Methods and compositions for treatment or diagnosis of inflammatory bowel disease (IBD)

Abstract

The present disclosure relates to: a method for treating IBD using digestive tract content or excreta modified with an IgA antibody, a composition, and a method for producing the same; a method for obtaining an IgA antibody that makes the flora in the digestive tract of an IBD patient sound; methods, compositions, and methods of making the same for modifying the contents or excreta of the digestive tract of an IBD patient using an IgA antibody; a method for testing a patient treated with an IBD therapeutic agent containing digestive tract contents or excreta using a diagnostic agent containing an IgA antibody; a composition; and a method for producing the composition.

Description

Technical Field

[0001] The present disclosure relates to: a method, a composition and a method for producing the same for treating inflammatory bowel disease (IBD) using digestive tract contents or excreta modified with IgA antibodies; a method for obtaining IgA antibodies that improve the flora in the digestive tract of IBD patients; a method, a composition and a method for producing the same for modifying the digestive tract contents or excreta of IBD patients using IgA antibodies; and a method, a composition and a method for producing the same for testing a diagnostic drug containing IgA antibodies in patients treated with an IBD therapeutic drug containing digestive tract contents or excreta. Background Art

[0002] It is reported that dysbiosis of intestinal flora is associated with the onset of various diseases including inflammatory bowel disease (IBD), and improving intestinal flora is considered to be important for maintaining health. It is reported that in the onset of inflammatory bowel disease, in a natural model of enteritis in mice, enteritis does not occur in the absence of bacteria, but occurs when intestinal bacteria are present (Sandra C. Kim, et al: Non-patent Document 1, Monika Schaubeck, et al: Non-patent Document 2). This suggests that in inflammatory bowel disease, intestinal flora is also one of the environmental factors significantly associated with the onset of the disease. As bacteria that cause inflammatory bowel disease, Escherichia coli (E. coli) (Arlette Darfeuille-Michaud, et al: Non-patent document 3, Adeline Sivignon, et al: Non-patent document 4), Fusobacterium varium (F. varium) (Toshifumi Ohkusa, et al: Non-patent document 5) and the like, which have adhesion and invasion to intestinal epithelial cells, can be cited as candidate bacteria, but clear pathogenic bacteria are still unclear. Therefore, it is expected that the disease can be prevented or treated by controlling the intestinal flora. At present, as one of the treatment methods for controlling the intestinal flora, sterilization of intestinal bacteria by administering antibiotics is used. The purpose is to remove pathogenic bacteria by administering antibiotics. However, there are also antibiotic-resistant bacteria among the enteritis-causing bacteria known as pathogenic bacteria. Therefore, as a result, not only the fermentation bacteria are also eliminated together with the enteritis-causing bacteria, but also the antibiotic-resistant bacteria are dominant, which may lead to aggravation of the disease or a susceptible state. As a new treatment method based on this problem, it is believed that it is best to exclude only enteritis-causing bacteria while retaining the fermentative bacteria that improve intestinal function. Inflammatory bowel disease (IBD) is a disease that is increasing, and only in Japan, it exceeds 200,000 people every year. The disease is characterized by having two periods, an active phase and a remission phase, and these two periods recur. The current status is to prevent deterioration by suppressing the inflammation of the active phase using anti-inflammatory drugs such as steroids or Asacol. However, these are only symptomatic treatments, and improving the intestinal environment that causes inflammation is a radical treatment, but when the patient's sample is actually investigated, the intestinal flora is varied, and it is believed that it is difficult to suppress using a single agent. Therefore, in the treatment of IBD, it is important to properly diagnose the suffering state and implement the best treatment for each patient.

[0003] Although administration of bacterial preparations and fecal transplantation have been reported to improve intestinal flora for the treatment of diseases such as inflammatory bowel disease (IBD) and have shown some effects, new therapeutic methods are still desired.

