.2 μl), and crystallization conditions were explored using the sitting drop vapor diffusion method. The protein solution used in the crystallization screening had previously been centrifuged to remove insoluble fractions. The incubation temperature conditions were 4°C and 20°C.

[0183] (7-5)RS_H000_L000GR Optimization of crystallization conditions for Fab-E. coli SHMT(25-45) complex Precipitants with different pH and PEG concentrations were prepared based on the crystallization conditions obtained by crystallization screening, and the sitting drop vapor diffusion method was performed. The crystallization conditions were 0.2M magnesium acetate, 0.1M sodium cacodylate pH 6.3, and 22% PEG10000 as the reservoir solution. The protein solution mixed with RS_H000_L000GR Fab: 0.2mM and E. coli SHMT(25-45): 0.6mM was mixed with the reservoir at a ratio of 0.2μl:0.2μl, and the temperature condition was 20℃, and crystallization was performed by sitting drop vapor diffusion method, and observation was performed after 7 days. The scale bar indicates 100μm (Figure 9).

[0184] (7-6)RS_H000_L000GR X-ray diffraction experiment and three-dimensional structure determination of Fab-E. coli SHMT(25-45) complex The RS_H000_L000GR Fab-E. coli SHMT(25-45) complex crystal obtained by optimizing the crystallization conditions was frozen in liquid nitrogen using 0.2M magnesium acetate, 0.1M sodium cacodylate pH6.3, 22.0% PEG10000, and 20.0% glycerol as cryoprotectants. The crystals were then measured at BL44XU of the large-scale synchrotron radiation facility SPring-8 in Hyogo Prefecture. After processing the acquired diffraction data with the XDS program, the phases were determined by molecular replacement using the structural information of RS_H000_L000GR Fab. The space group was P212121, the lattice constants were a = 60.4 Å, b = 140.7 Å, c = 180.7 Å, α = 90°, β = 90°, γ = 90°, and there were three molecules of RS_H000_L000GR Fab-E. coli SHMT(25-45) in the asymmetric unit. The structure was refined using a combination of the programs refmac 5, phenix refine, and coot. work / R freeThe refinement was completed at 22.8% and 25.7%, respectively. The structure diagram was created using the program PyMOL.

[0185] (7-7)RS_H000_L000GR Optimization of crystallization conditions for Fab-C. difficile iPGM(486-509) complex Crystallization of IgG W27 Fab C. difficile iPGM(486-509) was performed by the sitting drop vapor diffusion method using precipitants with different pH and PEG10000 concentrations based on the crystallization conditions for the structurally analyzed IgG W27 Fab-E. coli SHMT(25-45) (0.2M magnesium acetate, 0.1M sodium cacodylate pH 6.3, 22% PEG10000). The crystallization conditions were 0.2 M magnesium acetate, 0.1 M sodium cacodylate pH 6.5, and 23% PEG10000 as the reservoir solution. The mixing ratio of the protein solution and the reservoir was 0.2 μl:0.2 μl, with RS_H000_L000GR Fab: 0.25 mM and C. difficile iPGM (486-509): 1.0 mM, and the temperature condition was 20°C. The scale bar is 100 μm. Crystallization was performed by the sitting drop vapor diffusion method, and the observation was performed after 10 days (Figure 10).

[0186] (7-8)RS_H000_L000GR X-ray diffraction experiment and three-dimensional structure determination of Fab-C. difficile iPGM(486-509) complex The RS_H000_L000GR Fab-C. difficile iPGM(486-509) complex crystal obtained by optimizing the crystallization conditions was frozen in liquid nitrogen using 0.2M magnesium acetate, 0.1M sodium cacodylate pH6.5, 23.0% PEG10000, and 20.0% glycerol as cryoprotectants. It was then measured at BL41XU of the large-scale synchrotron radiation facility SPring-8 in Hyogo Prefecture. After processing the acquired diffraction data with the XDS program, the phase was determined by molecular replacement method using the structural information of IgG W27 Fab. The space group was P212121, the lattice constants were a = 60.423 Å, b = 140.185 Å, c = 185.956 Å, α = 90°, β = 90°, γ = 90°, and there were three molecules of RS_H000_L000GR Fab-C. difficile iPGM(486-509) in the asymmetric unit. The structure was refined using a combination of the programs refmac 5, phenix refine, and coot. work / R free The refinement was completed at 22.2% and 25.2%, respectively. The structure diagram was created using the program PyMOL.

