Methods and compositions for treating or diagnosing inflammatory bowel disease (IBD)

Abstract

The present disclosure relates to: a method and a composition both for treating IBD using digestive tract contents or an excrement each modified with an IgA antibody, and a method for producing the composition; a method for obtaining an IgA antibody capable of making a bacterial flora in a digestive tract of an IBD patient normal; a method and a composition both for modifying digestive tract contents or an excrement of an IBD patient using an IgA antibody, and a method for producing the composition; and a method and a composition both for testing using a diagnostic medicine containing an IgA antibody in a patient who is treated with an IBD therapeutic agent containing digestive tract contents or an excrement, and a method for producing the composition.

Description

[Technical Field]

[0001] This disclosure relates to methods, compositions, and methods for manufacturing the same for treating inflammatory bowel disease (IBD) using gastrointestinal contents or excrement modified with IgA antibodies; methods for obtaining IgA antibodies that restore the microbiota in the gastrointestinal tract of IBD patients; methods, compositions, and methods for manufacturing the same for modifying gastrointestinal contents or excrement of IBD patients using IgA antibodies; and methods, compositions, and methods for manufacturing the same for testing patients treated with an IBD therapeutic agent containing gastrointestinal contents or excrement with a diagnostic agent containing IgA antibodies. [Background technology]

[0002] Abnormalities in the gut microbiota (dysbiosis) have been reported to be associated with the development of many diseases, including inflammatory bowel disease (IBD), suggesting that improving the gut microbiota is important for maintaining health. In the development of inflammatory bowel disease, there are reports that in spontaneously developing colitis model mice, colitis does not develop when germ-free, but develops when gut bacteria are present (Sandra C. Kim, et al: Non-Patent Literature 1, Monika Schaubeck, et al: Non-Patent Literature 2). From this, it is suggested that the gut microbiota is one of the environmental factors that greatly plays a role in the development of inflammatory bowel disease. As bacteria that cause inflammatory bowel disease, Escherichia coli (E. coli) (Arlette Darfeuille-Michaud, et al: Non-Patent Literature 3, Adeline Sivignon, et al: Non-Patent Literature 4) and Fusobacterium varium (F. varium) (Toshifumi Ohkusa, et al: Non-Patent Literature 5), which adhere to and invasive to intestinal epithelial cells, have been listed as candidates, but the clear causative bacteria remain unknown. Based on the above, it is expected that controlling the gut microbiota will lead to the prevention and treatment of diseases. Currently, one treatment method for controlling the gut microbiota is the elimination of intestinal bacteria by administering antibiotics. The aim is to eliminate pathogenic bacteria by administering antibiotics. However, since there are antibiotic-resistant strains among the enteritis-causing bacteria known as pathogenic bacteria, this not only eliminates fermenting bacteria along with enteritis-causing bacteria, but also leads to the dominance of antibiotic-resistant bacteria, potentially resulting in the worsening of the disease and an increased susceptibility to infection. As a new treatment approach that takes this problem into account, it is considered desirable to eliminate only enteritis-causing bacteria while leaving behind fermenting bacteria that improve intestinal function. Inflammatory bowel disease (IBD) affects more than 200,000 people annually in Japan alone and is on the rise. This disease is characterized by two phases: an active phase and a remission phase, which repeat. Currently, the worsening of the disease is stopped by suppressing inflammation during the active phase with steroid drugs and anti-inflammatory drugs such as Asacol.However, these are merely symptomatic treatments. The true cure lies in improving the gut microbiome that causes inflammation. But when examining patient samples, it becomes clear that gut microbiota are diverse, making it difficult to control with a single drug. Therefore, in treating IBD, it is crucial to accurately diagnose the disease state and provide the most appropriate treatment for each individual patient.

[0003] While bacterial preparations and fecal transplantation are used to improve the gut microbiota in order to treat diseases such as inflammatory bowel disease (IBD), and have been reported to have some effect, there is still a need for new treatment methods. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Sandra C. Kim, et al.,Gastroenterology 128,891-906,2005 [Non-Patent Document 2] Monika Schaubeck, et al.,Gut 65,225-237,2016 [Non-Patent Document 3] Arlette Darfeuille-Michaud, et al.,Gastroenterology 127,412-421,2004 [Non-Patent Document 4] Adeline Sivignon, et al.,Inflamm Bowel Dis 21, 276-286,2015 [Non-Patent Document 5] Toshifumi Ohkusa, et al.,Journal of Medical Microbiology 58,535-545,2009 [Overview of the project] [Problems that the invention aims to solve]

[0005] This disclosure aims to provide a method for obtaining gastrointestinal contents or excrement that improves the gastrointestinal microbiota, by treating the gastrointestinal contents or excrement with an IgA antibody in a therapeutic method for administering gastrointestinal contents or excrement to treat IBD, a method for obtaining an IgA antibody related to this method, and a pharmaceutical composition to be used in these methods. [Means for solving the problem]

[0006] This disclosure provides a method for treating IBD using gastrointestinal contents or excrement modified with an IgA antibody, as well as compositions for use in the method and methods for producing the same. In another embodiment, this disclosure provides a method for obtaining IgA antibodies that restore the gut microbiota of patients with IBD. In another embodiment, this disclosure provides a method for modifying the gastrointestinal contents or excretions of an IBD patient using an IgA antibody, as well as compositions for use in the method and methods for producing the same. This disclosure also, in another form, includes gastrointestinal contents or excrement. This invention provides a method for testing patients treated with IBD therapeutic drugs using a diagnostic pharmaceutical containing IgA antibodies, as well as a pharmaceutical used in this method and a method for manufacturing the same.

[0007] More specifically, this disclosure describes, in one manner, The present invention provides a pharmaceutical composition for treating IBD, which contains gastrointestinal contents or excrement treated with an IgA antibody that promotes the health of the gut microbiota.

[0008] This disclosure is made in a manner that, (A1) The process of preparing the gastrointestinal contents or excrement of an IBD patient. (A2) A step of modifying the contents of the gastrointestinal tract or excrement with an IgA antibody that promotes a healthy bacterial flora in the gastrointestinal tract, and (A3) A method for treating IBD is provided, comprising the step of administering modified gastrointestinal contents or excrement to an IBD patient.

[0009] In one aspect, the present disclosure (A1´) Preparing the gastrointestinal contents or excrement of an IBD patient (A2´) Administering the gastrointestinal contents or excrement to the IBD patient. (A3´) A method for treating IBD is provided, which includes modifying the gastrointestinal contents or excrement by administering an IgA antibody that normalizes the gut microbiota to the IBD patient.

[0010] In one aspect, the present disclosure (B1) Preparing the gastrointestinal contents or excrement of an IBD patient, (B2) Modifying the gastrointestinal contents or excrement with a candidate IgA antibody, (B3) Confirming that the modified gastrointestinal contents or excrement can normalize the gut microbiota, A method for screening an IgA antibody that normalizes the gut microbiota is provided, which includes the above steps.

[0011] In one aspect, the present disclosure A composition containing an IgA antibody for treating the gastrointestinal contents or excrement of an IBD patient is provided.

[0012] In one aspect, the present disclosure (C1) Preparing the gastrointestinal contents or excrement of an IBD patient (C2) Contacting the gastrointestinal contents or excrement with an IgA antibody that normalizes the gut microbiota, A method for treating the gastrointestinal contents or excrement of an IBD patient is provided, which includes the above steps.

[0013] In one aspect, the present disclosure (D1) Preparing an IgA antibody that normalizes the gut microbiota in the digestive tract of an IBD patient; (D2) Contacting the gastrointestinal contents or excrement of the subject with the IgA antibody; and (D3) Identifying bacteria that bind to the IgA antibody, If the IgA antibody shows binding to IBD-related bacteria, the subject is treated as having IBD or being at risk of developing IBD. This invention provides a method for testing whether a subject has IBD or is at risk of developing it.

[0014] This disclosure is, in one manner, This invention provides a pharmaceutical product for diagnosing the presence or risk of developing IBD, including an IgA antibody that restores the gut microbiota in IBD patients.

[0015] This disclosure is, in one manner, (E1) The process of obtaining gastrointestinal contents or excrement from patients who are currently receiving, are scheduled to receive, or have received IBD treatments containing gastrointestinal contents or excrement; (E2) A step of bringing the contents of the gastrointestinal tract or excrement into contact with IgA antibodies; and (E3) The step includes analyzing bacteria that bind to the IgA antibody, If the analysis results indicate a healthy gastrointestinal microbiota, treatment with the IBD drug will be continued. This provides a method for testing the therapeutic effects of IBD medications.

[0016] This disclosure is, in one manner, The present invention provides an IgA antibody-containing pharmaceutical for testing the gastrointestinal contents or excrement of patients who are currently receiving, are scheduled to receive, or have received an IBD treatment drug containing gastrointestinal contents or excrement.

[0017] More specifically, this disclosure provides the following: [Item 1] A pharmaceutical composition for treating inflammatory bowel disease (IBD), comprising gastrointestinal contents or excrement treated with IgA antibodies that promote the healthy bacterial flora of the gastrointestinal tract. [Item 2] A pharmaceutical composition for treating IBD, containing an IgA antibody that promotes the health of the gut microbiota, to be used in combination with a composition containing gastrointestinal contents or excrement. [Item 3] The pharmaceutical composition according to item 1 or 2, wherein the IgA antibody that restores the bacterial flora in the gastrointestinal tract has one or more of the following characteristics: (1) IgA antibodies that, when in contact with gastrointestinal contents or excrement from IBD patients, or from gastrointestinal contents or excrement from healthy individuals, bind to a greater number of IBD-related bacteria when in contact with gastrointestinal contents or excrement from IBD patients; (2) When a first binding profile is created based on the types and amounts of bacteria bound to the IgA antibody after contacting the IgA antibody with gastrointestinal contents or excrement from an IBD patient, and a second binding profile is created based on the types and amounts of bacteria bound to the IgA antibody after contacting the IgA antibody with gastrointestinal contents or excrement from a healthy person, the IgA antibody that shows binding to a wider variety of IBD-related bacteria or a larger amount of binding to IBD-related bacteria in the first binding profile is compared with the first binding profile. (3) IgA antibodies that increase the bacterial diversity of gastrointestinal contents or excrement from IBD patients after treatment with IgA antibodies, when compared to the bacterial diversity of gastrointestinal contents or excrement after treatment with IgA antibodies; (4) When gastrointestinal contents or excrement modified with the IgA antibody is administered to a patient, and the bacterial diversity of the gastrointestinal contents or excrement after administration is compared with the bacterial diversity of the gastrointestinal contents or excrement before administration, the IgA antibody increases the bacterial diversity of the gastrointestinal contents or excrement after administration; (5) An IgA antibody that increases the short-chain fatty acid content after administration when gastrointestinal contents or excretions modified with the IgA antibody are administered to a patient, and the short-chain fatty acid content in the patient's gastrointestinal tract before administration is compared with the short-chain fatty acid content in the gastrointestinal contents or excretions after administration; (6) An IgA antibody that reduces the amount of Enterobacteriaceae in the gastrointestinal contents or excrement of a patient after administration, by administering the gastrointestinal contents or excrement modified with the IgA antibody modified with the IgA antibody modified with the gastrointestinal contents or excrement of the patient before administration and comparing the amount of Enterobacteriaceae in the gastrointestinal contents or excrement after administration. [Item 4] The pharmaceutical composition according to item 1 or 2, wherein the gastrointestinal contents or excrement are derived from a subject to whom the composition is administered. [Item 5] (B1) The process of preparing the gastrointestinal contents or excrement of an IBD patient. (B2) A step of modifying the gastrointestinal contents or excreta with a candidate IgA antibody. (B3) A step to confirm that the modified gastrointestinal contents or excrement can restore the bacterial flora in the gastrointestinal tract. A method for screening IgA antibodies that promote a healthy gut microbiome, including [specific antibody]. [Item 6] The method according to item 5, wherein the step of confirming that the modified gastrointestinal contents or excrement can restore the bacterial flora in the gastrointestinal tract is carried out by any of the following: (1) When the IgA antibody is brought into contact with gastrointestinal contents or excrement from an IBD patient, or from gastrointestinal contents or excrement from a healthy person, it is confirmed that it binds to more IBD-related bacteria when it is brought into contact with gastrointestinal contents or excrement from an IBD patient; (2) When a first binding profile is created based on the types and amounts of bacteria bound to the IgA antibody after contacting the IgA antibody with gastrointestinal contents or excrement from an IBD patient, and a second binding profile is created based on the types and amounts of bacteria bound to the IgA antibody after contacting the IgA antibody with gastrointestinal contents or excrement from a healthy person, it is confirmed that the first binding profile shows binding to a wider variety of IBD-related bacteria or a larger amount of binding to IBD-related bacteria; (3) When comparing the bacterial diversity of gastrointestinal contents or excrement from IBD patients before treatment with IgA antibodies with the bacterial diversity of gastrointestinal contents or excrement after treatment with IgA antibodies, confirm that the bacterial diversity of gastrointestinal contents or excrement increases after treatment with IgA antibodies; (4) Administer the gastrointestinal contents or excrement modified with the IgA antibody to the patient, and confirm that the bacterial diversity of the gastrointestinal contents or excrement increases after administration when comparing the bacterial diversity of the gastrointestinal contents or excrement in the patient before administration with that of the gastrointestinal contents or excrement after administration; (5) When gastrointestinal contents or excrement modified with the IgA antibody is administered to a patient, and the short-chain fatty acid content in the patient's gastrointestinal tract before administration is compared with the short-chain fatty acid content in the gastrointestinal contents or excrement after administration, the short-chain fatty acid content increases after administration; (6) The gastrointestinal contents or excrement modified with the IgA antibody is administered to the patient, and the Enterobacteriaceae content of the gastrointestinal contents or excrement of the subject before administration is compared with the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration, and the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration is reduced. [Item 7] (C1) Process of preparing gastrointestinal contents or excrement from an IBD patient. (C2) A step of bringing the contents of the digestive tract or excrement into contact with IgA, which promotes the health of the bacterial flora in the digestive tract, outside of the body. A method for processing the gastrointestinal contents or excrement of an IBD patient, including [specific example]. [Item 8] A composition containing IgA, which promotes a healthy bacterial flora in the gastrointestinal tract, for modifying the bacterial flora of gastrointestinal contents or excrement in vitro. [Item 9] (D1) A step to prepare IgA antibodies to restore the gut microbiota of IBD patients; (D2) A step of bringing the contents of the gastrointestinal tract or excrement of the subject into contact with the IgA antibody; and (D3) Includes the step of identifying bacteria that bind to the IgA antibody, If the IgA antibody shows binding to IBD-related bacteria, the subject is treated as having IBD or being at risk of developing IBD. A method of testing to diagnose whether a subject has IBD or is at risk of developing IBD. [Item 10] A pharmaceutical product containing IgA antibodies that restore the gut microbiota of IBD patients, used to diagnose the presence or risk of developing IBD. [Item 11] (E1) The process of obtaining gastrointestinal contents or excrement from patients who are currently receiving, are scheduled to receive, or have received IBD treatments containing gastrointestinal contents or excrement; (E2) A step of bringing the contents of the gastrointestinal tract or excrement into contact with IgA antibodies; and (E3) The step includes analyzing bacteria that bind to the IgA antibody, If the analysis results indicate a healthy gastrointestinal microbiota, treatment with the IBD drug will be continued. Methods for testing the therapeutic effects of IBD medications. [Item 12] The method according to item 11, wherein the contents of the gastrointestinal tract or excrement are modified with IgA. [Item 13] A diagnostic device containing IgA antibodies for testing the therapeutic effect of IBD medications in patients who are currently receiving, are scheduled to receive, or have received IBD medications containing gastrointestinal contents or excrement. [Item 14] The diagnostic agent according to item 13, wherein the contents of the gastrointestinal tract or excretion are modified with IgA. [Effects of the Invention]

[0018] This disclosure provides a method for obtaining gastrointestinal contents or excrement that improves the gastrointestinal microbiota, by treating the gastrointestinal contents or excrement with an IgA antibody, in a therapeutic method for administering gastrointestinal contents or excrement to treat IBD, and a method for obtaining an IgA antibody related to this method, as well as a pharmaceutical composition used in these methods. [Brief explanation of the drawing]