[0004] Prior art literature

[0005] Non-patent literature

[0006] Non-patent document 1: Sandra C. Kim, et al., Gastroenterology 128, 891-906, 2005

[0007] Non-patent document 2: Monika Schaubeck, et al., Gut 65, 225-237, 2016

[0008] Non-patent document 3: Arlette Darfeuille-Michaud, et al., Gastroenterology 127, 412-421, 2004

[0009] Non-patent document 4: Adeline Sivignon, et al., Inflamm Bowel Dis 21, 276-286, 2015

[0010] Non-patent document 5: Toshifumi Ohkusa, et al., Journal of Medical Microbiology 58, 535-545, 2009 Summary of the invention

[0011] Problems to be solved by the invention

[0012] The subject of the present disclosure is to provide: a method for obtaining digestive tract contents or excreta with improved digestive tract flora by treating the digestive tract contents or excreta with IgA antibodies in a therapeutic method of administering digestive tract contents or excreta for the treatment of IBD; and a method for obtaining IgA antibodies related to the method; and pharmaceutical compositions used in these methods.

[0013] Means for solving problems

[0014] The present disclosure provides a method for treating IBD using digestive tract contents or excreta modified with an IgA antibody, and a composition used in the method and a method for producing the same.

[0015] In another aspect, the present disclosure provides a method for obtaining IgA antibodies that improve the flora in the digestive tract of IBD patients.

[0016] In another aspect, the present disclosure provides a method for modifying the digestive tract contents or excreta of an IBD patient using an IgA antibody, and a composition used in the method and a method for producing the composition.

[0017] In another aspect, the present disclosure provides a method for testing a patient treated with an IBD therapeutic drug containing digestive tract contents or excreta using a diagnostic drug containing IgA antibodies, and a drug used in the method and a method for producing the drug.

[0018] More specifically, the present disclosure provides, in one embodiment, a pharmaceutical composition for treating IBD, comprising digestive tract contents or excreta treated with IgA antibodies that improve the flora in the digestive tract.

[0019] The present disclosure provides, in one embodiment, a method for treating IBD, comprising the following steps:

[0020] (A1) Steps for preparing digestive tract contents or excreta of an IBD patient;

[0021] (A2) a step of modifying the digestive tract contents or excreta using IgA antibodies that improve the flora in the digestive tract; and

[0022] (A3) A step of administering the modified digestive tract contents or excreta to an IBD patient.

[0023] The present disclosure provides, in one embodiment, a method for treating IBD, comprising the following steps:

[0024] (A1') Steps for preparing digestive tract contents or excreta of an IBD patient;

[0025] (A2') a step of administering digestive tract contents or excreta to an IBD patient;

[0026] (A3′) A step of modifying the above-mentioned digestive tract contents or excreta by administering an IgA antibody that improves the flora in the digestive tract to an IBD patient.

[0027] The present disclosure provides, in one embodiment, a method for screening IgA antibodies that improve the flora in the digestive tract, comprising the following steps:

[0028] (B1) Steps for preparing digestive tract contents or excreta of an IBD patient;

[0029] (B2) using the candidate IgA antibody to modify the digestive tract contents or excreta;

[0030] (B3) A step of confirming that the modified digestive tract contents or excreta can improve the bacterial flora in the digestive tract.

[0031] The present disclosure provides, in one embodiment, a composition containing an IgA antibody for treating digestive tract contents or excreta of an IBD patient.

[0032] The present disclosure provides, in one embodiment, a method for treating the digestive tract contents or excrement of an IBD patient, comprising the following steps:

[0033] (C1) Steps for preparing digestive tract contents or excreta from an IBD patient;

[0034] (C2) a step of contacting the digestive tract contents or excreta with IgA antibodies that improve the bacterial flora in the digestive tract.

[0035] The present disclosure provides, in one embodiment, a method for testing whether a subject suffers from IBD or the risk of developing the disease, comprising the following steps:

[0036] (D1) a step of preparing IgA antibodies that improve the flora in the digestive tract of IBD patients;

[0037] (D2) contacting the digestive tract contents or excreta of the subject with the IgA antibody; and

[0038] (D3) a step of identifying bacteria that bind to the above-mentioned IgA antibody,

[0039] When the IgA antibody shows binding to IBD-associated bacteria, the subject is regarded as suffering from IBD or having a risk of developing IBD and is treated.

[0040] In one embodiment, the present disclosure provides a drug for diagnosing the presence or absence of IBD or the risk of developing IBD, the drug comprising an IgA antibody that improves the flora in the digestive tract of an IBD patient.