[0187] As a result of the above series of experiments, the details of the interaction between the epitope peptide and the RS_H000_L000GR antibody were revealed (Figures 11 and 12). Specifically, the epitope peptides of SHMT and iPGM bound in opposite directions to each other, and the amino acid sequences of the epitopes showed the following similarities: TIFF2024041901000040.tif9140 Based on this information, it became possible to determine not only the type of amino acid residue but also the effect of the amino acid side chain on the interaction. Based on this information obtained from the structural analysis, amino acid mutations that do not affect antigen binding were selected to create a series of RS mutant antibodies, and it was revealed that all of them retained activity equivalent to that of the W27G2 antibody, as described in detail below. Therefore, using the structural analysis results of the present invention, it became possible to create various RS mutant recombinant antibodies that retain activity equivalent to that of the W27G2 antibody.

[0188] Example 8: Preparation of genetically modified recombinant antibodies To create RS mutants, PCR was performed using the H-chain expression vector and L-chain vector as templates, with mutagenesis primers containing the base sequences corresponding to each mutation, and the full length of the vector was amplified. The template vector DNA was then treated with DpnI, the DNA was purified by isopropanol precipitation, and transformed into DH5α competent cells. Plasmids were extracted from the resulting clones, clones with the desired mutations were selected, and the clones were transfected into ExpiCHO to obtain mutant recombinant antibodies. The amino acid sequences of the heavy and light chain variable regions of some of the mutant recombinant antibodies produced are shown in Figures 13 and 14. Non-reducing SDS-PAGE and Coomassie blue staining of each of the RS mutant recombinant purified antibodies confirmed that dimers were mainly produced (FIG. 15).

[0189] Example 9: Antigen recognition activity of recombinant RS modified antibodies The binding ability of the recombinant RS modified antibodies to Escherichia coli serine hydroxymethyltransferase (SHMT), which is the antigen molecule of the W27G2 antibody, was measured by ELISA. Materials and Methods The wild-type SHMT and SHMT mutant of E. coli were overexpressed in E. coli as GST fusion proteins and purified. They were suspended in 0.05 M Na2CO3 buffer at 2 μg / ml and immobilized on an ELISA plate. A dilution series of purified antibodies of each mutant type was prepared and added, and the binding ability to the bacteria was detected by the same method as the ELISA that measures the binding ability to the bacteria described above. (result) As representative examples, six types of purified mutant antibodies (RS_H00_L001, RS_H007_L001, RS_H007_L002, RS_H00_L005, RS_H007_L005 and RS_H007_L004) were tested, and it was confirmed that these antibodies bind to E. coli wild-type SHMT to almost the same extent as the hybridoma-derived W27G2 antibody (gel filtration purified), and further that they do not bind to SHMT mutant (Figure 16).

[0190] The W27G2 antibody is known to recognize different antigen molecules of multiple bacteria. As shown in the Western analysis in Figure 17, total proteins were extracted from multiple bacteria (including pathogenic bacteria) and transferred to a nitrocellulose membrane after reducing SDS-PAGE. As primary antibodies, W27G2CHT (W27G2 hybridoma culture supernatant crudely purified with a hydroxyapatite column), W27G2GF (W27G2CHT further purified into a multimer fraction with a gel filtration column), and three recombinant purified antibodies (RS_H000_L001, RS_H000_L005, RS_H007_L005) were reacted at a concentration of 2 μg / ml each. The secondary antibody was Goat anti-mouse IgA (Southern Biotech), and the tertiary antibody was IR800-conjugated anti-goat IgG (LI-COR), and the signals were detected with an Odyssey scanner. To measure the protein amount of the sample loaded in each well, the gel after SDS-PAGE electrophoresis was stained with Coomassie brilliant blue (Nacalai). The hybridoma-derived W27G2 antibody and the recombinant purified antibody recognized the same pattern of antigen molecules, confirming that there was no change in antigen recognition after the introduction of mutations.

[0191] Example 10: Confirmation of the E. coli growth inhibitory effect of recombinant genetically modified antibodies E. coli BW38029 strain was cultured in LB medium for 16 hours at 37°C under static anaerobism. The bacteria were collected by centrifugation at 8,000g for 5 minutes. After washing with sterile anaerobic LB, the viable cell count was measured using a flow cytometer. Based on the above measurement results, the bacteria were diluted with LB medium to 300 bacteria / 5 μl, 25 μl of each antibody or PBS was added, and 30 μl of M9 minimal medium was added. After 20 hours, the number of bacteria was counted using the flow cytometer as described above. The final antibody concentration was 0.42 mg / ml.

[0192] (result) All of the various mutant recombinant antibodies inhibited the growth of E. coli compared to the negative control (PBS-added) sample (FIG. 18).