[0019] [Figure 1] Figure 1 shows the results of testing the binding characteristics of endogenous polyclonal IgA antibody-bound bacteria collected from healthy individuals and IBD patients against various intestinal bacteria contained in feces collected from healthy individuals and IBD patients, respectively. The vertical axis represents the IgA Index. [Figure 2] Figure 2 shows the results of testing the binding of IgA antibodies obtained from the gastrointestinal tract of IBD patients to IBD-related bacteria. The binding affinity of IgA antibodies to IBD-related bacteria was evaluated by ELISA assay. [Figure 3] Figure 3 shows that endogenous polyclonal IgA antibodies have a high binding affinity to IBD-related bacteria. [Figure 4] Figure 4 shows that the RS_H000_L001 IgA antibody has a high binding affinity to IBD-related bacteria. [Figure 5] Figure 5 shows that the RS_H007_L004 IgA antibody, a variant of the RS_H000_L001 IgA antibody, has a high binding affinity to IBD-related bacteria. [Figure 6] Figure 6 shows that the SNK0003A IgA antibody has a high binding affinity to IBD-related bacteria. [Figure 7] Figure 7 shows that the SNK0001A IgA antibody has a high binding affinity to IBD-related bacteria. [Figure 8] Figure 8 shows that the SNK0002A IgA antibody has a high binding affinity to IBD-related bacteria. [Figure 9]Figure 9 shows the results of confirming the binding characteristics of RS_H000_L001 IgA antibodies against bacteria in feces obtained from healthy individuals or IBD patients. [Figure 10] Figure 10 shows that the RS_H000_L001 antibody exhibits a potent growth inhibitory effect against Gemella morbillorum in an in vitro growth inhibition study. [Figure 11] Figure 11 is a schematic diagram showing the scheme of an oral bacterial transplantation study in mice using intestinal bacteria derived from IBD patients (P10). [Figure 12] Figure 12 shows the changes in body weight in two groups in a mouse oral bacterial transplantation study using intestinal bacteria derived from IBD patients (P10): (1) the whole bacterial community / no antibody administration group and (2) the whole bacterial community / RS_H000_L001 antibody oral administration group. [Figure 13] Figure 13 shows the changes in body weight in two groups in a mouse oral bacterial transplantation study using enterobacteria derived from IBD patients (P10): (3) the group with bacteria not bound to RS_H000_L001 antibody / no antibody administration, and (4) the group with bacteria not bound to RS_H000_L001 antibody / oral administration of RS_H000_L001 antibody. [Figure 14] Figure 14 shows the results of the diversity analysis of the gut microbiota of mice after treatment in an oral bacterial transplantation study using gut bacteria derived from IBD patients (P10). [Figure 15] Figure 15 is a schematic diagram showing the scheme of an oral bacterial transplantation study in mice using intestinal bacteria derived from IBD patients (P13). [Figure 16] Figure 16 shows the bacterial flora from the stool of an IBD patient (P13) and the binding of the RS_H000_L001 antibody. [Figure 17] Figure 17 shows the changes in body weight in two groups in a mouse oral bacterial transplantation study using intestinal bacteria derived from IBD patients (P13): (1) the whole bacterial community / no antibody administration group and (2) the whole bacterial community / RS_H000_L001 antibody oral administration group. [Figure 18]Figure 18 shows the results of the Shannon index analysis of the diversity of the gut microbiota of mice after treatment in an oral bacterial transplantation study using gut bacteria derived from IBD patients (P13). [Figure 19] Figure 19 shows the results of the diversity analysis of the gut microbiota of mice after treatment using gut bacteria derived from IBD patients (P13), as shown by the change in the relative abundance ratio of Fusobacteriaceae. [Figure 20] Figure 20 shows the results of the diversity analysis of the gut microbiota of mice after treatment using gut bacteria derived from IBD patients (P13), as shown by the change in the relative abundance ratio of Lachnospiraceae. [Figure 21] Figure 21 shows the results of the diversity analysis of the gut microbiota of mice after treatment using gut bacteria derived from IBD patients (P13), as shown by the change in the relative abundance ratio of Bacteroidaceae. [Figure 22] Figure 22 shows the staining results of colon tissue sections after treatment in a mouse oral bacterial transplantation study using intestinal bacteria derived from IBD patients (P13). [Figure 23] Figure 23 shows the results of testing the binding affinity of SNK0004 IgA antibody to bacteria in feces obtained from healthy individuals and IBD patients. [Figure 24] Figure 24 shows the results of testing the binding affinity of SNK0005 IgA antibodies to bacteria in feces obtained from healthy individuals and IBD patients. [Figure 25] Figure 25 shows the results of confirming the binding characteristics of SNK0004 IgA antibodies against bacteria in feces obtained from IBD patients. [Figure 26] Figure 26 shows the results of confirming the binding characteristics of SNK0005 IgA antibodies against bacteria in feces obtained from IBD patients. [Figure 27] Figure 27 shows the results of confirming the binding characteristics of SNK0001 IgA antibodies against bacteria in feces obtained from IBD patients. [Figure 28] Figure 28 shows the results of confirming the binding characteristics of SNK0002 IgA antibodies against bacteria in feces obtained from IBD patients. [Figure 29] Figure 29 shows the results of confirming the binding characteristics of SNK0003 IgA antibodies against bacteria in feces obtained from IBD patients. [Figure 30] Figure 30 shows the results of comparing the proportion of bacteria from IBD patient stools that bind to the RS_H000_L001, SNK0001, SNK0002, SNK0003, SNK0004, and SNK0005 antibodies. [Modes for carrying out the invention]

[0020] Immunoglobulin A (IgA) antibodies, one of the antibody molecular isotypes, are known to be important for maintaining a normal gut microbiota and protecting against infection by pathogenic microorganisms that invade the intestinal tract. IgA antibodies are antibodies that primarily function not only in serum but also on mucosal surfaces such as the intestinal tract. IgA antibodies are produced by IgA antibody-producing cells located in the lamina propria. IgA antibodies are secreted into the lamina propria as dimeric IgA antibodies via a J chain. Dimeric IgA antibodies are transported into the intestinal lumen through mucosal epithelial cells. This transport is carried out by multimeric Ig receptors expressed on mucosal epithelial cells. Dimeric IgA antibodies bind to these receptors, are endocytized into vesicles, and transported to the intestinal lumen surface by transcytosis. At the intestinal lumen surface, the extracellular domain of the receptor is cleaved by proteolytic enzymes, and the dimeric IgA antibody is secreted into the intestinal lumen while retaining its extracellular domain (secretory factor). Secretory factors attached to dimeric IgA antibodies protect them from degradation by proteolytic enzymes in the intestinal lumen. Through this process, the secreted dimeric IgA antibodies bind to bacteria in the intestinal lumen, preventing their adhesion to and invasion of the epithelium.

[0021] The inventors of this disclosure have diligently studied the role of intestinal IgA antibodies in the regulation of the gut microbiota, and have elucidated that the host recognizes and controls intestinal bacteria through IgA antibodies secreted into its own intestinal tract. They have further elucidated that some intestinal IgA antibodies work to identify and bind to bacteria and eliminate those bacteria from the intestinal lumen, while others work to maintain the diversity of the gut microbiota by identifying, binding to, and retaining those bacteria in the intestinal mucosa. Thus, it was thought that intestinal IgA antibodies control the overall bacterial flora composition by interacting with intestinal bacteria in a complex manner, retaining bacteria that benefit the host and eliminating bacteria that harm the host. However, there were still unanswered questions regarding what each IgA antibody identifies and acts upon in each intestinal bacterium, and how it acts on target molecules.

[0022] As a result of diligent research by the inventors of this disclosure, they found that the state of the intestinal microbiota of patients with inflammatory bowel disease (IBD) differs from that of healthy individuals, with an increase in bacteria associated with IBD exacerbations (IBD-related bacteria), and that the binding ability of IgA antibodies to these IBD-related bacteria is reduced in IBD patients. Based on this, they conceived the idea that IBD could be improved by treating the gastrointestinal contents or excrement of IBD patients with IgA antibodies that promote a healthy intestinal microbiota, and then administering the treated material to IBD patients via fecal transplantation, and they succeeded in actually confirming this effect.

[0023] (definition) Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which this disclosure pertains.

[0024] In this specification, when multiple numerical ranges are given, a range consisting of any combination of the lower and upper limits of those ranges is also meant.

[0025] In this specification, the term “substantially” has the same meaning as commonly understood by those skilled in the art to which this disclosure belongs, but is used to include, for example, the desired state and the state that is inevitably not achieved due to the biological or chemical properties, considering that biological or chemical phenomena may not completely achieve the desired state.

[0026] In this specification, when the term "approximately" is used in relation to a numerical value, it means that the value may vary within a range such as 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01%.

[0027] In this specification, the term “contains” has the same meaning as commonly understood by those skilled in the art in the field to which this disclosure belongs, but for example, it includes “contains” and “consists of,” and specifically, a composition “contains” A may contain A alone, as well as other components, such as B.

[0028] In this specification, the terms “consisting of” or “composed of” used in reference to a composition have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs, but are used to indicate the components that constitute the composition exclusively. For example, a composition “consisting of” A contains exclusively A. However, in one embodiment, a composition “consisting of” A includes embodiments that contain impurities other than A that are unavoidable in the manufacture based on their biological and chemical properties.

[0029] In this specification, the term "antibody" is used in its broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, and antibody fragments that exhibit intended antigen-binding activity. Full-length antibodies consist mainly of a heavy chain and a light chain composed of polypeptides. The heavy chain and light chain each contain a region called a variable region that recognizes the antigen, and these regions are generally referred to as the heavy chain variable region and the light chain variable region, respectively. These variable regions have, in more detail, regions called CDR1-3, starting from the amino terminus, which are identified as antigen-recognizing regions. These CDR1-3 are also more specifically called heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, light chain CDR3, etc. Furthermore, regions of the heavy chain and light chain other than CDR1-3 are called heavy chain FR1-4 and light chain FR1-4, starting from the amino terminus, respectively. Antibodies may be in the form of two heavy chains and two light chains, or in the form of one heavy chain and one light chain (also called a single-chain antibody).

[0030] Antibodies are classified into classes such as IgG, IgE, IgM, IgD, IgA, or IgY, and these are further classified into subclasses such as IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

[0031] In this specification, the “antigen-binding fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It is known that the ability of an antibody to specifically bind to an antigen can also be maintained by fragments consisting of a part thereof. In one embodiment, the “antigen-binding fragment” of an antibody may 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 which is part of the heavy chain constant region; an F(ab')2 fragment containing two Fab fragments linked by disulfide crosslinking at a hinge region; an Fd fragment consisting of VH and CH1 domains; an Fv fragment consisting of VL and VH domains of a single arm of the antibody; a dAb fragment containing a single variable domain; and an isolated complementarity-determining region (CDR).

[0032] The antibodies described herein may be CDR-grafted antibodies. In one example, in a CDR-grafted antibody, part or all of the CDR region sequence of an antibody derived from one animal species is replaced with a CDR sequence from another animal species. For example, the CDR of one or more mouse antibodies may be replaced with a CDR sequence from a human antibody.

[0033] Methods 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, USD Department of Health and Human Services, NIH Publication, pp. 91-3242), which are well known to those skilled in the art, can be used. To identify CDR sequences within antibodies, CDRs may be identified based on information from public databases (e.g., https: / / www.ncbi.nlm.nih.gov / igblast).

[0034] In this specification, "identity" refers to the degree to which two or more comparable amino acid sequences or nucleotide sequences are identical to each other. Therefore, the higher the identity of two amino acid sequences or nucleotide sequences, the higher their identity or similarity. The level of identity of amino acid sequences 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 have been developed based on this BLAST algorithm (e.g., Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. J Mol Biol. 215:403-10 (1990), etc.). The specific methods of these analyses are publicly known and can be found on the NCBI website. For example, when an amino acid sequence A is said to be identical to another amino acid sequence B by a certain percentage, it means that amino acid sequences A and B have that percentage of identity.

[0035] In this specification, "monoclonal" is a modifier describing the characteristics of antibodies obtained from a substantially homogeneous population of antibodies. Individual antibodies in such a population are identical except for possible naturally occurring mutations that may be present in trace amounts.

[0036] In this specification, "conservative substitution technique" means a technique in which an amino acid residue is substituted with an amino acid residue having a similar side chain.

[0037] For example, substitutions between amino acid residues with basic side chains, such as lysine, arginine, and histidine, are considered conservative substitution techniques. Similarly, substitutions between amino acid residues with acidic side chains, such as aspartic acid and glutamic acid; amino acid residues with non-charged polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues with non-polar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues with β-branched side chains, such as threonine, valine, and isoleucine; and amino acid residues with aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine, are also considered conservative substitution techniques.

[0038] In this specification, “gastrointestinal tract” has the common meaning in the art of this disclosure and typically refers to the tubular tissue through which food enters the body and is expelled as waste, from the mouth to the rectum. For example, “gastrointestinal tract” includes the mouth, pharynx, esophagus, stomach, duodenum, small intestine, and large intestine.

[0039] In this specification, “gastrointestinal contents” includes all substances present in the lumen of the “gastrointestinal tract.” For example, this includes ingested food and beverages, substances of host tissue discharged into the lumen, their metabolites, and mixtures thereof.

[0040] In this specification, “excrement” includes all substances expelled from the “digestive tract.” Typically, such excrement includes feces.

[0041] 1. A method of treating IBD using gastrointestinal contents or excrement modified with IgA antibodies. This disclosure provides, in one embodiment, a method for treating IBD using gastrointestinal contents or excretions modified with an IgA antibody. The method for treating IBD according to this disclosure includes, in one embodiment, the following steps. (A1) The process of preparing the gastrointestinal contents or excrement of an IBD patient. (A2) A step of modifying the contents of the gastrointestinal tract or excrement with an IgA antibody that promotes a healthy bacterial flora in the gastrointestinal tract, and (A3) The process of administering modified gastrointestinal contents or excrement to an IBD patient.

[0042] The method for treating IBD according to this disclosure includes, in one embodiment, the following steps. (A1') The process of preparing the gastrointestinal contents or excrement of an IBD patient. (A2') The process of administering gastrointestinal contents or excrement to an IBD patient. (A3') A step of modifying the contents of the gastrointestinal tract or excretion by administering an IgA antibody that promotes a healthy bacterial flora in the gastrointestinal tract to an IBD patient.

[0043] (IgA antibody) The gastrointestinal microbiota of IBD patients is distinctly different from that of healthy individuals and has a composition that exacerbates IBD. Furthermore, the specific composition of the microbiota varies from patient to patient. Therefore, the IgA antibody used in this method is obtained specifically for each IBD patient, with the aim of restoring a healthy gastrointestinal microbiota.

[0044] Confirmation that the IgA antibody can improve the gastrointestinal microbiota of IBD patients can be carried out by methods such as analyzing the amount of IBD-related bacteria that bind to the IgA, analyzing the binding profiles of multiple bacteria that bind to the IgA antibody, testing the physical, chemical, or biological properties of gastrointestinal contents or excrement modified with the IgA antibody, for example, confirming that the bacterial diversity in the gastrointestinal contents or excrement is increased, confirming that the bacterial diversity in the intestinal tract is increased by administering the gastrointestinal contents or excrement, confirming that short-chain fatty acids in the intestinal tract are increased by administering the gastrointestinal contents or excrement, confirming that Enterobacteriaceae present in the intestines are reduced by administering the gastrointestinal contents or excrement, and confirming that the symptoms of IBD are improved by administering the gastrointestinal contents or excrement.

[0045] To confirm that an IBD-related antibody can improve the gastrointestinal microbiota of an IBD patient by analyzing IBD-related bacteria that bind to the antibody, this can be done, for example, by comparing the amount of binding of the IgA antibody to intestinal bacteria (IBD-related bacteria) known to be associated with the worsening of IBD in gastrointestinal contents or excrement from an IBD patient and from gastrointestinal contents or excrement from a healthy individual, and confirming that the IgA antibody binds to a larger amount of the IBD-related bacteria in gastrointestinal contents or excrement from an IBD patient. This confirmation test can be performed using methods known to those skilled in the art, such as FACS analysis or ELISA analysis. For the purposes of this disclosure, IBD-related bacteria refer to bacteria known to those skilled in the art as intestinal bacteria known to be associated with the worsening of IBD. Non-limiting examples of IBD-related bacteria include Enterobacteriaceae.

[0046] To confirm that an IgA antibody can improve the gastrointestinal microbiota of IBD patients by analyzing the binding profiles of multiple bacteria that bind to the IgA antibody, for example, this can be done by contacting the IgA antibody with gastrointestinal contents or excrement from an IBD patient, creating a binding profile based on the types and amounts of bacteria bound to the IgA antibody, and comparing this profile with a binding profile created by contacting the IgA antibody with gastrointestinal contents or excrement from a healthy individual, and confirming that the binding profile created from gastrointestinal contents or excrement from an IBD patient shows binding to a wider variety of IBD-related bacteria or a larger amount of IBD-related bacteria. In one example, the binding profile can be represented by quantitative data using the IgA Index, which is determined from the amount of IgA that each bacterium binds to and does not bind to IgA (Andrew L. Kau, et al. Sci Transl Med 7, 2015; Hirosuke Sugahara, et al., Frontiers in Microbiology 8, 1757, 2017). This confirmation test can be carried out using methods known to those skilled in the art, such as FACS analysis, ELISA analysis, and next-generation sequencing analysis.

[0047] To confirm that an IgA antibody can improve the health of the gastrointestinal microbiota in IBD patients, for example, by comparing the bacterial diversity of gastrointestinal contents or excrement before and after treatment with the IgA antibody, it is possible to confirm that the bacterial diversity of gastrointestinal contents or excrement after treatment with the IgA antibody has increased. This confirmation test can be performed using methods known to those skilled in the art, such as FACS analysis, ELISA analysis, and next-generation sequencing analysis.

[0048] To confirm that the IgA antibody can improve the health of the gastrointestinal microbiota of IBD patients, for example, by administering gastrointestinal contents or excrement treated with the IgA antibody to a subject and confirming an increase in the diversity of the intestinal microbiota, it is possible to compare the diversity of the intestinal microbiota of the subject before administration with the diversity of the intestinal microbiota after administration and confirm that the diversity of the intestinal microbiota of the gastrointestinal contents or excrement after administration has increased. This confirmation test can be performed using the gastrointestinal contents or excrement of the subject after administration as a sample, using methods known to those skilled in the art, such as FACS analysis, ELISA analysis, or next-generation sequencing analysis. In this test, IBD patients or IBD model animals may be used as subjects.

[0049] To confirm that the IgA antibody can improve the gastrointestinal microbiota of IBD patients by confirming an increase in short-chain fatty acids in the intestinal tract after administration of the gastrointestinal contents or excrement, for example, by comparing the short-chain fatty acid content of the gastrointestinal contents or excrement of the subject before administration with the short-chain fatty acid content of the gastrointestinal contents or excrement after administration, and confirming that the short-chain fatty acid content of the gastrointestinal contents or excrement has increased after administration, it can be confirmed that the IgA antibody can improve the gastrointestinal microbiota of IBD patients. This confirmation test can be carried out using methods known to those skilled in the art, such as chromatographic analysis. In this test, IBD patients or IBD model animals may be used as subjects.