[0041] The present disclosure provides, in one embodiment, a method for testing the therapeutic effect of an IBD therapeutic drug, comprising the following steps:

[0042] (E1) a step of obtaining digestive tract contents or excreta of a patient who is currently receiving, plans to receive, or has received an IBD therapeutic drug containing digestive tract contents or excreta;

[0043] (E2) contacting the digestive tract contents or excreta with IgA antibodies; and

[0044] (E3) a step of analyzing bacteria bound to the above-mentioned IgA antibody,

[0045] When the above analysis results show that the bacterial flora in the digestive tract has become healthy, the treatment with the above IBD therapeutic drug is continued.

[0046] In one embodiment, the present disclosure provides a drug containing IgA antibodies for testing digestive tract contents or excreta of a patient who is currently receiving, plans to receive, or has received an IBD therapeutic drug containing digestive tract contents or excreta.

[0047] Still more specifically, the present disclosure provides the following items.

[0048] [Project 1]

[0049] A pharmaceutical composition for treating inflammatory bowel disease (IBD) comprising digestive tract contents or excreta treated with IgA antibodies that improve the flora in the digestive tract.

[0050] [Project 2]

[0051] A pharmaceutical composition for treating IBD, which is used in combination with a composition containing digestive tract contents or excreta, and contains IgA antibodies that improve the flora in the digestive tract.

[0052] [Item 3]

[0053] The pharmaceutical composition according to item 1 or 2, wherein the IgA antibody that improves the flora in the digestive tract has one or more of the following characteristics:

[0054] (1) when the IgA antibody comes into contact with digestive tract contents or excreta from an IBD patient or digestive tract contents or excreta from a healthy person, the IgA antibody binds to more IBD-associated bacteria when the IgA antibody comes into contact with digestive tract contents or excreta from an IBD patient;

[0055] (2) contacting the digestive tract contents or excreta from an IBD patient with the IgA antibody, and preparing a first binding profile based on the types and amounts of bacteria bound to the IgA antibody; contacting the digestive tract contents or excreta from a healthy person with the IgA antibody, and preparing a second binding profile based on the types and amounts of bacteria bound to the IgA antibody, and when comparing the first binding profile with the second binding profile, in the first binding profile, it is observed that the IgA antibody binds to a greater number of IBD-related bacteria or has a greater amount of binding to IBD-related bacteria;

[0056] (3) When the bacterial diversity of the digestive tract contents or excreta from IBD patients before treatment with IgA antibodies is compared with the bacterial diversity of the digestive tract contents or excreta after treatment with IgA antibodies, the bacterial diversity of the digestive tract contents or excreta after treatment with IgA antibodies increases;

[0057] (4) when digestive tract contents or excreta modified with the IgA antibody are administered to a patient, and when the bacterial diversity in the digestive tract of the patient before administration is compared with the bacterial diversity in the digestive tract contents or excreta after administration, the bacterial diversity in the digestive tract contents or excreta after administration increases;

[0058] (5) when the digestive tract contents or excreta modified with the IgA antibody are administered to a patient, and when the short-chain fatty acid content in the digestive tract of the patient before administration is compared with the short-chain fatty acid content in the digestive tract contents or excreta after administration, the short-chain fatty acid content after administration increases;

[0059] (6) Gastrointestinal contents or excreta modified with the IgA antibody are administered to a patient, and the Enterobacteriaceae content in the digestive tract contents or excreta of the subject before administration is compared with the Enterobacteriaceae content in the digestive tract contents or excreta after administration, whereby the Enterobacteriaceae content in the digestive tract contents or excreta after administration is reduced.

[0060] [Item 4]

[0061] The pharmaceutical composition according to item 1 or 2, wherein the digestive tract contents or excreta are from a subject to which the composition is administered.

[0062] [Item 5]

[0063] A method for screening IgA antibodies that improve the flora in the digestive tract, comprising the following steps:

[0064] (B1) Steps for preparing digestive tract contents or excreta of an IBD patient;

[0065] (B2) using the candidate IgA antibody to modify the digestive tract contents or excreta;

[0066] (B3) A step of confirming that the modified digestive tract contents or excreta can improve the bacterial flora in the digestive tract.

[0067] [Item 6]

[0068] The method according to item 5, wherein the step (B3) of confirming that the modified digestive tract contents or excreta can improve the bacterial flora in the digestive tract is performed by any of the following methods.