[0193] All of the above experiments demonstrated that the RS mutant recombinant antibody prepared based on the results of the above three-dimensional structure analysis retains properties similar to those of the original hybridoma W27G2 antibody.

[0194] Example 11: Effect of recombinant IgA antibody on C. difficile-infected mice In Example 3, the effect of the hybridoma-derived antibody on mice infected with C. difficile was examined, and a similar experiment to confirm the effect of the recombinant IgA antibody on mice infected with C. difficile was also carried out. As the recombinant IgA antibody, the antibody RS_H000_L001 (shown as rW27 in FIG. 19) and the antibody SNK0002AR were used. When the recombinant IgA antibody, the antibody RS_H000_L001 (rW27) and the antibody SNK0002AR were administered to mice infected with C. difficile, respectively, both of them significantly suppressed the weight loss of the mice infected with C. difficile (FIG. 19). Interestingly, mice administered Vancomycin instead of the antibody showed no weight loss during administration, but showed a significant weight loss after the end of administration, and the survival rate also decreased (FIG. 19). One possible reason for this decrease may be that the administration of Vancomycin, to which Gram-positive bacteria are sensitive, selectively increases Gram-negative bacteria in the intestinal tract, causing a state of dysbiosis, and that the remaining C. difficile bacteria proliferated after Vancomycin administration was discontinued, resulting in the above-mentioned pathological condition.

[0195] To confirm the growth of C. difficile bacteria, the diluted stool suspensions at each time point after administration were inoculated onto C. difficile selective medium, cultured anaerobically, and colonies were counted. As a result, as shown in Figure 20, as expected, C. difficile increased in the mice administered Vancomycin on Day 14 after administration was stopped. On the other hand, in the mice administered each of the two types of IgA antibodies, C. difficile was not detected in the stool on Day 14, confirming that C. difficile had been successfully eliminated.

[0196] Next, we used 16S rRNA analysis to examine whether dysbiosis actually occurred as a result of vancomycin administration. Feces were collected at the time of death for mice that died during the treatment. Feces were collected on day 14 for mice that survived and analyzed. In addition, rW27 IgG antibody was produced and administered to mice, and the bacterial flora was compared. The results are shown in Figures 21 and 22. Figure 21 shows the results of an analysis of the relative abundance ratio at order level. In this analysis, data obtained from the administration of recombinant IgG antibody rW27 IgG antibody (a recombinant IgG antibody in which the sequences of the variable regions of heavy chain H000 and light chain L001 are linked to the sequences of mouse IgG1 and mouse Igk (kappa)) as a control was also included in the analysis.

[0197] The biggest change was a significant increase in Enterobacterales in the rW27 IgG antibody and Vancomycin administration groups. As mentioned above, it is believed that the administration of Vancomycin increased the number of Gram-negative bacteria. In contrast, there was no significant change in the bacterial flora in the groups not administered with antibodies or administered with either of the two recombinant IgA antibodies, and the original dominant species, Lachnospirales, was maintained as the dominant flora. Figure 22 shows the results of this change in β-diversity. It can be seen that the Vancomycin and rW27IgG groups showed a clear change in the bacterial flora compared to the PBS, recombinant IgA antibody RS_H000_L001 (rW27), and SNK0002AR groups. Vancomycin, the first-choice drug for human C. difficile enteritis, is clearly effective in alleviating symptoms, but it may also cause dysbiosis, which may lead to repeated enteritis. Combination therapy with IgA antibodies and vancomycin is expected to become a curative treatment for C. difficile enteritis. [Industrial Applicability]

[0198] The present invention provides antibodies that bind to and inhibit the growth of C. difficile bacteria, and can therefore be used to treat diseases associated with C. difficile bacteria, as well as in tests for detecting C. difficile bacteria in biological samples, etc.

Claims

1. Binds to Clostridium difficile bacteria, a) 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:7; and an antibody or antigen-binding fragment thereof comprising 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, the amino acid sequence of which is represented by SEQ ID NO:8; b) 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: 17; and an antibody or antigen-binding fragment thereof comprising 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:18; c) 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:27; and An antibody or antigen-binding fragment thereof comprising 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:28; or d) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 37, and For a reference antibody comprising a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 38, having at least one amino acid mutation in at least one region selected from heavy chain CDR1-3, light chain CDR1-3, and light chain FR1; An antibody or antigen-binding fragment thereof that binds to the amino acid sequence RQEEHIELIAS in the E. coli SHMT protein and the amino acid sequence VLDMMMKLEKPE in the iPGM protein of C. difficile.