[0050] To confirm that the IgA antibody can restore the gastrointestinal microbiota of IBD patients by confirming that administering the gastrointestinal contents or excrement reduces the amount of Enterobacteriaceae present in the intestinal tract, for example, the short-chain fatty acid content of the subject's gastrointestinal contents or excrement before administration and the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration can be compared, and the decrease in the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration can be confirmed, thereby confirming that the IgA antibody can restore the gastrointestinal microbiota of IBD patients. This confirmation test can be carried out using methods known to those skilled in the art, such as bacterial colony culture tests. In this test, IBD patients or IBD model animals may be used as subjects.

[0051] To confirm that the IgA antibody can restore the gastrointestinal microbiota of IBD patients, by confirming that the symptoms of IBD improve upon administration of the gastrointestinal contents or excretions in question, for example, by comparing the IBD symptoms of the subject before administration with those of the subject after administration and confirming that the symptoms have improved after administration, it can be confirmed that the IgA antibody can restore the gastrointestinal microbiota of IBD patients. This confirmation test can be performed using IBD diagnostic indicators known to those skilled in the art. In this test, IBD patients or IBD model animals may be used as subjects.

[0052] Confirmation that the above-mentioned IgA antibody can restore the gastrointestinal microbiota of IBD patients can be carried out in advance as a method separate from the treatment method disclosed herein, or it may be carried out as a step (A1-2) using the gastrointestinal contents or excrement obtained in step (A1) as one of the steps constituting the treatment method herein.

[0053] The IgA antibody used in the method of this disclosure may be further conjugated with other compounds or fused with heterologous peptides, provided that it does not lose its binding properties.

[0054] The IgA antibody used in the method of this disclosure may have an amino acid sequence derived from the same species as the IBD patient, or it may have amino acids derived from a different species, such as a closely related organism. Specifically, examples include those derived from humans, mice, rats, hamsters, rabbits, goats, donkeys, pigs, cattle, horses, chickens, monkeys, chimpanzees, camels, llamas, etc.

[0055] The IgA antibody of this disclosure may be produced from antibody-producing cells derived from B cells such as hybridomas, or it may be produced by introducing the nucleic acid encoding the antibody into cells other than those of the immune system using genetic engineering technology, and used as a recombinant antibody.

[0056] In one embodiment, the IgA antibody disclosed herein is The heavy chain variable region containing 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, as represented by SEQ ID NO: 7, The light chain variable region includes 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, which are part of the light chain variable region containing the amino acid sequence represented by Sequence ID No. 8. TIFF0007862032000001.tif50159

[0057] In one embodiment, the IgA antibody disclosed herein is Heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 1, Heavy chain CDR2 containing the amino acid sequence represented by Sequence ID No. 2, and Heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 3, Heavy chain variable region including, Light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 4, Light chain CDR2 containing the amino acid sequence represented by Sequence ID No. 5, Light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 6 Includes a light chain variable region. TIFF0007862032000002.tif53160

[0058] In one embodiment, the IgA antibody disclosed herein is A heavy chain variable region including the amino acid sequence represented by Sequence ID No. 7 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and Light chain variable region containing the amino acid sequence represented by Sequence ID No. 8 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. Includes.

[0059] In one embodiment, the IgA antibody of this disclosure is a recombinant purified antibody SNK0001AR obtained by determining the nucleic acid sequence encoding the IgM antibody SNK0001M produced by hybridomas obtained from spleen-derived B cells and applying recombinant technology to produce an IgA antibody. The antibody SNK0001AR has the following amino acid sequence structure. TIFF0007862032000003.tif176169

[0060] In one embodiment, the IgA antibody disclosed herein is The heavy chain variable region includes 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, which are represented by the amino acid sequence of SEQ ID NO: 17, and The light chain variable region includes 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, which are part of the light chain variable region containing the amino acid sequence represented by Sequence ID No. 18. TIFF0007862032000004.tif45155

[0061] In one embodiment, the IgA antibody disclosed herein is Heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 11, A heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 12, and Heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 13, Heavy chain variable region including, Light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 14, Light chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 15, Light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 16 Includes a light chain variable region. TIFF0007862032000005.tif51155

[0062] In one embodiment, the IgA antibody disclosed herein is A heavy chain variable region including the amino acid sequence represented by Sequence ID No. 17 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and Light chain variable region containing the amino acid sequence represented by Sequence ID No. 18 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. Includes.

[0063] In one embodiment, the IgA antibody of this disclosure is a recombinant purified antibody SNK0002AR obtained by determining the nucleic acid sequence of the IgM antibody SNK0002M produced by hybridomas obtained from spleen-derived B cells and applying recombinant technology to produce an IgA antibody. The antibody SNK0002AR has the following amino acid sequence structure. TIFF0007862032000006.tif172169

[0064] In one embodiment, the IgA antibody disclosed herein is The heavy chain variable region includes 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, which are represented by the amino acid sequence of SEQ ID NO: 27, and The light chain variable region includes 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, which are part of the light chain variable region containing the amino acid sequence represented by Sequence ID No. 28. TIFF0007862032000007.tif48155

[0065] In one embodiment, the IgA antibody disclosed herein is Heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 21, Heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 22, and Heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 23, Heavy chain variable region including, Light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 24, Light chain CDR2 containing the amino acid sequence represented by Sequence ID No. 25, Light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 26 Includes a light chain variable region. TIFF0007862032000008.tif50154

[0066] In one embodiment, the IgA antibody disclosed herein is A heavy chain variable region including the amino acid sequence represented by Sequence ID No. 27 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and Light chain variable region containing the amino acid sequence represented by Sequence ID No. 28 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. Includes.

[0067] In one embodiment, the IgA antibody of this disclosure is IgA antibody SNK0003A produced by hybridomas obtained from B cells derived from the lamina propria of the intestinal mucosa. A recombinant purified antibody SNK0003AR, produced by determining the nucleic acid sequence encoding antibody SNK0003A and applying recombinant technology, can also be used in the same manner. The antibody SNK0003A has the following amino acid sequence structure. TIFF0007862032000009.tif167168

[0068] In one embodiment, the IgA antibody disclosed herein is Monoclonal antibodies that bind to Clostridium difficile bacteria, Heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 31, Heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 32, and Heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 33 A heavy chain variable region including, Light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 41, Light chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 42, Light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 43, This antibody contains a light chain variable region that includes [specific component].

[0069] In one embodiment, the IgA antibody disclosed herein is A heavy chain variable region comprising the amino acid sequence represented by Sequence ID No. 37 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and Light chain variable region containing the amino acid sequence represented by Sequence ID No. 38 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. Includes.

[0070] In one embodiment, the IgA antibody disclosed herein is A heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 37, and, For a reference antibody containing a light chain variable region that includes the amino acid sequence represented by SEQ ID NO: 38, In at least one region selected from the heavy chain CDR1-3, light chain CDR1-3, and light chain FR1, there is at least one amino acid mutation, This antibody binds to the amino acid sequence RQEEHIELIAS (SEQ ID NO: 72) in the SHMT protein of E. coli and the amino acid sequence VLDMMKLEKPE (SEQ ID NO: 73) in the iPGM protein of C. difficile. TIFF0007862032000010.tif53154

[0071] At least one amino acid mutation in the above antibody can be identified by applying techniques available to those skilled in the art, by utilizing the results of structural analysis of the antibody having the amino acid sequence of the reference antibody and the E. coli SHMT protein, and the conjugate of the antibody having the amino acid sequence of the reference antibody and the 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.

[0072] Such reference antibodies are not particularly limited as long as they have W27G2_HV (SEQ ID NO: 37) as the heavy chain variable region and W27G2_LV (SEQ ID NO: 38) as the 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 hybridomas obtained from B cells derived from the lamina propria of the intestinal mucosa. TIFF0007862032000011.tif90169

[0073] The fact that antibodies possessing the identified mutations actually bind to the amino acid sequence RQEEHIELIAS in the E. coli SHMT protein and the amino acid sequence VLDMMKLEKPE in the C. difficile iPGM protein can be confirmed by methods well known to those skilled in the art, such as ELISA and Western blotting.

[0074] In one embodiment, the antibody that binds to the C. difficile bacterial cell of this disclosure is Heavy chain CDR1 containing the amino acid sequence X1YYIH, Heavy chain CDR2 containing the amino acid sequence RIDPENX2X3TTYAPKFQ Heavy chain CDR3 containing the amino acid sequence of YCARSTVL, Heavy chain variable region including, Light chain CDR1 containing the amino acid sequence RX4SQSIVHTNG, Light chain CDR2 containing the amino acid sequence of KLLIYKV, Light chain CDR3 containing the amino acid sequence GVYYFQGS, Includes a light chain variable region, Light chain FR1 The amino acid sequence of TPLSLPVSLGDQA or It contains the amino acid sequence SPASX5SVSLGDRX6, X1, X2, and X3 are antibodies that are independently either neutral polar amino acids or acid polar amino acids, X4 is an antibody that is either a nonpolar amino acid or a neutral polar amino acid, and X5 and X6 are antibodies that are independently either nonpolar amino acids. be.

[0075] The above antibody was designed, based on three-dimensional structural analysis, to have a binding mode similar to that of the W27G2 antibody to the amino acid sequence RQEEHIELIAS in the SHMT protein of E. coli and the amino acid sequence VLDMMKLEKPE in the iPGM protein of C. difficile. In fact, it exhibits a binding mode and physiological activity similar to that of the W27G2 antibody against these bacteria.

[0076] In one embodiment, the IgA antibody disclosed herein is Binds to Clostridium difficile bacteria, Heavy chain CDR1 containing the amino acid sequence X1YYIH, Heavy chain CDR2 containing the amino acid sequence RIDPENX2X3TTYAPKFQ Heavy chain CDR3 containing the amino acid sequence of YCARSTVL, Heavy chain variable region including, Light chain CDR1 containing the amino acid sequence RX4SQSIVHTNG, Light chain CDR2 containing the amino acid sequence of KLLIYKV, Light chain CDR3 containing the amino acid sequence GVYYFQGS, Includes a light chain variable region, Light chain FR1, The amino acid sequence of TPLSLPVSLGDQA or The amino acid sequence SPASX5SVSLGDRX6 is included. An antibody in which X1, X2, and X3 are each independently neutral polar amino acids or acid polar amino acids, X4 is a nonpolar amino acid or neutral polar amino acid, and X5 and X6 are each independently nonpolar amino acids. This antibody consists of X1 aspartic acid, X2 aspartic acid, X3 glutamic acid, and X4 alanine.

[0077] In one embodiment, the heavy chain of the IgA antibody of this disclosure is Heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 31 or 41, Heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NOs. 32, 42-44, A heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 33, Includes. TIFF0007862032000012.tif57154

[0078] In one embodiment, the heavy chain of the IgA antibody disclosed herein is Heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 31, Heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 32, A heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 33, Includes.

[0079] In one embodiment, the heavy chain of the IgA antibody of this disclosure is Heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 31, Heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 42, A heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 33, Includes.

[0080] In one embodiment, the heavy chain of the IgA antibody of this disclosure is Heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 31, Heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 43, A heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 33, Includes.

[0081] In one embodiment, the heavy chain of the IgA antibody of this disclosure is Heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 31, Heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 44, A heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 33, Includes.

[0082] In one embodiment, the heavy chain of the IgA antibody of this disclosure is Heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 41, Heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 32, A heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 33, Includes.

[0083] In one embodiment, the heavy chain of the IgA antibody of this disclosure is Heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 41, Heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 42, A heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 33, Includes.

[0084] In one embodiment, the heavy chain of the IgA antibody of this disclosure is Heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 41, Heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 43, A heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 33, Includes.

[0085] In one embodiment, the heavy chain of the IgA antibody of this disclosure is Heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 41, Heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 44, A heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 33, Includes.

[0086] In one embodiment, the light chain of the IgA antibody of this disclosure is Light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 34 or 52, Light chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 35, Light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 36, Includes. TIFF0007862032000013.tif38155

[0087] In one embodiment, the light chain of the IgA antibody of this disclosure is Light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 34, Light chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 35, Light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 36, Includes.

[0088] In one embodiment, the light chain of the IgA antibody of this disclosure is Light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 52, Light chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 35, Light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 36, Includes.

[0089] In one embodiment, the IgA antibody of this disclosure includes a sequence that binds to Protein L in the light chain variable region. The antibody molecule or its antigen-binding fragment containing the sequence that binds to Protein L can be purified using a Protein L column.

[0090] In one embodiment, the IgA antibody comprising the Protein L-binding sequence of the present disclosure comprises a light chain variable region comprising the SPASX5SVSLGDRX6 amino acid sequence, where X5 and X6 are independently nonpolar amino acids.

[0091] In one embodiment, the IgA antibody comprising the Protein L-binding sequence of the present disclosure comprises a light chain variable region comprising the amino acid sequence SPASX5SVSLGDRX6, where X5 is leucine or methionine and X6 is alanine or valine.

[0092] In one embodiment, the IgA antibody comprising a Protein L-binding sequence of the present disclosure comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 53 to 55. TIFF0007862032000014.tif39158

[0093] In one embodiment, an IgA antibody comprising a Protein L-binding sequence of the present disclosure comprises a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 54. This sequence was created by introducing a mutation into the FR1 region of the light chain of the W27G2 antibody, specifically the sequence TPLSLPVSLGDQA (Sequence ID 56), so that Protein L can bind to the antibody molecule.

[0094] In one embodiment, an IgA antibody or its antigen-binding fragment containing a sequence that binds to Protein L of the present disclosure includes a light chain variable region containing the amino acid sequence represented in SEQ ID NO: 55. This sequence is a modified version of the sequence in which further mutations have been introduced into the FR1 region of the light chain variable region, which includes the amino acid sequence represented by SEQ ID NO: 54, to allow Protein L and the antibody molecule to bind more strongly.

[0095] In one embodiment, an IgA antibody or its antigen-binding fragment containing a sequence that binds to Protein L of the present disclosure includes a light chain variable region containing the amino acid sequence represented in SEQ ID NO: 58. This sequence is a modified version of the sequence in which further mutations have been introduced into the FR1 region of the light chain variable region, which includes the amino acid sequence represented by SEQ ID NO: 54, to allow Protein L and the antibody molecule to bind more strongly.

[0096] The IgA antibody of this disclosure may have any combination of the heavy chain and light chain described above, and may or may not include a sequence that binds to Protein L.

[0097] In one embodiment, the IgA antibody or its antigen-binding fragment of the present disclosure is Heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 31 or 41, Heavy chain CDR2 containing an amino acid sequence represented by one of SEQ ID NOs: 32, 42-44, and A heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 33, Heavy chain variable region including; Light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 34 or 52, Light chain CDR2 containing the amino acid sequence represented by Sequence ID No. 35, and A light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 36; and, Light chain FR1 containing the amino acid sequence represented by one of sequence numbers 53-56, This antibody contains a light chain variable region that includes [specific component].

[0098] For example, the IgA antibody of this disclosure includes a combination of any of the following heavy chain variable regions and any of the light chain variable regions. TIFF0007862032000015.tif165169TIFF0007862032000016.tif119163

[0099] Preferably, the IgA antibody of this disclosure comprises the following combination of heavy chain variable region and light chain variable region. TIFF0007862032000017.tif113153

[0100] In one embodiment, the IgA antibody and related antibodies used as IgA in this disclosure are shown below. TIFF0007862032000018.tif83159TIFF0007862032000019.tif232156TIFF0007862032000020.tif231157TIFF0007862032000021.tif231156TIFF0007862032000022.tif232158TIFF0007862032000023.tif148156Antibodies produced in CHO cells by introducing mutations into the heavy or light chain of the W27G2 antibody in a manner that does not substantially alter the antigen specificity are called RS mutant recombinant purified antibodies. These RS mutant recombinant purified antibodies also have mutations for binding to protein L.

[0101] The IgA antibody of this disclosure may have mutations in its constituent amino acid sequence, such as in its heavy chain variable region, light chain variable region, or heavy chain CDR1-3 or light chain CDR1-3, as long as it does not lose its antigen-binding properties. Such mutations may be, but are not limited to, substitutions, deletions, or insertions. For example, if a substitution occurs, a conservative substitution technique can be employed. Furthermore, by analyzing the binding mode between the antibody and antigen in detail using methods such as three-dimensional structural analysis, the IgA antibody of this disclosure can be given various mutations as long as it does not lose its antigen-binding properties.

[0102] (nucleic acid) The nucleic acid encoding the antibody or its antigen-binding fragment of this disclosure may be a ribonucleotide or a deoxynucleotide. Furthermore, 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, depending on the type of host cell used during manufacturing, codon frequencies and other factors can be appropriately selected. In one embodiment, the nucleic acid encoding the antibody or its antigen-binding fragment of this disclosure is used to express and manufacture the antibody or its antigen-binding fragment according to this disclosure.

[0103] When the base nucleic acid encoding the antibody or its antigen-binding fragment encodes the antigen-binding fragment, for example, the two domains of the Fv fragment, VL and VH, may be encoded by separate nucleic acid molecules, or a single nucleic acid may be used to encode a single protein chain (single-stranded Fv(scFv)) in which the VL and VH region pair forms a monovalent molecule.

[0104] Sequence information relating to the base nucleic acid encoding the antibody or its antigen-binding fragment of this disclosure is exemplified below. TIFF0007862032000024.tif79156TIFF0007862032000025.tif235161TIFF0007862032000026.tif228156TIFF00078620320 00027.tif226155TIFF0007862032000028.tif228157TIFF0007862032000029.tif222156TIFF0007862032000030.tif230162 TIFF0007862032000031.tif224157TIFF0007862032000032.tif225157TIFF0007862032000033.tif232154TIFF0007862032 000034.tif231155TIFF0007862032000035.tif232155TIFF0007862032000036.tif233155TIFF0007862032000037.tif83157

[0105] (Gastrointestinal contents or excrement) The gastrointestinal contents or excrement used in the method of this disclosure are not particularly limited, and those skilled in the art can appropriately select specific gastrointestinal contents or excrement for use, taking into consideration the purpose and workability. In a preferred embodiment, the gastrointestinal contents or excrement used in the method of this disclosure is feces. The gastrointestinal contents or excrement may be obtained by methods known in the art, for example, from inside the lumen of the gastrointestinal tract using a laparoscope, or a portion of the excrement may be obtained.