[0069] (1) when the IgA antibody is brought into contact with digestive tract contents or excreta from an IBD patient or digestive tract contents or excreta from a healthy person, it is confirmed that the IgA antibody binds to more IBD-associated bacteria when brought into contact with digestive tract contents or excreta from an IBD patient;

[0070] (2) contacting the digestive tract contents or excreta from an IBD patient with the IgA antibody, and preparing a first binding profile based on the types and amounts of bacteria bound to the IgA antibody; contacting the digestive tract contents or excreta from a healthy person with the IgA antibody, and preparing a second binding profile based on the types and amounts of bacteria bound to the IgA antibody, and when comparing the first binding profile with the second binding profile, confirming that the IgA antibody binds to a greater number of IBD-related bacteria or a greater amount of IBD-related bacteria in the first binding profile;

[0071] (3) When the bacterial diversity of the digestive tract contents or excreta from IBD patients before treatment with IgA antibodies was compared with the bacterial diversity of the digestive tract contents or excreta after treatment with IgA antibodies, it was confirmed that the bacterial diversity of the digestive tract contents or excreta after treatment with IgA antibodies increased;

[0072] (4) when the digestive tract contents or excreta modified with the above-mentioned IgA antibody are administered to a patient, and when the bacterial diversity in the digestive tract of the patient before administration is compared with the bacterial diversity in the digestive tract contents or excreta after administration, it is confirmed that the bacterial diversity in the digestive tract contents or excreta after administration increases;

[0073] (5) When the digestive tract contents or excreta modified with the above-mentioned IgA antibody are administered to a patient, and the short-chain fatty acid content in the digestive tract of the patient before administration is compared with the short-chain fatty acid content in the digestive tract contents or excreta after administration, the short-chain fatty acid content after administration increases;

[0074] (6) Gastrointestinal contents or excreta modified with the IgA antibody are administered to a patient, and the Enterobacteriaceae content in the digestive tract contents or excreta of the subject before administration is compared with the Enterobacteriaceae content in the digestive tract contents or excreta after administration, whereby the Enterobacteriaceae content in the digestive tract contents or excreta after administration is reduced.

[0075] [Item 7]

[0076] A method for treating digestive tract contents or excrement of an IBD patient, comprising the following steps:

[0077] (C1) Steps for preparing digestive tract contents or excreta from an IBD patient;

[0078] (C2) A step of bringing the digestive tract contents or excreta into contact with IgA that improves the bacterial flora in the digestive tract in vitro.

[0079] [Item 8]

[0080] A composition for modifying the flora of digestive tract contents or excreta in vitro, comprising IgA that improves the flora in the digestive tract.

[0081] [Item 9]

[0082] A method for conducting a test to diagnose whether a subject suffers from IBD or is at risk of developing IBD, comprising the following steps:

[0083] (D1) a step of preparing IgA antibodies that improve the flora in the digestive tract of IBD patients;

[0084] (D2) contacting the digestive tract contents or excreta of the subject with the IgA antibody; and

[0085] (D3) a step of identifying bacteria that bind to the above-mentioned IgA antibody,

[0086] When the IgA antibody shows binding to IBD-associated bacteria, the subject is regarded as suffering from IBD or having a risk of developing IBD and is treated.

[0087] [Item 10]

[0088] A drug for diagnosing the presence or absence of IBD or the risk of developing IBD, comprising IgA antibodies that improve the flora in the digestive tract of IBD patients.

[0089] [Item 11]

[0090] A method for testing the therapeutic effect of an IBD therapeutic drug, comprising the following steps:

[0091] (E1) a step of obtaining digestive tract contents or excreta of a patient who is currently receiving, plans to receive, or has received an IBD therapeutic drug containing digestive tract contents or excreta;

[0092] (E2) contacting the digestive tract contents or excreta with IgA antibodies; and

[0093] (E3) a step of analyzing bacteria bound to the above-mentioned IgA antibody,

[0094] When the above analysis results show that the bacterial flora in the digestive tract has become healthy, the treatment with the above IBD therapeutic drug is continued.

[0095] [Item 12]

[0096] The method according to item 11, wherein the digestive tract contents or excreta are modified using the IgA.