2. A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:1; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:2; and Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:3 and a heavy chain variable region comprising A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:4; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:5; and Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:6 a light chain variable region comprising: A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 11; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 12; and Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:13 and a heavy chain variable region comprising A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 14; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 15; and Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 16 or a light chain variable region comprising A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:21; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:22; and Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:23 and a heavy chain variable region comprising A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:24; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:25; and Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:26 A light chain variable region comprising The antibody or antigen-binding fragment thereof according to claim 1 .

3. A heavy chain variable region comprising a sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO:7; and The antibody or antigen-binding fragment thereof according to claim 1, comprising a light chain variable region comprising an sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO:

8.

4. A heavy chain variable region comprising a sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 17; and The antibody or antigen-binding fragment thereof according to claim 1, comprising a light chain variable region comprising an sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO:

18.

5. A heavy chain variable region comprising a sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO:27; and The antibody or antigen-binding fragment thereof according to claim 1, comprising a light chain variable region comprising an sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO:

28.

6. X １ A heavy chain CDR1 comprising the amino acid sequence of YYIH; RIDPENX ２ X ３ a heavy chain CDR2 comprising the amino acid sequence of TTYAPKFQ; A heavy chain CDR3 comprising the amino acid sequence of YCARSTTVL; and a heavy chain variable region comprising R.X. ４ A light chain CDR1 comprising the amino acid sequence of SQSIVHTNG; a light chain CDR2 comprising the amino acid sequence of a light chain CDR3 comprising the amino acid sequence of GVYYFQGS; and a light chain variable region comprising Light chain FR1 is The amino acid sequence of TPLSLPVSLGDQA or SPASX ５ SVSLGDRX ６ comprising the amino acid sequence X １ , X ２ , X ３ are each independently a neutral polar amino acid or an acidic polar amino acid, X ４ is a nonpolar amino acid or a neutral polar amino acid, X ５ , X ６ are each independently a non-polar amino acid, X １ is aspartic acid, X ２ is aspartic acid, X ３ is glutamic acid, and X ４ is alanine and the light chain FR1 comprises the sequence TPLSLPVSLGDQA.

7. X １ A heavy chain CDR1 comprising the amino acid sequence of YYIH; RIDPENX ２ X ３ a heavy chain CDR2 comprising the amino acid sequence of TTYAPKFQ; and A heavy chain CDR3 comprising the amino acid sequence of YCARSTTVL; and a heavy chain variable region comprising R.X. ４ A light chain CDR1 comprising the amino acid sequence of SQSIVHTNG; a light chain CDR2 comprising the amino acid sequence of KLLIYKV; and a light chain CDR3 comprising the amino acid sequence of GVYYFQGS; and a light chain variable region comprising Light chain FR1 The amino acid sequence of TPLSLPVSLGDQA or SPASX ５ SVSLGDRX ６ comprising the amino acid sequence X １ , X ２ are each independently asparagine or aspartic acid; X ３ is glutamine or glutamic acid, and X ４ is alanine or serine, X ５ is leucine or methionine, X ６ is alanine or valine, X １ is aspartic acid, X ２ is aspartic acid, X ３ is glutamic acid and the light chain FR1 comprises the sequence TPLSLPVSLGDQASISCRA.

8. A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31 or 41; A heavy chain CDR2 comprising an amino acid sequence represented by any one of SEQ ID NOs: 32, 42 to 44, and A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; a heavy chain variable region comprising: A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34 or 52; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:35; and A light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:36; and A light chain FR1 comprising an amino acid sequence represented by any one of SEQ ID NOs: 53 to 56; 1. An antibody comprising a light chain variable region comprising: A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:37, and The antibody or antigen-binding fragment thereof according to claim 1 , excluding one comprising a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:

38.

9. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

10. An expression vector comprising the nucleic acid of claim 9.

11. A cell comprising the nucleic acid of claim 9 or the expression vector of claim 10.

12. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and a pharma- ceutically acceptable carrier or excipient.

13. The pharmaceutical composition of claim 12, which is in a lyophilized form.

14. 1) preparing antibody-producing immortalized cells from B cells collected from the intestinal lamina propria or the spleen; 2) culturing the antibody-producing immortalized cells and determining which cells produce antibodies that bind to Clostridium difficile bacteria; and 3) recovering antibodies from cells that produce antibodies that bind to the Clostridium difficile bacteria. A method for producing an antibody that binds to Clostridium difficile bacteria, comprising:

15. The method for producing an antibody that binds to Clostridium difficile bacterial bodies according to claim 14, wherein step 2 is carried out by determining cells that produce antibodies that bind to the bacterial bodies immobilized on a carrier.

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