[0106] (Modification of gastrointestinal contents or excrement by IgA antibodies) In the methods disclosed herein, modification of gastrointestinal contents or excrement with an IgA antibody encompasses a variety of processes including contacting the IgA antibody with the gastrointestinal contents or excrement.

[0107] In one embodiment, gastrointestinal contents or excrement are modified by contact with an IgA antibody that restores the gut microbiota of an IBD patient before administration to a subject. In one embodiment, following the contact of the IgA antibody with the gastrointestinal contents or excrement, the IgA antibody is separated from the gastrointestinal contents or excrement along with its conjugates. In one embodiment, after contact with the IgA antibody with the gastrointestinal contents or excrement, the IgA antibody is used without being separated from the gastrointestinal contents or excrement. In a preferred embodiment, after contact with the IgA antibody with the gastrointestinal contents or excrement, bacteria that bind to the IgA antibody are removed from the gastrointestinal contents or excrement along with the IgA antibody. The gastrointestinal contents or excrement may undergo further processing before or after modification with the IgA antibody. For example, a bacterial flora can be purified from the gastrointestinal contents or excrement, and this bacterial flora can be modified with the IgA antibody and used as modified gastrointestinal contents or excrement. In another embodiment, after modifying gastrointestinal contents or excrement with an IgA antibody, a bacterial flora can be purified from the modification product and used as the modified gastrointestinal contents or excrement.

[0108] In one embodiment, gastrointestinal contents or excrement are modified by contact with IgA antibodies that restore the gut microbiota of IBD patients after administration to the subject. For example, by co-administering a composition containing gastrointestinal contents or excrement and a composition containing IgA antibodies, the gastrointestinal contents or excrement after administration to the subject can be modified with IgA antibodies. The composition containing gastrointestinal contents or excrement and the composition containing IgA antibodies may be contained in separate dose units or in a single dose unit. If the composition containing gastrointestinal contents or excrement and the composition containing IgA antibodies are contained in separate dose units, these compositions can be administered in any order, frequency, schedule, and dosage combination depending on the purpose. In a preferred embodiment, the composition containing gastrointestinal contents or excrement is administered first, followed by the composition containing IgA antibodies.

[0109] The amount of IgA antibody used to modify gastrointestinal contents or excrement is not particularly limited and is adjusted by those skilled in the art, taking into account the purpose, workability, etc. For example, when applied to human excrement, the amount of IgA antibody can be applied in the range of 1 mg to 1 g per gram of excrement. The amount of IgA antibody applied can be further adjusted according to the number of bacteria contained in the excrement.

[0110] (Method of administering modified gastrointestinal contents or excrement) In the methods of the present disclosure, the method of administering the IgA antibody-modified gastrointestinal contents or excrement to an IBD patient is not particularly limited as long as it can affect the patient's intestinal microbiota. For example, it may be administered by general fecal microbiota transplantation. The IgA antibody-modified gastrointestinal contents or excrement of the present disclosure may be administered, for example, by colonoscopy, suppositories, enemas, rectal administration via upper endoscopy, upper push-type enteroscopy, or intubation through the nose or mouth via a nasogastric tube, nasojejunal tube, or nasojejunal tube.

[0111] Furthermore, the gastrointestinal contents or excretions modified with the IgA antibody of this disclosure may also be administered orally in the form of solids such as pills, tablets, suspensions, gels, gel tablets, semi-solids, tablets, sachets, lozenges, or capsules or microcapsules, or as intestinal preparations, or as compositions such as liquids, suspensions, gels, gel tablets, semi-solids, tablets, sachets, lozenges, or capsules.

[0112] Compositions containing gastrointestinal contents or excrement modified with the IgA antibody of this disclosure may undergo further processing before administration. Furthermore, compositions containing gastrointestinal contents or excrement modified with the IgA antibody of this disclosure may be lyophilized, freeze-dried, frozen, or processed into powder.

[0113] The compositions of this disclosure contain, in one embodiment, one or more pharmaceutically acceptable carriers. Carriers known in the art may be used and are appropriately selected depending on the dosage form and mode of administration of the composition. For example, the carrier may be a diluent or excipient, such as a bulking agent, binder, wetting agent, disintegrant, surfactant, flow enhancer, or lubricant. Generally, the carrier may be a solid (including powder), liquid, or a combination thereof. Each carrier is preferably acceptable in that it is compatible with the other components of the composition and is harmless to the target. The carrier may be biocompatible and inert. For example, the carrier is formulated to allow the composition to maintain the viability of its biological material until it is delivered to the appropriate site.

[0114] When the compositions of this disclosure are used as oral compositions, they may contain an inert diluent or an edible carrier. For example, they may contain a sweetener such as sucrose or saccharin, or a flavoring agent such as peppermint, methyl salicylate, or orange flavor. Alternatively, the oral compositions of this disclosure may be prepared by combining gastrointestinal contents or excrement modified with an IgA antibody with a food. In one embodiment, the food used for administration is chilled and provided in the form of, for example, ice cream. In one embodiment, an oral composition containing a bacterial flora purified from gastrointestinal contents or excrement can be provided as a food composition.

[0115] The amount of gastrointestinal contents or excrement contained per dose unit of a composition containing gastrointestinal contents or excrement modified with the IgA antibody of this disclosure can be determined by a person skilled in the art, taking into consideration the route of administration, the form of administration, the patient's condition, the frequency of administration, etc. For example, the daily dose of the active ingredient (modified gastrointestinal contents or excrement) can be approximately 0.001 to 1000 milligrams (mg) / kilogram (kg) of body weight, but is not limited thereto. For example, it can be used in amounts such as 0.01 to 100 mg / kg of body weight, 0.1 to 50 mg / kg of body weight, or 1 to 10 mg / kg of body weight.

[0116] 2. A composition for use in a method of treating IBD using gastrointestinal contents or excrement modified with IgA antibodies. In one embodiment, this disclosure provides a composition for treating IBD, comprising gastrointestinal contents or excretions modified with an IgA antibody, and a method for producing the same.

[0117] The composition for treating IBD according to this disclosure is, in one embodiment, a composition comprising gastrointestinal contents or excrement modified with an IgA antibody, as described in "1. Method for treating IBD using gastrointestinal contents or excrement modified with an IgA antibody" above. The gastrointestinal contents or excrement contained in the compositions for treating IBD of this disclosure are those described in (Gastrointestinal contents or excrement) of "1. Method for treating IBD using gastrointestinal contents or excrement modified with IgA antibodies" above. The gastrointestinal contents or excrement are modified with IgA antibodies that can restore the gastrointestinal microbiota of an IBD patient, in accordance with the descriptions of (IgA antibodies) and (Modification of gastrointestinal contents or excrement with IgA antibodies) above. In one embodiment, gastrointestinal contents or excretions modified with IgA antibodies are included in the composition as a purified and isolated bacterial flora.

[0118] In one embodiment, the composition for treating IBD according to this disclosure is a composition comprising an IgA antibody that modifies gastrointestinal contents or excrement after administration, as described in "1. Method for treating IBD using gastrointestinal contents or excrement modified with an IgA antibody" above.

[0119] The compositions for treating IBD according to this disclosure are formulated either mixed with a carrier or without a carrier, so as to have properties suitable for the various administration routes and dosages described in "1. Method for treating IBD using modified gastrointestinal contents or excrement" (method for administering modified gastrointestinal contents or excrement and compositions containing the same) above.

[0120] 3. Method for obtaining IgA antibodies to restore the gut microbiota in IBD patients. . In one aspect, this disclosure provides a method for screening IgA antibodies that promote a healthy gastrointestinal microbiota in patients with IBD. The screening method described herein includes, in one embodiment, the following steps: (B1) The process of preparing the gastrointestinal contents or excrement of an IBD patient. (B2) A step of modifying the gastrointestinal contents or excreta with a candidate IgA antibody, and (B3) A step to confirm that the modified gastrointestinal contents or excrement can restore the health of the bacterial flora in the gastrointestinal tract.

[0121] In the method of this disclosure, (B1) the step of preparing the gastrointestinal contents or excrement of an IBD patient is performed in accordance with the description of (gastrointestinal contents or excrement) in "1. Method for treating IBD using gastrointestinal contents or excrement modified with IgA antibody" above.

[0122] In the method of this disclosure, the candidate IgA antibody used in the step of modifying the gastrointestinal contents or excreta with the candidate IgA antibody (B2) may have an amino acid sequence derived from the same species as the IBD patient, or it may have amino acids derived from a different species such as a closely related organism. Specifically, examples include human-derived, mouse-derived, rat-derived, hamster-derived, rabbit-derived, goat-derived, donkey-derived, pig-derived, cattle-derived, horse-derived, chicken-derived, monkey-derived, chimpanzee-derived, camel-derived, llama-derived, etc.

[0123] The candidate IgA antibody may also be conjugated with other compounds or fused with heterologous peptides.

[0124] In the method disclosed herein, the modification in the step of modifying the gastrointestinal contents or excrement with a candidate IgA antibody (B2) is carried out in accordance with the description of (Modification of gastrointestinal contents or excrement with IgA antibody) in "1. Method for treating IBD using gastrointestinal contents or excrement modified with an IgA antibody" above.

[0125] In the method of this disclosure, the step of confirming (B3) that the modified gastrointestinal contents or excrement can restore the gut microbiota is carried out by the various methods described in (IgA antibody) of "1. Method for treating IBD using gastrointestinal contents or excrement modified with IgA antibody" above, in the method for confirming that IgA antibody can restore the gut microbiota of IBD patients.

[0126] In one embodiment, this disclosure provides a method for producing an IgA antibody, comprising each step of the above-described screening method.

[0127] 4. A method for processing the contents or excrement of the gastrointestinal tract of IBD patients in vitro using IgA antibodies. This disclosure provides, in one embodiment, a method for modifying the gastrointestinal contents or excretions of an IBD patient using an IgA antibody. The method disclosed herein includes, in one embodiment, the following steps. (C1) Process of preparing gastrointestinal contents or excrement from an IBD patient. (C2) A step of contacting the contents of the digestive tract or excrement with an IgA antibody that restores the bacterial flora in the digestive tract in vitro.

[0128] The step of (C1) preparing gastrointestinal contents or excrement of an IBD patient in the method of this disclosure is carried out in accordance with the description of (gastrointestinal contents or excrement) in "1. Method for treating IBD using gastrointestinal contents or excrement modified with IgA antibody" above. The IgA antibody used in step (C2) of the method of this disclosure, in vitro, in which the gastrointestinal contents or excrement are brought into contact with an IgA antibody that restores the gastrointestinal bacterial flora, is one that has been confirmed to restore the gastrointestinal bacterial flora of IBD patients by various methods described in (IgA antibody) of "1. Method for treating IBD using gastrointestinal contents or excrement modified with an IgA antibody" above.

[0129] Contact between the gastrointestinal contents or excrement and the IgA antibody is typically carried out by mixing the two. Before mixing, the gastrointestinal contents or excrement may or may not be diluted with a diluent.

[0130] The gastrointestinal contents or excretions of IBD patients modified with IgA antibodies obtained as a result of the method disclosed herein can be administered to IBD patients as an active ingredient in a pharmaceutical product for improving IBD.

[0131] 5. Compositions containing IgA antibodies for modifying the gastrointestinal contents or excretions of IBD patients in vitro. In one embodiment, this disclosure provides a composition containing an IgA antibody for processing the contents or excretions of the gastrointestinal tract of IBD patients in vitro, and a method for producing the same. The IgA antibody contained in the composition of this disclosure has been confirmed to be able to restore the gastrointestinal microbiota of IBD patients by various methods described in "1. Method for treating IBD using gastrointestinal contents or excrement modified with IgA antibody" above, as a method for confirming that the IgA antibody can restore the gastrointestinal microbiota of IBD patients.

[0132] The compositions of this disclosure are formulated in one embodiment with one or more pharmaceutically acceptable carriers in various forms suitable for modifying the gastrointestinal contents or excretions of IBD patients. For example, they may be formulated in the form of solutions, suspensions, gels, capsules or microcapsules. Diluents or excipients, such as volume extenders, binders, wetting agents, disintegrants, surfactants, flow enhancers, and lubricants, may be used as carriers.

[0133] The amount of IgA antibody contained in the composition of this disclosure is not particularly limited and can be adjusted by those skilled in the art considering the purpose, workability, etc. For example, when applied to human excrement, the formulation can be prepared to apply IgA antibody in a range of 1 mg to 1 g per gram of excrement. The amount of IgA antibody applied can be further adjusted according to the number of bacteria contained in the excrement.

[0134] 6. A method for testing a subject using a pharmaceutical product containing IgA antibodies to diagnose the presence or absence of IBD or the risk of developing IBD. This disclosure provides a method for testing a subject using an IgA antibody that promotes the health of the gastrointestinal bacterial flora, in order to diagnose whether or not the subject has IBD or is at risk of developing IBD. The method disclosed herein includes, in one embodiment, the following steps: (D1) A step to prepare IgA antibodies to restore the gut microbiota of IBD patients; (D2) A step of bringing the contents of the gastrointestinal tract or excrement of the subject into contact with the IgA antibody; and (D3) A step to identify bacteria that bind to the IgA antibody. Furthermore, if the IgA antibody shows binding to IBD-related bacteria, the subject is treated as having IBD or being at risk of developing IBD.

[0135] The IgA antibody used in this study is an IgA antibody that has been confirmed to be able to restore the gut microbiota in IBD patients. The ability of this IgA antibody to restore the gut microbiota can be confirmed, for example, by following the same method as described above for the method of confirming that the IgA antibody can restore the gut microbiota in IBD patients in the case of the method of treating IBD using gut contents or excrement modified with the IgA antibody.

[0136] The step of (D2) of this disclosure, in which the gastrointestinal contents or excrement are brought into contact with IgA antibody, is typically carried out by mixing the two. Before mixing, the gastrointestinal contents or excrement may or may not be diluted with a diluent. The amount of IgA antibody used can be appropriately determined by those skilled in the art depending on the characteristics of the sample, the desired sensitivity, etc. For example, when applied to human excrement, the amount of IgA antibody applied can be in the range of 1 mg to 1 g per gram of excrement. The amount of IgA antibody applied can be further adjusted depending on the number of bacteria contained in the excrement.

[0137] The step of (D3) of this disclosure to identify bacteria that bind to the IgA antibody is carried out, for example, by comparing the amount of the IgA antibody bound to intestinal bacteria known to exacerbate IBD in gastrointestinal contents or excrement from the patient with that from gastrointestinal contents or excrement from a healthy person. In another embodiment, the analytical step can be carried out, for example, by contacting the patient's gastrointestinal contents or excrement with the IgA antibody and comparing the binding profile created based on the types and amounts of bacteria bound to the IgA antibody with the gastrointestinal contents or excrement from a healthy person and comparing the binding profile created based on the types and amounts of bacteria bound to the IgA antibody. In one example, the binding profile can be represented by quantitative data using the IgA Index, which is determined from the amount of IgA that each bacterium binds to and does not bind to IgA (Andrew L. Kau, et al Sci Transl Med 7, 2015; Hirosuke Sugahara, et al., Frontiers in Microbiology 8, 1757, 2017). For example, the IgA Index is calculated according to the following formula: TIFF0007862032000038.tif43139

[0138] The method for testing subjects for the treatment of IBD according to this disclosure can use multiple types of IgA antibodies. In one embodiment, the test according to this disclosure is performed using multiple types of IgA antibodies that have been confirmed to be able to restore the gastrointestinal microbiota in different IBD patients, and if one or more of these antibodies show specific binding to IBD-related bacteria, the subject is treated as having IBD or being at risk of developing IBD. If multiple types of IgA among the tested IgA antibodies show specific binding to IBD-related bacteria, it can be more reliably inferred that the subject has IBD or is at risk of developing IBD. For example, if one or more, two or more, three or more, four or more, or five or more IgA capable of restoring the gastrointestinal microbiota of IBD patients bind to IBD-related bacteria, the subject is treated as having IBD or being at risk of developing IBD.

[0139] Whether a subject has IBD or is at risk of developing IBD can be expressed using qualitative or quantitative indicators. For example, this can be quantified using indicators representing the amount or specificity of IgA antibody binding to IBD-related bacteria, or the quantitative data can be categorized and presented qualitatively.

[0140] When a subject is treated as having IBD or being at risk of developing IBD, such treatment includes any treatment related to maintaining or improving the subject's health. This includes, but is not limited to, classifying or tagging the patient as having IBD or being at risk of developing IBD in an information processing system, providing lifestyle improvement guidance to the patient as having IBD or being at risk of developing IBD, and treating and preventing IBD.

[0141] When treating a subject for the treatment of IBD according to this disclosure, the treatment method is not particularly limited, and known IBD treatment methods may be used. In one embodiment, the IBD treatment method includes administering to a subject gastrointestinal contents or excretions modified with an IgA antibody that showed binding to IBD-associated bacteria in the study.

[0142] 7. Pharmaceuticals containing IgA antibodies, used to test subjects to determine whether or not IBD treatment is necessary. In one aspect, this disclosure provides a pharmaceutical product for diagnosing the presence or risk of developing IBD, comprising an IgA antibody that promotes the health of the gastrointestinal microbiota in IBD patients.

[0143] The diagnostic agent disclosed herein is used in "6. A method for testing a subject for the treatment of IBD using a diagnostic agent containing an IgA antibody," and contains an IgA antibody having the properties described in that method.

[0144] The compositions of this disclosure are formulated in one embodiment with one or more pharmaceutically acceptable carriers in various forms suitable for testing the gastrointestinal contents or excretions of IBD patients. For example, they may be formulated in the form of solutions, suspensions, gels, capsules, or microcapsules. Diluents or excipients, such as volume extenders, binders, wetting agents, disintegrants, surfactants, flow enhancers, and lubricants, may be used as carriers.