[0097] [Item 13]

[0098] A diagnostic drug containing IgA antibodies is used to test the therapeutic effect of an IBD therapeutic drug on a patient who is currently receiving, is planning to receive, or has received an IBD therapeutic drug containing digestive tract contents or excreta.

[0099] [Item 14]

[0100] The diagnostic drug according to Item 13, wherein the digestive tract contents or excreta are modified by the IgA.

[0101] Effects of the Invention

[0102] The effects of the present disclosure are to provide: a method for obtaining digestive tract contents or excreta with improved digestive tract flora by treating the digestive tract contents or excreta with IgA antibodies in a therapeutic method of administering digestive tract contents or excreta for the treatment of IBD; and a method for obtaining IgA antibodies related to the method; and pharmaceutical compositions used in these methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 The graph shows the results of testing the binding properties of endogenous polyclonal IgA antibodies to various intestinal bacteria contained in stool collected from healthy individuals or IBD patients. The vertical axis represents the IgA index.

[0104] Figure 2 This is a graph showing the results of a test on the binding of IgA antibodies obtained from the digestive tract of IBD patients to IBD-related bacteria. The binding ability of IgA antibodies to IBD-related bacteria was evaluated by ELISA analysis.

[0105] Figure 3 This is a graph showing that endogenous polyclonal IgA antibodies have high binding affinity to IBD-associated bacteria.

[0106] Figure 4This is a graph showing that the RS_H000_L001 IgA antibody has a high binding ability to IBD-related bacteria.

[0107] Figure 5 This is a graph showing that RS_H007_L004 IgA antibody, a variant of RS_H000_L001 IgA antibody, has high binding ability to IBD-associated bacteria.

[0108] Figure 6 This is a graph showing that the SNK0003A IgA antibody has a high binding ability to IBD-related bacteria.

[0109] Figure 7 This is a graph showing that the SNK0001A IgA antibody has a high binding ability to IBD-related bacteria.

[0110] Figure 8 This is a graph showing that the SNK0002A IgA antibody has a high binding ability to IBD-related bacteria.

[0111] Fig. 9 The graph shows the results of confirming the binding properties of RS_H000_L001 IgA antibody to bacteria in stool obtained from healthy individuals or IBD patients.

[0112] Fig.10 The graph shows that the RS_H000_L001 antibody exerts a strong growth inhibition effect on Gemella morbillorum in an in vitro growth inhibition test.

[0113] Fig.11 Schematic diagram showing the protocol of a mouse oral bacterial transplantation experiment using intestinal bacteria from IBD patients (P10).

[0114] Fig.12 The graphs show changes in body weight of (1) the total flora / antibody non-administration group and (2) the total flora / RS_H000_L001 antibody oral administration group in a mouse oral bacterial transplantation experiment using intestinal bacteria from IBD patients (P10).

[0115] Fig.13 The graphs show changes in body weight of (3) the RS_H000_L001 antibody-unbound bacteria / antibody-unadministered group and (4) the RS_H000_L001 antibody-unbound bacteria / RS_H000_L001 antibody-orally administered group in a mouse oral bacterial transplantation experiment using intestinal bacteria from IBD patients (P10).

[0116] Fig.14The graph shows the results of the diversity analysis of the intestinal flora of mice after treatment in a mouse oral bacteria transplantation experiment using intestinal bacteria from IBD patients (P10).

[0117] Fig.15 Schematic diagram showing the protocol of a mouse oral bacterial transplantation experiment using intestinal bacteria from an IBD patient (P13).

[0118] Fig.16 This is a diagram showing the binding between the fecal flora from an IBD patient (P13) and the RS_H000_L001 antibody.

[0119] Fig.17 The graphs show changes in body weight of (1) the total flora / antibody non-administration group and (2) the total flora / RS_H000_L001 antibody oral administration group in a mouse oral bacterial transplantation experiment using intestinal bacteria from an IBD patient (P13).

[0120] Fig.18 This is a graph showing the results of the analysis of the diversity of the intestinal flora of mice treated in a mouse oral bacteria transplantation experiment using intestinal bacteria from IBD patients (P13) using the Shannon index.

[0121] Fig.19 The graph shows the results of the diversity analysis of the intestinal flora of mice after treatment in a mouse oral bacteria transplantation experiment using intestinal bacteria from IBD patients (P13), using changes in the relative abundance ratio of Fusobacteriaceae.

[0122] Fig. 20 The graph shows the results of the diversity analysis of the intestinal flora of mice after treatment in a mouse oral bacteria transplantation experiment using intestinal bacteria from IBD patients (P13), using changes in the relative abundance ratio of Lachnospiraceae.

[0123] Fig.21 The graph shows the results of the diversity analysis of the intestinal flora of mice after treatment in a mouse oral bacteria transplantation experiment using intestinal bacteria from IBD patients (P13), using changes in the relative abundance ratio of Bacteroidaceae.

[0124] Fig. 22 The graph shows the staining results of large intestinal tissue sections after treatment in a mouse oral bacteria transplantation experiment using intestinal bacteria from an IBD patient (P13).

[0125] Fig.23The graph shows the results of confirming the binding property of SNK0004 IgA antibody to bacteria in stool obtained from healthy individuals or IBD patients.

[0126] Fig.24 The graph shows the results of confirming the binding property of SNK0005 IgA antibody to bacteria in stool obtained from healthy individuals or IBD patients.

[0127] Fig.25 The graph shows the results of confirming the binding properties of the SNK0004 IgA antibody to bacteria in stool obtained from IBD patients.

[0128] Fig.26 The graph shows the results of confirming the binding properties of SNK0005 IgA antibody to bacteria in stool obtained from IBD patients.

[0129] Fig. 27 The graph shows the results of confirming the binding properties of SNK0001 IgA antibody to bacteria in stool obtained from IBD patients.

[0130] Fig.28 The graph shows the results of confirming the binding properties of SNK0002 IgA antibody to bacteria in stool obtained from IBD patients.

[0131] Fig.29 The graph shows the results of confirming the binding properties of SNK0003 IgA antibody to bacteria in stool obtained from IBD patients.

[0132] Fig.30 This is a graph showing the results of comparing the ratios of bacteria derived from IBD patient stool that bind to RS_H000_L001, SNK0001, SNK0002, SNK0003, SNK0004, and SNK0005 antibodies. DETAILED DESCRIPTION

[0133] It is known that immunoglobulin A (IgA) antibody, one of the isotypes of antibody molecules, is important in the maintenance of normal intestinal flora and in the defense against infection by pathogenic microorganisms invading the intestinal tract. IgA antibody is an antibody that works not only in serum but also mainly on the mucosal surface of the intestinal tract. IgA antibody is produced by IgA antibody-producing cells present in the mucosal lamina propria. IgA antibody is secreted into the mucosal lamina propria in the form of dimeric IgA antibody with the help of J chain. Dimeric IgA antibody is transported to the intestinal cavity through mucosal epithelial cells. This transport is carried out by the polymer Ig receptor expressed in the mucosal epithelial cells. Dimeric IgA antibody binds to the receptor, is endocytosed into the vesicle, and is transported to the intestinal cavity surface by transcytosis. On the intestinal cavity surface, the extracellular domain of the receptor is cut by protease, and the dimeric IgA antibody is secreted into the intestinal cavity while maintaining the extracellular domain (secretory factor). The secretory factors attached to the dimeric IgA antibodies protect the antibodies from being degraded by the proteolytic enzymes in the intestinal cavity. After the above process, the secreted dimeric IgA antibodies bind to the bacteria in the intestinal cavity, preventing the adhesion and invasion of bacteria to the epithelium.

[0134] The inventors of the present disclosure have conducted in-depth research on the role of intestinal IgA antibodies in intestinal flora control, and found that the host identifies and controls intestinal bacteria by secreting IgA antibodies into its own intestine. And it was found that there are antibodies in the intestinal IgA antibodies that work in the direction of identifying bacteria and excluding the bound bacteria from the intestinal cavity, and there are also antibodies that play a role in maintaining the diversity of intestinal flora by identifying, binding bacteria and keeping the bound bacteria in the intestinal mucosal layer. In this way, it is believed that intestinal IgA antibodies have complex interactions with intestinal bacteria in the direction of maintaining bacteria that are beneficial to the host on the one hand and excluding bacteria that are harmful to the host on the other hand, while controlling the overall flora composition. However, there are still unclear points about what each IgA antibody recognizes each intestinal bacterium to play a role and how to act on the target molecule.