[0145] The amount of IgA antibody contained in the composition of this disclosure is not particularly limited and can be adjusted by those skilled in the art considering the purpose, workability, etc. For example, when applied to human excrement, the formulation can be prepared to apply IgA antibody in a range of 1 mg to 1 g per gram of excrement. The amount of IgA antibody applied can be further adjusted according to the number of bacteria contained in the excrement.

[0146] 8. A method for testing patients treated with IBD therapeutic drugs containing gastrointestinal contents or excrement using a diagnostic agent containing IgA antibodies. In one embodiment, this disclosure provides a method for testing patients treated with an IBD therapeutic agent containing gastrointestinal contents or excrement using a diagnostic agent containing an IgA antibody. The method disclosed herein includes, in one embodiment, the following steps: (E1) The process of obtaining gastrointestinal contents or excrement from patients who are currently receiving, are scheduled to receive, or have received IBD treatments containing gastrointestinal contents or excrement; (E2) A step of bringing the contents of the gastrointestinal tract or excrement into contact with IgA antibodies; and (E3) A step of analyzing bacteria that bind to the IgA antibody. Furthermore, if the analysis results indicate a healthy gastrointestinal microbiota, treatment with the IBD drug will be continued.

[0147] In the method of this disclosure, the patients being studied are those who are currently receiving, are scheduled to receive, or have previously received an IBD treatment containing gastrointestinal contents or excrement.

[0148] The IBD therapeutic agent containing gastrointestinal contents or excrement of this disclosure is preferably an IBD therapeutic agent containing gastrointestinal contents or excrement modified with the IgA antibody used in this study. In one embodiment, the gastrointestinal contents or excrement contained in the IBD therapeutic agent of this disclosure is gastrointestinal contents or excrement obtained from the patient himself. Preferably, the IgA antibody used in this study is an IgA antibody that has been confirmed to be able to restore the gastrointestinal microbiota of the patient being tested.

[0149] The step of (D2) of this disclosure, in which the gastrointestinal contents or excrement are brought into contact with IgA antibody, is typically carried out by mixing the two. Before mixing, the gastrointestinal contents or excrement may or may not be diluted with a diluent. The amount of IgA antibody used can be appropriately determined by those skilled in the art depending on the characteristics of the sample, the desired sensitivity, etc. For example, when applied to human excrement, the amount of IgA antibody applied can be in the range of 1 mg to 1 g per gram of excrement. The amount of IgA antibody applied can be further adjusted depending on the number of bacteria contained in the excrement.

[0150] The step of (D3) of this disclosure, which involves analyzing bacteria that bind to the IgA antibody, is carried out, for example, by comparing the amount of IgA antibody bound to intestinal bacteria known to exacerbate IBD in gastrointestinal contents or excrement from the patient with that from gastrointestinal contents or excrement from a healthy person. In another embodiment, the analysis step can be carried out, for example, by contacting gastrointestinal contents or excrement from the patient with the IgA antibody and comparing a binding profile created based on the types and amounts of bacteria bound to the IgA antibody with gastrointestinal contents or excrement from a healthy person and comparing a binding profile created based on the types and amounts of bacteria bound to the IgA antibody.

[0151] In the method of this disclosure, the analysis results of bacteria binding to the IgA antibody indicate a healthy gastrointestinal microbiota, which means, for example, that when comparing the amount of IgA antibody bound to intestinal bacteria known to exacerbate IBD in gastrointestinal contents or excrement from the patient and gastrointestinal contents or excrement from a healthy person, there is no significant difference between the two. In another embodiment, the analysis results of bacteria binding to the IgA antibody indicate a healthy gastrointestinal microbiota, which means, for example, when comparing a binding profile created based on the type and amount of bacteria bound to the IgA antibody after contacting gastrointestinal contents or excrement from the patient with the IgA antibody, with a binding profile created based on the type and amount of bacteria bound to the IgA antibody after contacting gastrointestinal contents or excrement from a healthy person with the IgA antibody, there is no significant difference in the profile of IBD-related bacteria.

[0152] 9. Diagnostic medical devices containing IgA antibodies for testing patients treated with IBD therapeutic drugs containing gastrointestinal contents or excrement.

[0153] In one aspect, this disclosure provides a diagnostic agent containing an IgA antibody for testing patients being treated with an IBD therapeutic agent containing gastrointestinal contents or excrement.

[0154] The diagnostic agent disclosed herein is used in "8. A method for testing patients treated with an IBD therapeutic agent containing gastrointestinal contents or excrement using a diagnostic agent containing IgA antibodies," and contains IgA antibodies having the properties described in that method.

[0155] The compositions of this disclosure are formulated in one embodiment with one or more pharmaceutically acceptable carriers in various forms suitable for testing the gastrointestinal contents or excretions of IBD patients. For example, they may be formulated in the form of solutions, suspensions, gels, capsules, or microcapsules. Diluents or excipients, such as volume extenders, binders, wetting agents, disintegrants, surfactants, flow enhancers, and lubricants, may be used as carriers.

[0156] The amount of IgA antibody contained in the composition of this disclosure is not particularly limited and can be adjusted by those skilled in the art considering the purpose, workability, etc. For example, when applied to human excrement, the formulation can be prepared to apply IgA antibody in a range of 1 mg to 1 g per gram of excrement. The amount of IgA antibody applied can be further adjusted according to the number of bacteria contained in the excrement.

[0157] In one embodiment, the methods described herein—1. A method for treating IBD using gastrointestinal contents or excrement modified with IgA antibodies, 6. A method for testing a subject for IBD treatment using a diagnostic agent containing IgA antibodies, and 8. A method for testing a patient treated with an IBD therapeutic agent containing gastrointestinal contents or excrement using a diagnostic agent containing IgA antibodies—and the compositions used in these methods can be used in combination, in whole or in part. For example, a patient designated for IBD treatment using "6. A method for testing a subject for IBD treatment using a diagnostic agent containing IgA antibodies" can be treated using "1. A method for treating IBD using gastrointestinal contents or excrement modified with IgA antibodies," and the course of treatment can be diagnosed using "8. A method for testing a patient treated with an IBD therapeutic agent containing gastrointestinal contents or excrement using a diagnostic agent containing IgA antibodies." The IgA antibodies used in these methods may be the same or different. [Examples]

[0158] 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.

[0159] Example 1: Binding characteristics of endogenous IgA antibodies

[0160] (Methods and Materials) • Bacterial extraction from human stool samples Human stool samples from patients with inflammatory bowel disease (n=12, of which 7 had ulcerative colitis and 5 had Crohn's disease, P1-P12) and healthy individuals (n=12, C1-C12) were provided by Shiga University of Medical Science and Kanazawa University. Healthy individuals were defined as volunteers who showed no abnormal values ​​in their medical checkup results. Stool samples were immediately placed in anaerobic packs after collection, stored frozen (-80°C), and sent to the University of Tokyo. In addition, a human stool sample from one patient with ulcerative colitis (P13) was provided by the Department of Gastroenterology, University of Tokyo, and used in the experiment on the same day. The samples received were prepared by preparing bacterial suspensions in an anaerobic chamber (80% N2, 10% H2, 10% CO2, Coy Laboratory Products) using the following method, then portioned and stored at -80°C for subsequent experiments. All reagents used in the anaerobic chamber were sterilized by passing them through a 0.22 μm filter and allowed to stand overnight in the anaerobic chamber to undergo anaerobic conditions. Frozen stool samples were dissolved in 30 ml of PBS in the anaerobic chamber, then suspended and centrifuged at 50 g for 15 minutes at 4°C. Samples provided by the University of Tokyo were weighed, suspended in 9 times the volume of PBS, and centrifuged at 50 g for 15 minutes at 4°C. The supernatant was collected in a separate 50 ml tube, thoroughly suspended, and then dispensed into 1.5 ml tubes in 1 ml portions. These were stored at -80°C as bacterial freeze stocks of human stool samples.

[0161] • Measurement of bacterial count concentration in bacterial freeze stocks of human stool samples The bacterial freeze stock of human fecal samples was thawed in an anaerobic chamber. 10 μl of the bacterial freeze stock was taken and diluted 1,000-fold with PBS. The measurement of the bacterial count was performed according to the protocol of the Cell Viability kit (BD). In this study, since all bacteria contained in human fecal samples were targeted, Propidium iodide (PI) for detecting dead bacteria was not used, and only Thiazole orange (TO) for staining nucleic acids and detecting bacteria was used to measure the bacterial count. PBS, Liquid Counting Beads (BD), and Thiazole orange (final concentration 42 nM, BD) were added to the bacterial dilution, and the mixture was reacted on ice for 10 minutes. The bacterial count measurement solution was subjected to flow cytometry analysis using a SONY SA3800 Analyzer (SONY). The bacterial count concentration of the bacterial dilution was calculated based on the number of Counting Beads detected in the analysis.

[0162] · 16S rRNA gene amplicon sequencing analysis of bacterial DNA Bacterial DNA was subjected to polymerase chain reaction (PCR) targeting the V3-V4 region of the variable region of the 16S rRNA gene. Primers were prepared for next-generation sequencers using Miseq and used with an index sequence added to distinguish the samples to be analyzed (see below). After the PCR reaction, 10× Loading Buffer (TAKARA) was added and agarose electrophoresis was performed. The gel for running the PCR samples was a 1.5% agarose gel (VWR life science) with LED StainG (LABTAS+). A 100 bp DNA Ladder (TAKARA) was used as the DNA size marker. Electrophoresis was performed at 100 V for 30 minutes. After electrophoresis, the PCR product of approximately 500 bp was excised and purified using the Fast Gene gel extraction kit (Nippon Genetics). The purified PCR product was electrophoresed again to confirm whether purification was successful. Sequence analysis was requested from TAK-Circulator Co., Ltd. using the Mi-seq Reagent Kit V3 (Illumina). The sequence data obtained by sequence analysis was subjected to bacterial flora analysis using Qiime2 software (ver. 2020-2).

[0163] The primer sequences for 16S rRNA gene amplicon sequence analysis are shown below. TIFF0007862032000039.tif16168XXX represents the index sequence, and the gray indicates the sequence targeting 16S rRNA, which is the template.

[0164] ·Calculation method of IgA Index The IgA Index indicates that a high value suggests that the bacteria are strongly bound to the IgA antibody, while a low value suggests that the bacteria are weakly bound to the IgA antibody. Each individual's IgA Index was calculated considering that the proportion of the total bacterial flora bound to the IgA antibody differs for each individual. IgA binding ability is the proportion of bacteria bound to the IgA antibody relative to the total bacterial flora. Using this, the proportion of IgA antibody-bound or non-bound bacteria (IgA+ taxon abundance, IgA- taxon abundance) was calculated for each individual, and then the IgA Index was determined (Andrew L. Kau, et al Sci Transl Med 7, 2015; Hirosuke Sugahara, et al., Frontiers in Microbiology 8, 1757, 2017). Specifically, the relative abundance of IgA-binding bacteria obtained by 16S rRNA analysis was multiplied by the IgA binding ability (the proportion of IgA antibody-binding bacteria to the total bacterial community) to obtain the taxon abundance of IgA antibody-binding bacteria. On the other hand, for non-antibody-binding bacteria, the relative abundance was multiplied by 1-IgA binding ability (the number obtained by subtracting the proportion of IgA antibody-binding bacteria from 100%) to obtain the taxon abundance of non-IgA antibody-binding bacteria. The IgA index was calculated using each taxon abundance.

[0165] • Isolation of RS_H000_L001 IgA antibody-bound and unbound bacteria from human stool samples using Magnetic Cell Sorter (MACS) and cell sorter. The procedure was performed in an anaerobic chamber. After bacterial count measurement, the bacteria from the human stool sample were mixed with PBS and incubated at 6 × 10⁻⁶ times. 7The bacterial count was adjusted to 10⁴ cells / ml. The mixture was then centrifuged at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. 100 μl of PBS containing 20% ​​normal rat serum (Wako) was added to the remaining bacterial cells, and the mixture was reacted on ice for 20 minutes. This inhibited nonspecific binding of the antibody used. FACS buffer was added to the bacterial suspension, and the mixture was centrifuged at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. Bacterial count: 6 × 10⁴ 6 15 μg of biotin-containing RS_H000_L001 IgA antibody was added to each cell, and the mixture was reacted on ice for 20 minutes. FACS buffer was added to the antibody reaction mixture, and the mixture was centrifuged at 8,000 g for 5 minutes at 4°C. The supernatant was removed. Bacterial count: 6 × 10⁶ 6 Microbeads-streptavidin (Milteny) was added to each cell and reacted on ice for 20 minutes. Bacterial isolation was performed according to the MACS column (LS column, Milteny) kit protocol. Bacteria adsorbed to the MACS column were collected as RS_H000_L001 IgA antibody-conjugated bacteria, and bacteria that passed through the column were collected as RS_H000_L001 IgA antibody-unconjugated bacteria. RS_H000_L001 IgA antibody-conjugated bacteria were concentrated again using the MACS column. The collected RS_H000_L001 IgA antibody-conjugated and unconjugated bacteria were centrifuged at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. RS_H000_L001 IgA antibody-conjugated and unconjugated bacteria recovered by MACS were treated with FACS buffer, TO (final concentration 42 nM, BD), and Phycoerythrin (PE)-anti mouse IgA antibody (final concentration 5 μg / ml, Biolegend), and reacted on ice in the dark for 10 minutes. Subsequently, RS_H000_L001 IgA antibody-conjugated and unconjugated bacteria were separated by cell sorting using a SONY Cell Sorter SH800 (SONY). The bacteria from each fraction recovered by cell sorting were re-analyzed using the SONY Cell Sorter SH800, and it was confirmed that the target fraction was recovered at a rate of 90% or more.

[0166] • Isolation of endogenous IgA antibody-bound and unbound bacteria from human stool samples using Magnetic Cell Sorter (MACS) and cell sorter. Bacteria from human stool samples were thawed in an anaerobic chamber. All reagents used in the anaerobic chamber were sterilized by passing them through a 0.22 μm filter and allowed to stand overnight in the anaerobic chamber to undergo anaerobic conditions. After bacterial counting, PBS was added to the sample to form a 6 × 10⁻⁶ solution. 7 The bacterial count was adjusted to 10⁴ cells / ml. Then, the mixture was centrifuged at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. 100 μl of PBS containing 20% ​​normal rat serum (Wako) was added to the remaining bacterial cells, and the mixture was reacted on ice for 20 minutes. This inhibited nonspecific binding of the antibody used. FACS buffer was added to the bacterial suspension, and the mixture was centrifuged at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. Bacterial count: 6 × 10⁴ 6 15 μl of Anti-IgA-PE human antibody (Milteny) was added to each cell, and the mixture was reacted on ice for 20 minutes. FACS buffer was added to the antibody reaction mixture, and the mixture was centrifuged at 8,000 g for 5 minutes at 4°C. The supernatant was removed. Bacterial count: 6 × 10⁶ 6 15 μl of Anti-PE Microbeads (Milteny) was added to each cell, and the mixture was reacted on ice for 20 minutes. Bacterial isolation was performed according to the MACS column (LS column, Milteny) kit protocol. Bacteria adsorbed to the MACS column were collected as human endogenous IgA antibody-conjugated bacteria, and bacteria that passed through the column were collected as human endogenous IgA antibody-unconjugated bacteria. The human endogenous IgA antibody-conjugated bacteria were concentrated again using the MACS column. The collected human endogenous IgA antibody-conjugated and unconjugated bacteria were centrifuged at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. FACS buffer and TO (final concentration 42 nM, BD) were added to the human endogenous IgA antibody-conjugated and unconjugated bacteria collected by MACS, and the mixture was reacted on ice in the dark for 10 minutes. Subsequently, human endogenous IgA antibody-bound and non-bound bacteria were isolated by cell sorting using a SONY Cell Sorter SH800 (SONY). The bacteria in each fraction recovered by cell sorting were re-analyzed using the SONY Cell Sorter SH800, and it was confirmed that the target fraction was recovered at a rate of over 90%.

[0167] (result) Endogenous polyclonal IgA antibody-conjugated bacteria were collected from healthy individuals and IBD patients, and their binding characteristics to various intestinal bacteria contained in feces collected from healthy individuals and IBD patients were tested. As a result, as shown in Figure 1, endogenous polyclonal IgA from healthy individuals showed a median IgA index > 0 for Enterobacteriaceae, Erysipelotrichaceae, Gemellaceae, and Bacteroidaceae, confirming its binding to these bacteria. In particular, it showed high binding affinity to Enterobacteriaceae. This bacterium has been linked to deterioration of the intestinal environment, such as causing colitis, and this suggests that endogenous IgA from healthy individuals plays a role in the body's defense function by binding to these bacteria that exacerbate IBD and suppressing their activity. On the other hand, endogenous polyclonal IgA from IBD showed a median IgA index > 0 for Lactobacillaceae, Veillonellaceae, Enterococcaceae, Enterobacteriaceae, Actinomycetaceae, and Bacteroidaceae, confirming its binding to these bacteria. It is noteworthy that endogenous polyclonal IgA from IBD patients exhibits significantly different binding characteristics from endogenous polyclonal IgA from healthy individuals, showing the highest specificity for Lactobacillaceae, a fermenting bacterium not typically associated with a worsening of the gut environment. This result suggests that the body's defense mechanism mediated by endogenous polyclonal IgA is not functioning properly in IBD patients.

[0168] Example 2: Decreased binding affinity of IgA from IBD patients to IBD-related bacteria

[0169] (Methods and Materials) • Method for preparing human IgA antibodies derived from stool samples Stool samples from healthy individuals or patients (frozen and stored at -80°C after collection) were mixed with an appropriate amount of PBS, and the mixture was dissolved and suspended at room temperature. All procedures were performed under aerobic conditions. The suspended solution was centrifuged at 2,000 g for 10 minutes at 4°C, and the supernatant was collected. The supernatant was then centrifuged again at 8,000 g for 10 minutes at 4°C, and the supernatant was collected. This procedure was repeated once more. An equal volume of saturated ammonium sulfate was added to the supernatant while stirring on ice, and the mixture was left to stand overnight at 4°C. The next day, the mixture was centrifuged at 10,000 g for 10 minutes at 4°C to obtain an ammonium sulfate precipitate. A 50% saturated ammonium sulfate solution was added to this precipitate, and the mixture was suspended and washed by centrifugation at 10,000 g for 10 minutes at 4°C. The precipitate was then dissolved in PBS, and further dialyzed with PBS to obtain a human stool-derived IgA antibody solution, which was then divided into smaller portions and frozen and stored at -20°C.