[0135] The inventors of the present disclosure conducted in-depth research and found that the state of the intestinal flora of patients with inflammatory bowel disease (IBD) is different from that of healthy people, the bacteria associated with the worsening of IBD (IBD-related bacteria) increase, and the binding ability of IgA antibodies of IBD patients to these IBD-related bacteria decreases. Therefore, they came up with the idea of ​​using IgA antibodies that improve the intestinal flora to treat the digestive tract contents or excrement of IBD patients, and administering the treated products to IBD patients through fecal transplantation, etc., thereby improving IBD, and successfully confirmed its effect in practice.

[0136] (definition)

[0137] Unless otherwise defined, the meanings of technical and scientific terms used in this specification are the same as those commonly understood by those skilled in the art to which the present disclosure belongs.

[0138] In the present specification, when a plurality of numerical ranges are shown, a range consisting of a combination of arbitrary lower limits and upper limits of these plurality of ranges is also meaningful.

[0139] In this specification, the term "substantially" has the same meaning as generally understood by technicians in the technical field to which the present disclosure belongs. For example, considering that biological or chemical phenomena sometimes cannot completely achieve the target state, it is used with the intention of covering the target state as well as the state that cannot be inevitably achieved due to biological or chemical characteristics.

[0140] In this specification, when the term "about" is used with respect to a numerical value, it means that the value may vary within a range of, for example, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01%.

[0141] In this specification, the term "comprising" has the same meaning as that generally understood by those skilled in the art to which the present disclosure belongs, for example, including the cases of "comprising" and "consisting of..." Specifically, a composition "comprising" A sometimes also includes other constituent elements B in addition to the case where it only includes A.

[0142] In this specification, the term "consisting of" or "composed of" used for a composition has the same meaning as commonly understood by a person skilled in the art to which the present disclosure belongs, and is used to indicate constituent elements that exclusively constitute the composition. For example, a composition "consisting of A" exclusively contains A. However, in one embodiment, a composition "consisting of A" includes an embodiment that contains admixtures other than A that are unavoidable in manufacturing due to biological and chemical properties.

[0143] In this specification, "antibody" is used in the broadest sense, including monoclonal antibodies, polyclonal antibodies and antibody fragments that show the expected antigen binding activity, but is not limited to these. Full-length antibodies mainly include heavy chains and light chains composed of polypeptides. The heavy chain and light chain each contain a site called a variable region that recognizes the antigen, which is usually referred to as a heavy chain variable region and a light chain variable region, respectively. The variable region has sites that are determined to be sites for further detailed antigen recognition, respectively referred to as CDR1 to 3, starting from the amino terminus. In addition, these CDR1 to 3 are also referred to as heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, light chain CDR3, etc. in more detail. In addition, the regions other than CDR1 to 3 of the heavy chain and light chain are respectively referred to as heavy chain FR1 to 4 and light chain FR1 to 4 in sequence from the amino terminus. In addition to the form of an antibody containing two heavy chains and two light chains, an antibody (also called a single-chain antibody) can also be in the form of an antibody containing one heavy chain and one light chain.

[0144] Antibodies are classified into classes such as IgG, IgE, IgM, IgD, IgA, or IgY, which are further classified into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2, and the like.

[0145] 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 a fragment comprising a portion thereof. As one embodiment, the "antigen-binding fragment" of an antibody can be a Fab fragment comprising a light chain variable region (VL), a heavy chain variable region (VH), a light chain constant region (CL), and a CH1 region that is a part of the heavy chain constant region; a F(ab')2 fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; an Fd fragment comprising VH and CH1 regions; an Fv fragment comprising the VL and VH regions of a single arm of an antibody; a dAb fragment comprising a single variable region; and a separated complementary determining region (CDR), but is not limited to these.

[0146] The antibodies disclosed herein may also be CDR-grafted antibodies. In one example, in a CDR-grafted antibody, a portion or all of the CDR region of an antibody derived from one animal species is replaced by a CDR sequence from another animal species. For example, one or more CDRs of a mouse antibody are replaced by a CDR sequence of a human antibody.