[0170] • Measurement of human IgA antibody titers derived from stool samples The concentration of IgA antibody in IgA antibody solutions extracted from stool samples was measured using a standard sandwich ELISA. Goat anti-human IgA INLB (final concentration 2 μg / ml, Southern Biotech) was used as the anti-human IgA antibody coated onto ELISA plates (C96 MaxiSorp Nunc Immuno Plate (Thermo Fisher)). The ELISA plates with the immobilized anti-human IgA antibody were washed three times with PBS, and 150 μl of PBS supplemented with 1% bovine serum albumin (BSA, Wako) was added to each well. The plates were then incubated overnight at 4°C for blocking. Human IgA isotype control (Genway, 1 mg / ml) was used as the control IgA antibody, and dilution series of control IgA antibodies and human endogenous IgA antibody solutions extracted from healthy individuals or patients were prepared in 96-well plates. Dilution was performed using PBS with 1% BSA to serially dilute to 1x, 1 / 3x, 1 / 10x, 1 / 30x, 1 / 100x, 1 / 300x, 1 / 1,000x, and 1 / 3,000x. After blocking, the PBS with 1% BSA was removed from the ELISA plate, and 50 μl of the serially diluted antibody was added to each well. The mixture was then reacted at room temperature for 1 hour. Next, the ELISA plate was washed three times with PBS with 0.05% Tween 20 (Chem Cruz), and then Alkaline Phosphatase-conjugated goat anti-human IgA (final concentration 0.5 μg / ml, Southern Biotech) was added as the secondary antibody. The mixture was then reacted at room temperature for 1 hour. For the color development substrate reaction solution, magnesium chloride (final concentration 2 mM) and Phosphatase substrate (Sigma) were dissolved in a solution of sodium bicarbonate (final concentration 6.5 mM) and sodium carbonate (final concentration 18.5 mM). After the plates reacted with the secondary antibody were washed again with PBS containing 0.05% Tween 20, 50 μl of the substrate reaction solution was added to each well, and the plates were shielded from light with aluminum foil to allow color development. The absorbance (OD 405 nm) was then measured using Tristar2 LB942 (BERTHOLD TECHNOLOGIES).The IgA antibody concentration of each sample was calculated by comparing it with the reaction curve of the control IgA antibody.

[0171] • Measurement of the binding affinity of human IgA antibodies to bacteria (using the ELISA method) Each bacterium was cultured overnight under optimal conditions aerobically or anaerobicly. Then, PBS was added, and the mixture was centrifuged at 8,000 g for 5 minutes at 4°C, with the supernatant removed. After counting, each bacterium was placed on an ELISA plate (C96 MaxiSorp Nunc Immuno Plate (Thermo Fisher)) with a bacterial count of 1.0 x 10⁶. 7 The bacteria were suspended in 50 mM sodium carbonate solution at a concentration of 50 μl / well. 50 μl / well was added to an ELISA plate, and the plate was left to stand overnight at 4°C to allow the bacteria to become immobilized. ELISA plates were washed using a plate washer (Vaccu-Pette / 96 multiwell pipetter, Sigma-Aldrich). ELISA plates with immobilized bacteria were washed three times with PBS. 150 μl of PBS containing 1% bovine serum albumin (BSA, Wako) was added to each well, and the plates were allowed to stand overnight at 4°C for blocking. A dilution series of human endogenous IgA antibody samples (adjusted to a concentration of 30 μg / ml) extracted from healthy individuals or patients was prepared in a 96-well plate. Dilutions were performed using PBS containing 1% BSA to 1x, 1 / 3x, 1 / 10x, 1 / 30x, 1 / 100x, 1 / 300x, 1 / 1,000x, and 1 / 3,000x. After blocking, the 1% BSA-containing PBS was removed from the ELISA plate, 50 μl of the serially diluted antibody was added to each well, and the plates were allowed to react at room temperature for 1 hour. Next, after washing three times with PBS supplemented with 0.05% Tween 20 (Chem Cruz), Alkaline Phosphatase-conjugated goat anti-human IgA (final concentration 0.5 μg / ml, Southern Biotech) was added as the secondary antibody and reacted at room temperature for 1 hour. For the substrate reaction solution for color development, magnesium chloride (final concentration 2 mM) and Phosphatase substrate (Sigma) were dissolved in a solution of sodium bicarbonate (final concentration 6.5 mM) and sodium carbonate (final concentration 18.5 mM). After the plates reacted with the secondary antibody were washed again with PBS supplemented with 0.05% Tween 20, 50 μl of the substrate reaction solution was added to each well, and the plates were allowed to develop color under light protection. Absorbance (OD 405 nm) was measured using Tristar2 LB942 (BERTHOLD TECHNOLOGIES). After confirming that each sample developed color according to the dilution series, Figure 2 plots the absorbance (405 nm) values ​​for each sample at an IgA antibody concentration of 10 mg / ml. The x-axis represents the median. The p-value was calculated using a two-tailed Student's t-test.

[0172] (result) ELISA analysis of the binding affinity of human IgA antibodies to bacteria revealed that there was no difference in the binding affinity of IgA antibodies to probiotics (beneficial bacteria) such as Bifidobacterium bifidum between healthy individuals and patients. However, the response of IgA antibodies to bacteria belonging to the Proteobacteria group, such as E. coli and Pseudomonas fulva, was significantly reduced in IBD patients (Figure 2).

[0173] Example 3: IgA antibody with high binding affinity to IBD-related bacteria

[0174] (Methods and Materials) • Bacterial extraction from human stool samples As in Example 1, the provided samples were prepared in an anaerobic chamber (80% N2, 10% H2, 10% CO2, Coy Laboratory Products) using the following method, and the bacterial suspension was divided into smaller portions and stored at -80°C for subsequent experiments. All reagents used in the anaerobic chamber were sterilized by passing them through a 0.22 μm filter and allowed to stand overnight in the anaerobic chamber to undergo anaerobic conditions. Frozen stool samples were dissolved in 30 ml of PBS in the anaerobic chamber, then suspended and centrifuged at 50 g for 15 minutes at 4°C. Samples provided by the University of Tokyo were weighed, suspended in 9 times the volume of PBS, and centrifuged at 50 g for 15 minutes at 4°C. The supernatant was collected in a separate 50 ml tube, thoroughly suspended, and then dispensed into 1.5 ml tubes in 1 ml portions. These were stored at -80°C as bacterial freeze stocks of human stool samples.

[0175] • Measurement of bacterial count concentration in bacterial freeze stocks of human stool samples Similar to Example 1, the bacterial freeze stock of human fecal samples was thawed in an anaerobic chamber. 10 μl of the bacterial freeze stock was taken and diluted 1,000-fold with PBS. The measurement of the bacterial count was performed according to the protocol of the Cell Viability kit (BD). In this study, since all bacteria contained in human fecal samples were targeted, Propidium iodide (PI) for detecting dead bacteria was not used, and only Thiazole orange (TO) for staining nucleic acids to detect bacteria was used to measure the bacterial count. PBS, Liquid Counting Beads (BD), and Thiazole orange (final concentration 42 nM, BD) were added to the bacterial dilution, and the reaction was carried out on ice for 10 minutes. The bacterial count measurement solution was subjected to flow cytometry analysis using a SONY SA3800 Analyzer (SONY). The bacterial count concentration of the bacterial dilution was calculated based on the number of Counting Beads detected in the analysis.

[0176] · Analysis of the proportion of human endogenous IgA antibody-binding bacteria (Figure 3) to bacteria in human fecal samples The operation was carried out in an anaerobic chamber. After measuring the bacterial count, the bacteria derived from human feces were adjusted to 6×10 7 cells / ml by adding PBS. Then, centrifugation was performed at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. 100 μl of PBS containing 20% normal rat serum (Wako) was added to the bacterial cells after removing the supernatant, and the reaction was carried out on ice for 30 minutes. This inhibited the non-specific binding of antibodies. FACS buffer was added to the bacterial solution, and centrifugation was performed at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. 15 μl of Anti-IgA-PE human antibody (Milteny) was added to 6×10 6 bacteria, and the reaction was carried out on ice for 20 minutes. FACS buffer was added to the antibody reaction solution, and centrifugation was performed at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. FACS buffer and Thiazole Orange (final concentration 42 nM, BD) were added to the bacteria, and the reaction was carried out on ice for 10 minutes. Then, the proportion of all endogenous IgA antibody-binding bacteria was analyzed using a SONY Cell Sorter SH800 (SONY).

[0177] · Analysis of the ratios of bacteria in human fecal samples that bind to RS_H000_L001 IgA antibody (Figure 4), RS_H007_L004 antibody (Figure 5), SNK0003A antibody (Figure 6), SNK0001A antibody (Figure 7), and SNK0002A antibody (Figure 8) The operations were carried out inside an anaerobic chamber. After measuring the bacterial count, the bacteria in the human fecal sample were adjusted to 6×107 cells / ml by adding PBS. Then, they were centrifuged at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. 100 μl of PBS containing 20% normal rat serum (Wako) was added to the bacterial pellet from which the supernatant had been removed, and the mixture was reacted on ice for 30 minutes. This inhibited the non-specific binding of the antibody. The Fluorescence activated cell sorter (FACS) buffer for antibody staining was prepared by adding 10% fetal bovine serum (FBS, Nichirei) and EDTA (final concentration 5 μM, Nacalai Tesque) to PBS and sterilizing it through a 0.22-μm filter. FACS buffer was added to the bacterial solution, and the mixture was centrifuged at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. 15 μg of each biotinylated antibody was added to 6×106 bacteria, and the mixture was reacted on ice for 20 minutes. FACS buffer was added to the antibody reaction solution, and the mixture was centrifuged at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. 20 μl of PE / Cyanine7-Streptavidin (final concentration 10 μg / ml, BioLegend) was added to the bacterial pellet, and the mixture was reacted on ice in the dark for 20 minutes. FACS buffer was added to the reaction solution, and the mixture was centrifuged at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. FACS buffer and Thiazole Orange (final concentration 42 nM, BD) were added to the bacteria, and the mixture was reacted on ice for 10 minutes. Then, the ratios of bacteria bound to each antibody were analyzed using a SONY Cell Sorter SH800 (SONY).

[0178] · Analysis of the ratio of bacteria in human fecal samples that bind to RS_H000_L001 antibody The operations were carried out inside an anaerobic chamber. After measuring the bacterial count, the bacteria in the human fecal sample were adjusted to 6×10 7The bacterial count was adjusted to 6 × 10¹⁶ cells / ml. The mixture was then centrifuged at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. 100 μl of PBS containing 20% ​​normal rat serum (Wako) was added to the remaining bacterial cells, and the mixture was reacted on ice for 30 minutes. This inhibited nonspecific binding of the antibody. For antibody staining, a fluorescence-activated cell sorter (FACS) buffer was prepared by adding 10% fetal bovine serum (FBS, Nichirei) and EDTA (final concentration 5 μM, Nacalai Tesque) to PBS and sterilizing it through a 0.22 μm filter. The FACS buffer was added to the bacterial suspension, centrifuged at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. Bacterial count: 6 × 10¹⁶ 6 15 μg of biotin-conjugated RS_H000_L001 antibody was added to each cell, and the mixture was reacted on ice for 20 minutes. FACS buffer was added to the antibody reaction mixture, and the mixture was centrifuged at 8,000 g for 5 minutes at 4°C, with the supernatant removed. 20 μl of PE / Cyanine7-Streptavidin (final concentration 10 μg / ml, BioLegend) was added to the bacterial cells, and the mixture was reacted on ice, protected from light, for 20 minutes. FACS buffer was added to the reaction mixture, and the mixture was centrifuged at 8,000 g for 5 minutes at 4°C, with the supernatant removed. FACS buffer and Thiazole Orange (final concentration 42 nM, BD) were added to the bacteria, and the mixture was reacted on ice for 10 minutes. Subsequently, the percentage of RS_H000_L001 antibody-conjugated bacteria was analyzed using a SONY Cell Sorter SH800 (SONY).

[0179] Gemella morbillorum growth inhibition test The procedure was performed in an anaerobic chamber. Gemella morbillorum (G. morbillorum, strain ATCC27824) was cultured using Tryptic Soy Broth (BD) supplemented with defibrous sheep blood (Nippon Bioceam, final concentration 5%). G. morbillorum was started from a glycerol stock and incubated statically overnight at 37°C in 10 ml of medium. 50 μl of bacterial suspension was mixed with 450 μl of medium, centrifuged at 8,000 g for 5 minutes at room temperature, and the supernatant was removed. After adding 500 μl of medium and counting the bacteria, the bacterial suspension was mixed with medium and divided into 2 × 10⁶ units. 3The bacterial suspension was prepared at a concentration of cells / μl. 5 μl of bacterial suspension was mixed with 25 μl of PBS or RS_H000_L001 IgA antibody (1 mg / ml) and reacted at 37°C for 1 hour. After the reaction, 30 μl of culture medium was added and incubated at 37°C for 8 hours. After incubation, the bacterial suspension was seeded onto agar plates and incubated at 37°C overnight, and the number of colonies was measured.

[0180] (result) In a search for IgA antibodies with high binding affinity to IBD-related bacteria in the stool of IBD patients, it was found that the RS_H000_L001 antibody and its variant (RS_H007_L004), SNK0003A, SNK0001A, and SNK0002A exhibited high binding affinity to IBD-related bacteria obtained from the stool of IBD patients (Figures 3-8). Figure 3 shows the results obtained by FACS analysis of the proportion of bacteria that bind to the total endogenous IgA antibody (number of bound bacteria / total number of bacteria in the fecal sample) from bacteria obtained from the stool of healthy individuals or IBD patients (Crohn's disease: CD, ulcerative colitis: UC). Regarding the proportion of bacteria that bind to the total endogenous IgA antibody, no significant difference was observed in the binding rate to the antibody sample between bacteria from healthy individuals' stool and bacteria from IBD patients' stool (Figure 3). On the other hand, when RS_H000_L001 and its variant (RS_H007_L004), SNK0003A, SNK0001A, and SNK0002A were used as antibody samples, the binding rate to bacteria from healthy human stool was low, while bacteria from IBD patient stool showed a significantly higher binding rate to these antibody samples (Figures 4-8).

[0181] When the binding characteristics of the RS_H000_L001 IgA antibody to bacteria in feces obtained from healthy individuals and IBD patients were examined, no bacteria showing particularly specific binding were identified in feces from healthy individuals. However, bacteria collected from feces from IBD patients showed binding to Enterococcaceae, as well as Enterobacteriaceae, Gemellaceae, and Veillonellaceae, which are known to be associated with inducing colitis (Figure 9). Gemellaceae is one of the bacteria to which IgA antibodies derived from healthy individuals bind in feces derived from healthy individuals (Figure 1). In an in vitro growth inhibition test, the RS_H000_L001 antibody showed a strong growth inhibitory effect against Gemella morbillorum (Figure 10). These results suggest that IgA antibodies with high binding affinity to IBD-related bacteria may improve the gut microbiota and alleviate IBD disease by suppressing the growth of IBD-related bacteria in the gastrointestinal tract of IBD patients.

[0182] Example 4: Therapeutic effect of treating feces with an IgA antibody that has high binding affinity to IBD-related bacteria and oral administration of the said IgA antibody on IBD.

[0183] (Methods and Materials) • Mice used for human fecal sample transplantation We used germ-free mice with BALB / c background and deficiency of endogenous IgA, specifically activation-induced cytidine deaminase (AID) knockout. The mice were housed in sterile vinyl isolators. Male mice aged 5–19 weeks were used. At the end of the experiment, the mice were euthanized by cervical dislocation during sample collection.

[0184] • Oral administration of enteric bacteria derived from IBD patient samples (P10 and P13) to mice for bacterial transplantation. Similar to Example 3, the procedure was carried out in an anaerobic chamber. After bacterial counting, PBS was added to the bacterial suspension, and 3 × 10⁻⁶ of PBS was added. 8 The bacterial cultures were prepared in individual / tube portions. They were then centrifuged at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. 100 μl of sterile PBS was added, and the cultures were orally administered to mice. The number and intervals of oral administration of the bacterial culture differed between P10 and P13. Schematic diagrams and details of the experiments are shown in Figures 11 and 15.

[0185] • Removal of RS_H000_L001 antibody-bound bacteria from IBD patient samples (P10) using a column. The procedure was performed in an anaerobic chamber. After bacterial count measurement, PBS was added to the bacteria in the human stool sample, resulting in a concentration of 3 × 10⁻⁶. 8The samples were prepared in individual cells / tube. They were then centrifuged at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. 100 μl of PBS containing 20% ​​normal rat serum (Wako) was added to the remaining bacterial cells, and the mixture was reacted on ice for 30 minutes to block nonspecific binding. FACS buffer was added to the bacterial suspension, and it was centrifuged at 8,000 g for 5 minutes at 4°C, with the supernatant removed. Bacterial count: 6 × 10⁶ 6 15 μg of biotin-conjugated RS_H000_L001 IgA antibody was added to each cell and reacted on ice for 40 minutes. FACS buffer was added to the antibody reaction mixture and centrifuged at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. RS_H000_L001 antibody-conjugated bacteria were removed using the Veritas Magnetic Bead Separation (MACS) kit. For the removal of RS_H000_L001 IgA antibody-conjugated bacteria from the P10 stool sample, Myone C1-streptavidin beads (Veritas) were used. 15 μl of beads prepared according to the Veritas bead washing protocol were added to 15 μg of biotin-conjugated RS_H000_L001 IgA antibody and reacted on ice for 30 minutes. Bacterial isolation was performed according to the Veritas MACS bacterial isolation protocol. Bacteria adsorbed to a magnet were collected as RS_H000_L001 IgA antibody-conjugated bacteria, and bacteria collected as flow-through were collected as RS_H000_L001 antibody-unconjugated bacteria. Both were suspended in 500 μl of FACS buffer. 5 μl of RS_H000_L001 antibody-unconjugated bacteria were collected, and FACS buffer, TO (final concentration 42 nM, BD) and Phycoerythrin (PE)-anti mouse IgA antibody (final concentration 5 μg / ml, Biolegend) were added. The mixture was reacted on ice, protected from light, for 10 minutes. Subsequently, FACS analysis using a SONY Cell Sorter SH800 (SONY) confirmed that more than 90% of the bacteria were unconjugated. The remaining RS_H000_L001 antibody-unconjugated bacteria were centrifuged at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. This was prepared in 100 μl of PBS and administered orally to mice.

[0186] • Oral administration of RS_H000_L001 IgA antibody Starting from the last day of bacterial suspension administration, RS_H000_L001 antibody was orally administered to mice via a gastric tube once daily for 7 days (P10) or 8 days (P13). Each dose was prepared by adding 100 μg / mice of RS_H000_L001 antibody to 200 μl of PBS.

[0187] • Dextran sulfate sodium (DSS) induced colitis model mouse Starting the day after the final administration of RS_H000_L001 antibody, 4% dextran sulfate sodium (DSS (MP Biomedicals)) was added to a water bottle and orally administered to mice to induce enteritis. DSS was added to sterile water to a final concentration of 4% and administered to mice as free drinking water for 7 days. The mice's body weight and stool score were measured daily.

[0188] • Freezing and staining of colon tissue On day 7 of DSS administration, mice were euthanized due to cervical dislocation, and the mice were dissected to remove the large intestine. The contents of the large intestine tissue were washed, frozen with liquid nitrogen using Optimumcutting temperature (OCT) compound (Sakura), and then stored at -80°C. Tissue sections 6 μm thick were prepared from mouse colon tissue cryopreserved with OCT compound. The tissue sections were fixed with acetone for 15 minutes, stained with hematoxylin-eosin and Alcian blue, and observed under a light microscope. For hematoxylin-eosin staining, Hematoxylin solution modified acc. to Gill III (Merck) and eosin alcohol solution, acid extract (Wako) were used. For Alcian blue staining, Alcian blue solution, pH 2.5 (Wako) and Kern-Echtroth solution (Muto Chemical) were used.

[0189] • Method for extracting bacterial DNA from mouse feces Mouse stool samples were collected before (day 0) and after administration of RS_H000_L001 IgA antibody. The mouse stool samples were weighed and 9 times the volume of sterile PBS was added. The samples were suspended by vortexing and centrifuged at 50 g for 15 minutes at 4°C. The supernatant was collected in a 1.5 ml tube and centrifuged at 8,000 g for 5 minutes at 4°C, and the supernatant was removed. DNA Lysis Buffer for DNA extraction was prepared by adding Tris-HCl (final concentration 50 mM, Nacalai Tesque), NaCl (final concentration 300 mM, Nacalai Tesque), EDTA (final concentration 1 mM, Nacalai Tesque), and 0.5% SDS (Nacalai Tesque) to ultrapure water. After suspending the bacterial cells in 500 μl of DNA Lysis Buffer, the cells were transferred to a sterile 2.0 ml tube (TM-625S, TOMY) containing glass beads (GB-01, 0.1Φ, TOMY). Bead disruption was performed using a bead cell disruptor (MS-100, TOMY) at 3,500 rpm for 1 minute. Protein kinase K (final concentration 0.5 mg / ml, Nacalai Tesque) was added to the bead-disrupted bacterial suspension and vortexed. The mixture was left to stand overnight at 55°C to completely lyse the bacterial cells. The mixture was then returned to room temperature from 55°C, and 500 μl of phenol / chloroform / isoamyl alcohol = 24:25:1 (Nacalai Tesque) was added to the lysate. The mixture was vortexed, centrifuged at 13,000 rpm for 5 minutes at 4°C, and the upper layer was collected in a separate 1.5 ml tube. The collected liquid was mixed with an equal volume of chloroform (Nacalai Tesque) and vortexed, then centrifuged at 13,000 rpm for 5 minutes at 4°C. The upper layer was collected again in a separate 1.5 ml tube, and an equal volume of isopropanol (Nacalai Tesque) and glycogen (final concentration 0.04 mg / ml, Nacalai Tesque) was added, then mixed by inversion. The DNA lysate was allowed to stand at -20°C for 20 minutes, then centrifuged at 13,000 rpm for 10 minutes at 4°C, and the supernatant was removed. 500 μl of 70% ethanol was added to the precipitated DNA, and the mixture was centrifuged at 13,000 rpm for 5 minutes at 4°C, and the supernatant was removed. After air-drying the precipitated DNA, Ultra Pure Distilled Water (Invitrogen) was added to dissolve the DNA. The DNA concentration was measured using Bio Drop DUO (Biochrome).

[0190] (result) For specific IBD patients (P10), the entire bacterial flora obtained from the patients' stool, or bacteria that did not bind to the RS_H000_L001 IgA antibody (which showed high binding affinity to IBD-related bacteria), were orally administered multiple times to IBD model mice. Analysis of the RS_H000_L001 IgA antibody-binding bacteria in the bacterial flora of these IBD patients (P10) showed a high IgA index against Enterobacteriaceae and other organisms, and a low IgA index against Bifidobacteriaceae and other organisms. In addition to oral administration of bacteria, some of these mice were also orally administered the RS_H000_L001 IgA antibody. In other words, the test groups consisted of four groups: (1) whole bacterial flora / no antibody administration group, (2) whole bacterial flora / RS_H000_L001 IgA antibody oral administration group, (3) RS_H000_L001 IgA antibody-unbound bacteria / no antibody administration group, and (4) RS_H000_L001 IgA antibody-unbound bacteria / RS_H000_L001 IgA antibody oral administration group (Figure 11). As a result, (1) the whole bacterial community / non-antibody administration group showed weight loss after oral bacterial administration, which is thought to be associated with the development of DSS-induced colitis, whereas (2) weight loss was significantly suppressed in the whole bacterial community / RS_H000_L001 IgA antibody oral administration group (Figure 12). On the other hand, in the (3) RS_H000_L001 IgA antibody-unbound bacteria / antibody-free group, little weight loss, which is thought to be associated with the development of DSS-induced colitis, was observed after oral administration of bacteria, suggesting that modifying the bacterial flora of IBD patients' feces with RS_H000_L001 IgA antibody reduced the effect on the development of colitis. Furthermore, the weight change in the (4) RS_H000_L001 IgA antibody-unbound bacteria / RS_H000_L001 IgA antibody oral administration group, which combined oral administration of RS_H000_L001 IgA antibody, was almost the same as in the (3) RS_H000_L001 IgA antibody-unbound bacteria / antibody-free group (Figure 13). Furthermore, when the diversity of the gut microbiota of treated mice was analyzed, the following groups showed greater diversity compared to (1) the whole microbiota / no-antibody administration group: (2) the whole microbiota / RS_H000_L001 IgA antibody oral administration group, (3) the RS_H000_L001 IgA antibody-unbound bacteria / no-antibody administration group, and (4) the RS_H000_L001 IgA antibody-unbound bacteria / RS_H000_L001 IgA antibody oral administration group (Figure 14). These results demonstrate that when treating IBD patients with autologous fecal transplantation (microbiota transplantation), co-administering orally an IgA antibody with high binding affinity to IBD-related bacteria, or modifying the microbiota contained in the autologous fecal with an IgA antibody with high binding affinity to IBD-related bacteria, can improve the gut microbiota and alleviate IBD disease symptoms in IBD patients.

[0191] A similar test was conducted using the bacterial flora from the stool of a different IBD patient (P13) than the IBD patient (P10) mentioned above. As shown in Figure 15, this test aimed to examine in more detail the combined antibody effect of the entire bacterial flora and the RS_H000_L001 IgA antibody. FACS analysis revealed that approximately 70% of the bacterial flora from the stool of IBD patients (P13) bound to the RS_H000_L001 antibody (Figure 16). When we observed weight changes after oral administration of bacteria in (1) the whole bacterial community / no antibody administration group and (2) the whole bacterial community / RS_H000_L001 IgA antibody oral administration group, we found that for the bacterial community derived from the stool of IBD patients (P13), there was almost no weight change in (1) the whole bacterial community / no antibody administration group, and the same was true for (2) the whole bacterial community / RS_H000_L001 IgA antibody oral administration group (Figure 17). Furthermore, analysis of the diversity of the gut microbiota after treatment (Figures 18-21) showed almost no change in the Shannon index (Figure 18). On the other hand, when the relative abundance of various bacteria was examined, a decrease in the relative abundance of Fusobacteriaceae and Bacteroidaceae, which have been linked to IBD, was observed in both (1) the total bacterial flora / no antibody administration group and (2) the total bacterial flora / RS_H000_L001 IgA antibody oral administration group (Figures 19, 21), while conversely, an increase in the relative abundance of Lachnospiraceae was observed (Figure 20). Furthermore, observation of colon tissue sections after treatment revealed that (1) abnormalities in tissue and cell structure were observed in the whole bacterial community / antibody non-administered group, while (2) normal tissue and cell structure was observed in the whole bacterial community / RS_H000_L001 IgA antibody oral administration group (Figure 22). These results confirm that when treating IBD patients with autologous fecal transplantation (microbiota transplantation), co-administering IgA antibodies with high binding affinity to IBD-related bacteria can improve the gut microbiota and alleviate IBD disease symptoms. Furthermore, in a mouse administration study using microbiota derived from the feces of IBD patients (P10), significant weight loss, thought to be associated with the development of DSS-induced colitis, occurred when IgA antibodies were not co-administered. In contrast, no such significant weight loss was observed in a mouse administration study using microbiota derived from the feces of IBD patients (P13), suggesting that the microbiota configuration involved in the development of IBD differs among individual IBD patients. The present invention provides a more effective IBD improvement effect by using IgA antibodies that promote the health of the gastrointestinal microbiota in individual patients.

[0192] Example 5: Further IgA antibodies with high binding affinity to IBD-related bacteria Further IgA antibodies with high binding affinity to IBD-related bacteria were tested using the same method as in Example 3. For SNK0004 and SNK0005 antibodies, which are IgA antibody clones isolated from mouse intestinal mucosa, the binding rate of bacteria from the stool of healthy individuals that bind to these antibodies was compared with the binding rate of bacteria from the stool of IBD patients that bind to these antibodies. The amino acid sequences of the heavy and light chains of SNK0004 and SNK0005 are shown below. TIFF0007862032000041.tif175167TIFF0007862032000042.tif175167

[0193] The results are shown in Figures 23 and 24. For the SNK0004 antibody, the binding rate of bacteria from IBD patient stool was higher than that of bacteria from healthy individuals. On the other hand, for SNK0005, there was no clear difference between the binding rate of bacteria from healthy individuals and bacteria from IBD patient stool. On the other hand, when we examined the binding characteristics of individual bacterial species from BD patient stool samples that bind to these antibodies, it became clear that both SNK0004 and SNK0005 showed specific binding to Enterobacteriaceae, while other bacteria showed different binding characteristics (Figures 25 and 26).

[0194] The SNK0001, SNK0002, and SNK0003 antibodies used in Example 3 were also identified as being specifically bound to bacteria derived from the stool of IBD patients (Figures 27-29). These results indicate that even among antibodies that show a high binding rate to bacteria derived from the stool of IBD patients compared to those that show a high binding rate to bacteria derived from the stool of healthy individuals, the bacteria that show specific binding differ for each antibody.

[0195] Figure 30 shows the results of comparing the proportion of bacteria from IBD patient stool samples that bind to the RS_H000_L001, SNK0001, SNK0002, SNK0003, SNK0004, and SNK0005 antibodies. These results suggest that while all of these antibodies bind to Enterobacteriaceae, thus contributing to the healthy gut microbiota, they also indicate that each antibody binds to different bacterial species.

[0196] It is believed that the abnormal proportions of IBD-related bacteria differ from patient to patient. These results demonstrate that different types of IgA antibodies can be used to test the state of these abnormally present bacteria in IBD patients, and that, based on the test results, the most appropriate IgA antibody for each patient can be used as a drug for restoring the gastrointestinal microbiota and as a treatment for IBD. [Industrial applicability]

[0197] This disclosure provides a method for obtaining gastrointestinal contents or excrement that improves the gastrointestinal microbiota, by treating the gastrointestinal contents or excrement with an IgA antibody in a therapeutic method for administering gastrointestinal contents or excrement to treat IBD, a method for obtaining an IgA antibody related to this method, and a pharmaceutical composition used in these methods, which has extremely high industrial value.

Claims

1. A pharmaceutical composition for treating inflammatory bowel disease (IBD), comprising gastrointestinal contents or excrement treated with an IgA antibody that restores the bacterial flora in the gastrointestinal tract, The following IgA antibodies promote the health of the bacterial flora in the gastrointestinal tract: (1) to (6) (1) IgA antibodies that, when brought into contact with gastrointestinal contents or excrement from an IBD patient treated with the composition, or from gastrointestinal contents or excrement from a healthy person, bind to a greater number of IBD-related bacteria when brought into contact with gastrointestinal contents or excrement from an IBD patient; (2) When a first binding profile is created based on the types and amounts of bacteria bound to the IgA antibody after contacting the IgA antibody with gastrointestinal contents or excrement from an IBD patient treated with the composition, and a second binding profile is created based on the types and amounts of bacteria bound to the IgA antibody after contacting the IgA antibody with gastrointestinal contents or excrement from a healthy person, the IgA antibody that shows binding to a wider variety of IBD-related bacteria or a larger amount of binding to IBD-related bacteria in the first binding profile is compared with the first binding profile; (3) IgA antibodies that increase the bacterial diversity of gastrointestinal contents or excrement from IBD patients treated with the composition, when compared with the bacterial diversity of gastrointestinal contents or excrement after treatment with the IgA antibody; (4) An IgA antibody that, when administered to a patient being treated with the composition, increases the bacterial diversity of the gastrointestinal contents or excrement after administration, when the bacterial diversity of the gastrointestinal contents or excrement of the patient before administration is compared with the bacterial diversity of the gastrointestinal contents or excrement after administration; (5) An IgA antibody that, when administered to an IBD patient treated with the composition, increases the short-chain fatty acid content of the gastrointestinal contents or excrement after administration, when the short-chain fatty acid content of the gastrointestinal contents or excrement of the patient before administration is compared with the short-chain fatty acid content of the gastrointestinal contents or excrement after administration; (6) An IgA antibody modified with the IgA antibody is administered to an IBD patient being treated with the composition, and the Enterobacteriaceae content of the patient's gastrointestinal contents or excrement before administration is compared with the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration, and the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration is reduced. An IgA antibody having one or more characteristics selected from the following, The aforementioned digestive tract contents are the contents of the digestive tract from the large intestine to the rectum, in the pharmaceutical composition.

2. A pharmaceutical composition for treating IBD, which contains an IgA antibody that restores the bacterial flora in the gastrointestinal tract, and is used in combination with a composition containing gastrointestinal contents or excrement, The following IgA antibodies promote the health of the bacterial flora in the gastrointestinal tract: (1) to (6) (1) IgA antibodies that, when brought into contact with gastrointestinal contents or excrement from an IBD patient treated with the composition, or from gastrointestinal contents or excrement from a healthy person, bind to a greater number of IBD-related bacteria when brought into contact with gastrointestinal contents or excrement from an IBD patient; (2) When a first binding profile is created based on the types and amounts of bacteria bound to the IgA antibody after contacting the IgA antibody with gastrointestinal contents or excrement from an IBD patient treated with the composition, and a second binding profile is created based on the types and amounts of bacteria bound to the IgA antibody after contacting the IgA antibody with gastrointestinal contents or excrement from a healthy person, the IgA antibody that shows binding to a wider variety of IBD-related bacteria or a larger amount of binding to IBD-related bacteria in the first binding profile is compared with the first binding profile; (3) IgA antibodies that increase the bacterial diversity of gastrointestinal contents or excrement from IBD patients treated with the composition, when compared with the bacterial diversity of gastrointestinal contents or excrement after treatment with the IgA antibody; (4) An IgA antibody that, when administered to a patient being treated with the composition, increases the bacterial diversity of the gastrointestinal contents or excrement after administration, when the bacterial diversity of the gastrointestinal contents or excrement of the patient before administration is compared with the bacterial diversity of the gastrointestinal contents or excrement after administration; (5) An IgA antibody that, when administered to an IBD patient treated with the composition, increases the short-chain fatty acid content of the gastrointestinal contents or excrement after administration, when the short-chain fatty acid content of the gastrointestinal contents or excrement of the patient before administration is compared with the short-chain fatty acid content of the gastrointestinal contents or excrement after administration; (6) An IgA antibody modified with the IgA antibody is administered to an IBD patient being treated with the composition, and the Enterobacteriaceae content of the patient's gastrointestinal contents or excrement before administration is compared with the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration, and the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration is reduced. An IgA antibody having one or more characteristics selected from the following, The gastrointestinal contents or excrement used in combination are derived from the subject to whom the composition is administered. The aforementioned digestive tract contents are the contents of the digestive tract from the large intestine to the rectum, in the pharmaceutical composition.

3. The pharmaceutical composition according to claim 1, wherein the contents of the gastrointestinal tract or excrement are derived from a subject to whom the composition is administered.

4. (B1) The process of preparing the gastrointestinal contents or excrement of an IBD patient. (B2) A step of modifying the gastrointestinal contents or excreta with a candidate IgA antibody. (B3) A step to confirm that the modified gastrointestinal contents or excrement can restore the bacterial flora in the gastrointestinal tract. A method for screening IgA antibodies that promote a healthy bacterial flora in the gastrointestinal tract, including, The steps to confirm that the modified gastrointestinal contents or excrement described in (B3) can restore the bacterial flora in the gastrointestinal tract are as follows (1) to (6): (1) When the IgA antibody is brought into contact with gastrointestinal contents or excrement from an IBD patient, or from gastrointestinal contents or excrement from a healthy person, it is confirmed that it binds to a greater number of IBD-related bacteria when it is brought into contact with gastrointestinal contents or excrement from an IBD patient; (2) When a first binding profile is created based on the types and amounts of bacteria bound to the IgA antibody after contacting the IgA antibody with the gastrointestinal contents or excrement of an IBD patient, and a second binding profile is created based on the types and amounts of bacteria bound to the IgA antibody after contacting the IgA antibody with the gastrointestinal contents or excrement of a healthy person, it is confirmed that binding to a wider variety of IBD-related bacteria or a larger amount of binding to IBD-related bacteria is observed in the first binding profile; (3) When comparing the bacterial diversity of gastrointestinal contents or excrement from IBD patients before treatment with IgA antibody with the bacterial diversity of gastrointestinal contents or excrement after treatment with IgA antibody, confirm that the bacterial diversity of gastrointestinal contents or excrement increases after treatment with IgA antibody; (4) Administer the gastrointestinal contents or excrement modified with the IgA antibody to the IBD patient and compare the bacterial diversity of the gastrointestinal contents or excrement after administration with the bacterial diversity of the gastrointestinal contents or excrement after administration, confirming that the bacterial diversity of the gastrointestinal contents or excrement increases after administration; (5) When gastrointestinal contents or excrement modified with the IgA antibody is administered to an IBD patient, and the short-chain fatty acid content in the patient's gastrointestinal tract before administration is compared with the short-chain fatty acid content in the gastrointestinal contents or excrement after administration, the short-chain fatty acid content increases after administration; (6) Administering gastrointestinal contents or excrement modified with the IgA antibody to the IBD patient, comparing the Enterobacteriaceae content of the patient's gastrointestinal contents or excrement before administration with the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration, and confirming that the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration has decreased; It is done by one or more selected from, The aforementioned digestive tract contents are the contents of the digestive tract from the large intestine to the rectum. The aforementioned method.

5. A method for producing IgA antibodies that improve the bacterial flora in the gastrointestinal tract, comprising the screening method of claim 4.

6. (C1) Process of preparing the gastrointestinal contents or excrement of an IBD patient. (C2) A step of bringing the contents of the digestive tract or excrement into contact with an IgA antibody that restores the bacterial flora in the digestive tract outside of the body. A method for processing the gastrointestinal contents or excrement of an IBD patient, including, The IgA antibodies used in (C2) to restore the gut microbiota are as follows (1) to (6): (1) When the IgA antibody is brought into contact with gastrointestinal contents or excrement from an IBD patient, or from gastrointestinal contents or excrement from a healthy person, it is confirmed that it binds to a greater number of IBD-related bacteria when it is brought into contact with gastrointestinal contents or excrement from an IBD patient; (2) When a first binding profile is created based on the types and amounts of bacteria bound to the IgA antibody after contacting the gastrointestinal contents or excrement from an IBD patient with the IgA antibody, and a second binding profile is created based on the types and amounts of bacteria bound to the IgA antibody after contacting the gastrointestinal contents or excrement from a healthy person with the IgA antibody, it is confirmed that binding to a wider variety of IBD-related bacteria or a larger amount of binding to IBD-related bacteria is observed in the first binding profile; (3) When comparing the bacterial diversity of the gastrointestinal contents or excrement from the IBD patient before treatment with IgA antibody with the bacterial diversity of the gastrointestinal contents or excrement after treatment with IgA antibody, confirm that the bacterial diversity of the gastrointestinal contents or excrement increases after treatment with IgA antibody; (4) Administer the gastrointestinal contents or excrement modified with the IgA antibody to the patient, and confirm that the bacterial diversity of the gastrointestinal contents or excrement increases after administration when comparing the bacterial diversity of the gastrointestinal contents or excrement of the IBD patient before administration with that of the gastrointestinal contents or excrement after administration; (5) When gastrointestinal contents or excrement modified with the IgA antibody is administered to a patient, and the short-chain fatty acid content in the gastrointestinal contents or excrement of the IBD patient before administration is compared with the short-chain fatty acid content in the gastrointestinal contents or excrement after administration, the short-chain fatty acid content increases after administration; (6) Administering gastrointestinal contents or excrement modified with the IgA antibody to the IBD patient, comparing the Enterobacteriaceae content of the patient's gastrointestinal contents or excrement before administration with the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration, and confirming that the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration has decreased; Includes a process obtained by selecting one or more from, The aforementioned digestive tract contents are the contents of the digestive tract from the large intestine to the rectum. The aforementioned method.

7. A composition for modifying the bacterial flora of gastrointestinal contents or excrement in vitro, containing an IgA antibody that promotes the health of the bacterial flora in the gastrointestinal tract, The following IgA antibodies promote the health of the bacterial flora in the gastrointestinal tract: (1) to (6) (1) IgA antibodies that, when the bacterial flora is brought into contact with the modified gastrointestinal contents or excrement, or with the gastrointestinal contents or excrement of a healthy person, bind to a greater number of IBD-related bacteria when the bacterial flora is brought into contact with the modified gastrointestinal contents or excrement; (2) When the IgA antibody is brought into contact with the gastrointestinal contents or excrement in which the bacterial flora is modified, and a first binding profile is created based on the types and amounts of bacteria bound to the IgA antibody, and a second binding profile is created based on the types and amounts of bacteria bound to the IgA antibody when the gastrointestinal contents or excrement from a healthy person is brought into contact with the IgA antibody, an IgA antibody is observed to bind to a wider variety of IBD-related bacteria or to a larger amount of IBD-related bacteria in the first binding profile; (3) IgA antibodies that increase the bacterial diversity of the gastrointestinal contents or excrement after treatment with IgA antibodies, when comparing the bacterial diversity of the gastrointestinal contents or excrement after treatment with IgA antibodies with the bacterial diversity of the gastrointestinal contents or excrement before treatment with IgA antibodies; (4) An IgA antibody that, when gastrointestinal contents or excrement modified with the IgA antibody is administered to a subject from which the gastrointestinal contents or excrement originates, and the bacterial diversity of the gastrointestinal contents or excrement of the subject before administration is compared with the bacterial diversity of the gastrointestinal contents or excrement of the subject after administration, increases the bacterial diversity of the gastrointestinal contents or excrement after administration; (5) An IgA antibody that, when gastrointestinal contents or excrement modified with the IgA antibody is administered to a subject from which the gastrointestinal contents or excrement originates, increases the short-chain fatty acid content of the gastrointestinal contents or excrement after administration when the short-chain fatty acid content of the gastrointestinal contents or excrement of the subject before administration is compared with the short-chain fatty acid content of the gastrointestinal contents or excrement after administration; (6) An IgA antibody modified with the gastrointestinal contents or excrement is administered to a subject from which the gastrointestinal contents or excrement originates, and the Enterobacteriaceae content of the subject's gastrointestinal contents or excrement before administration is compared with the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration, and the IgA antibody reduces the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration; An IgA antibody having one or more characteristics selected from the following, The aforementioned digestive tract contents are the contents of the digestive tract from the large intestine to the rectum. The aforementioned composition.

8. (D1) A step to prepare IgA antibodies to restore the gut microbiota of IBD patients; (D2) A step of contacting the contents of the gastrointestinal tract or excrement of an in vivo subject with the IgA antibody; and (D3) The step of identifying bacteria that bind to the IgA antibody, If the IgA antibody shows binding to IBD-related bacteria, the subject is treated as having IBD or being at risk of developing IBD. A method for testing to diagnose whether a subject has IBD or is at risk of developing IBD, The steps for preparing IgA antibodies to restore the bacterial flora in the gastrointestinal tract of IBD patients (D1) are (1) to (6): (1) When the IgA antibody is brought into contact with gastrointestinal contents or excrement from an IBD patient, or from gastrointestinal contents or excrement from a healthy person, it is confirmed that it binds to a greater number of IBD-related bacteria when it is brought into contact with gastrointestinal contents or excrement from an IBD patient; (2) When a first binding profile is created based on the types and amounts of bacteria bound to the IgA antibody after contacting the gastrointestinal contents or excrement from an IBD patient with the IgA antibody, and a second binding profile is created based on the types and amounts of bacteria bound to the IgA antibody after contacting the gastrointestinal contents or excrement from a healthy person with the IgA antibody, it is confirmed that binding to a wider variety of IBD-related bacteria or a larger amount of binding to IBD-related bacteria is observed in the first binding profile; (3) When comparing the bacterial diversity of the gastrointestinal contents or excrement from the IBD patient before treatment with IgA antibody with the bacterial diversity of the gastrointestinal contents or excrement after treatment with IgA antibody, confirm that the bacterial diversity of the gastrointestinal contents or excrement increases after treatment with IgA antibody; (4) Administer the gastrointestinal contents or excrement modified with the IgA antibody to the IBD patient and compare the bacterial diversity of the gastrointestinal contents or excrement after administration with the bacterial diversity of the gastrointestinal contents or excrement after administration, confirming that the bacterial diversity of the gastrointestinal contents or excrement after administration increases; (5) When gastrointestinal contents or excrement modified with the IgA antibody is administered to an IBD patient, and the short-chain fatty acid content in the patient's gastrointestinal tract before administration is compared with the short-chain fatty acid content in the gastrointestinal contents or excrement after administration, the short-chain fatty acid content increases after administration; (6) The gastrointestinal contents or excrement modified with the IgA antibody is administered to the IBD patient, and the Enterobacteriaceae content of the gastrointestinal contents or excrement of the subject before administration is compared with the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration, and the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration is found to be decreased; Includes one or more selected from, The method wherein the contents of the digestive tract are the contents of the digestive tract from the large intestine to the rectum.

9. A pharmaceutical product for diagnosing the presence or absence of IBD or the risk of developing IBD, which contains an IgA antibody that restores the bacterial flora in the gastrointestinal tract of IBD patients. The following IgA antibodies promote a healthy gut microbiota in IBD patients: (1) to (6): (1) When the IgA antibody is brought into contact with gastrointestinal contents or excrement from an IBD patient, or from gastrointestinal contents or excrement from a healthy person, it is confirmed that it binds to a greater number of IBD-related bacteria when it is brought into contact with gastrointestinal contents or excrement from an IBD patient; (2) When a first binding profile is created based on the types and amounts of bacteria bound to the IgA antibody after contacting the IgA antibody with the gastrointestinal contents or excrement of an IBD patient, and a second binding profile is created based on the types and amounts of bacteria bound to the IgA antibody after contacting the IgA antibody with the gastrointestinal contents or excrement of a healthy person, it is confirmed that binding to a wider variety of IBD-related bacteria or a larger amount of binding to IBD-related bacteria is observed in the first binding profile; (3) When comparing the bacterial diversity of gastrointestinal contents or excrement from IBD patients before treatment with IgA antibody with the bacterial diversity of gastrointestinal contents or excrement after treatment with IgA antibody, confirm that the bacterial diversity of gastrointestinal contents or excrement increases after treatment with IgA antibody; (4) Administer the gastrointestinal contents or excrement modified with the IgA antibody to the IBD patient and compare the bacterial diversity of the gastrointestinal contents or excrement after administration with the bacterial diversity of the gastrointestinal contents or excrement after administration, confirming that the bacterial diversity of the gastrointestinal contents or excrement increases after administration; (5) When gastrointestinal contents or excrement modified with the IgA antibody is administered to an IBD patient, and the short-chain fatty acid content in the patient's gastrointestinal tract before administration is compared with the short-chain fatty acid content in the gastrointestinal contents or excrement after administration, the short-chain fatty acid content increases after administration; (6) Administering gastrointestinal contents or excrement modified with the IgA antibody to the IBD patient, comparing the Enterobacteriaceae content of the patient's gastrointestinal contents or excrement before administration with the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration, and confirming that the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration has decreased; An IgA antibody having one or more characteristics selected from the following, The aforementioned digestive tract contents are the contents of the digestive tract from the large intestine to the rectum, as described above.

10. (E1) A step of obtaining gastrointestinal contents or excrement from an IBD patient who is or has been administered an IBD therapeutic drug containing gastrointestinal contents or excrement; (E2) A step of contacting the gastrointestinal contents or excrement obtained from the IBD patient in (E1) with IgA antibodies; and (E3) The step of analyzing bacteria that bind to the IgA antibody, If the analysis results indicate a healthy gastrointestinal microbiota, treatment with the IBD drug will be continued. A method for testing the therapeutic effect of IBD drugs, The IgA antibodies are as follows (1) to (6): (1) IgA antibodies that, when brought into contact with gastrointestinal contents or excrement from an IBD patient or from a healthy person, bind to a greater number of IBD-related bacteria when brought into contact with the gastrointestinal contents or excrement from the IBD patient; (2) When a first binding profile is created based on the types and amounts of bacteria bound to the IgA antibody after contacting the IgA antibody with the gastrointestinal contents or excrement of an IBD patient being tested, and a second binding profile is created based on the types and amounts of bacteria bound to the IgA antibody after contacting the IgA antibody with the gastrointestinal contents or excrement of a healthy person, the IgA antibody that shows binding to a wider variety of IBD-related bacteria or a larger amount of binding to IBD-related bacteria in the first binding profile is compared with the first binding profile. (3) IgA antibodies that increase the bacterial diversity of gastrointestinal contents or excrement after treatment with IgA antibodies, when comparing the bacterial diversity of gastrointestinal contents or excrement from the IBD patient being tested before treatment with IgA antibodies with the bacterial diversity of gastrointestinal contents or excrement after treatment with IgA antibodies; (4) An IgA antibody that, when the gastrointestinal contents or excrement modified with the IgA antibody is administered to an IBD patient being tested, and the bacterial diversity of the gastrointestinal contents or excrement before administration is compared with the bacterial diversity of the gastrointestinal contents or excrement after administration, increases the bacterial diversity of the gastrointestinal contents or excrement after administration; (5) An IgA antibody that, when gastrointestinal contents or excretions modified with the IgA antibody are administered to an IBD patient being tested, and the short-chain fatty acid content of the gastrointestinal contents or excretions of the patient before administration is compared with the short-chain fatty acid content of the gastrointestinal contents or excretions after administration, increases the short-chain fatty acid content after administration; (6) An IgA antibody modified with the IgA antibody is administered to an IBD patient being tested, and the Enterobacteriaceae content of the patient's gastrointestinal contents or excrement before administration is compared with the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration, and the IgA antibody that reduces the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration; An IgA antibody having one or more characteristics selected from the above, wherein the improvement of the gastrointestinal bacterial flora is assessed by the decrease in the binding of the IgA antibody to gastrointestinal contents or excretions from IBD patients after administration of an IBD therapeutic agent. The method wherein the contents of the digestive tract are the contents of the digestive tract from the large intestine to the rectum.

11. The method according to claim 10, wherein the gastrointestinal contents or excretions contained in the IBD therapeutic agent are modified with IgA.

12. A diagnostic agent containing an IgA antibody for testing the therapeutic effect of an IBD drug in IBD patients who are or have been administered an IBD drug containing gastrointestinal contents or excrement, (1) IgA antibodies that, when brought into contact with gastrointestinal contents or excrement from an IBD patient being tested with the pharmaceutical product, or from gastrointestinal contents or excrement from a healthy person, bind to a greater number of IBD-related bacteria when brought into contact with the gastrointestinal contents or excrement from the IBD patient; (2) When the IgA antibody is brought into contact with gastrointestinal contents or excrement from an IBD patient to be tested with the pharmaceutical product, and a first binding profile is created based on the types and amounts of bacteria bound to the IgA antibody, and a second binding profile is created based on the types and amounts of bacteria bound to the IgA antibody when the IgA antibody is brought into contact with gastrointestinal contents or excrement from a healthy person, the IgA antibody that shows binding to a wider variety of IBD-related bacteria or a larger amount of binding to IBD-related bacteria in the first binding profile is compared with the first binding profile; (3) IgA antibodies that increase the bacterial diversity of gastrointestinal contents or excrement from IBD patients tested with the drug when the bacterial diversity of gastrointestinal contents or excrement is compared with the bacterial diversity of gastrointestinal contents or excrement after treatment with the IgA antibody; (4) An IgA antibody that, when administered to an IBD patient undergoing testing with the pharmaceutical product, increases the bacterial diversity of the gastrointestinal contents or excrement after administration, when the bacterial diversity of the gastrointestinal contents or excrement of the patient before administration is compared with the bacterial diversity of the gastrointestinal contents or excrement after administration; (5) An IgA antibody that, when administered to an IBD patient whose gastrointestinal contents or excretions modified with the IgA antibody are tested with the pharmaceutical product, increases the short-chain fatty acid content of the gastrointestinal contents or excretions after administration when the short-chain fatty acid content of the gastrointestinal contents or excretions of the patient before administration is compared with the short-chain fatty acid content of the gastrointestinal contents or excretions after administration; (6) An IgA antibody modified with the said IgA antibody is administered to an IBD patient to be tested with the pharmaceutical product, and the Enterobacteriaceae content of the patient's gastrointestinal contents or excrement before administration is compared with the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration, and the IgA antibody that reduces the Enterobacteriaceae content of the gastrointestinal contents or excrement after administration; An IgA antibody having one or more characteristics selected from the following, The aforementioned digestive tract contents are the contents of the digestive tract from the large intestine to the rectum, as described in the diagnostic drug.

13. The diagnostic pharmaceutical agent according to claim 12, wherein the contents of the gastrointestinal tract or excreta are modified with IgA.

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