[0147] To identify the heavy chain CDR1 to 3 in the heavy chain variable region and the light chain CDR1 to 3 in the light chain variable region of an antibody, methods known to those skilled in the art can be used. For example, the “Kabat definition” (Kabat et al., Ann. NY Acad, Sci. 1971, Vol. 190, pp. 382-391 and Kabat.EA et al., Sequences of Proteins of Immunological Interest, 5th edition, 1991, US Department of Health and Human Services, NIH Publication, pp. 91-3242), the “Chothia definition” (Chothia et al., Nature, 1989, Vol. 342, pp. 877-883), the “contact definition” (MacCallum et al., J. Mol. Biol., 1996, Vol. 262, pp. 732-745), the “IGMT” (Lefranc, M.-P., Nucl. Acids Res., 33, D593-D597 (2005)), etc., which are well known to those skilled in the art, can be used. To identify CDR sequences within antibodies, CDRs can also be identified based on information in public databases (eg, https: / / www.ncbi.nlm.nih.gov / igblast).

[0148] "Consistency" in this specification refers to the degree of the same amino acid sequence or base sequence of more than two comparable amino acid sequences or base sequences relative to each other. Therefore, the higher the consistency of a certain two amino acid sequences or base sequences, the higher the consistency or similarity of their sequences. The consistency level of amino acid sequences or base sequences is usually determined using the sequence analysis tool FASTA using default parameters. Alternatively, the algorithm BLAST proposed by Karlin and Altschul (for example, 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.) can be used to determine. Based on such BLAST algorithm, programs called BLASTN and BLASTX were developed (e.g., Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. J Mol Biol. 215: 403-10 (1990) etc.). The specific methods of these analysis methods are well known and can be found on the NCBI website. For example, a certain amino acid sequence A is identical to another amino acid sequence B by a certain percentage (%), which means that the amino acid sequence A and the amino acid sequence B have the same percentage (%).

[0149] In the present specification, "monoclonal" is a modifier that characterizes an antibody obtained from a substantially homogeneous antibody population. The individual antibodies included in such an antibody population are identical except for possible naturally occurring mutations that may exist in trace amounts.

[0150] The "conservative substitution technique" in the present specification refers to a technique of substituting an amino acid residue with an amino acid residue having a similar side chain.

[0151] For example, substitutions between amino acid residues with basic side chains such as lysine, arginine, and histidine belong to conservative substitution techniques. In addition, 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 also belong to conservative substitution techniques.

[0152] In this specification, "digestive tract" has the usual meaning in the technical field of the present disclosure, and typically refers to the tubular tissue from the mouth to the rectum through which food enters the body and is excreted from the body in the form of excrement. For example, the "digestive tract" includes the mouth, throat, esophagus, stomach, duodenum, small intestine, and large intestine.

[0153] In this specification, "digestive tract contents" include all substances present in the lumen of the "digestive tract", including, for example, ingested food and drink, substances derived from host tissues excreted into the lumen, their metabolites, and mixtures thereof.

[0154] In this specification, "excreta" includes all substances discharged from the above-mentioned "digestive tract". Typically, the excreta includes feces.

[0155] 1. Method for treating IBD using digestive tract contents or excreta modified with IgA antibodies

[0156] In one embodiment, the present disclosure provides a method for treating IBD using digestive tract contents or excreta modified with an IgA antibody.

[0157] In one embodiment, the method for treating IBD disclosed herein comprises the following steps.

[0158] (A1) Steps for preparing digestive tract contents or excreta of IBD patients,

[0159] (A2) a step of modifying the digestive tract contents or excreta using IgA antibodies that improve the flora in the digestive tract, and

[0160] (A3) A step of administering the modified digestive tract contents or excreta to an IBD patient.

[0161] In one embodiment, the method for treating IBD disclosed herein comprises the following steps.

[0162] (A1') Steps for preparing digestive tract contents or excreta of IBD patients,

[0163] (A2') a step of administering digestive tract contents or excreta to an IBD patient,

[0164] (A3′) A step of modifying the above-mentioned digestive tract contents or excreta by administering an IgA antibody that improves the flora in the digestive tract to an IBD patient.

[0165] (IgA antibody)

[0166] The flora in the digestive tract of IBD patients is significantly different from that of healthy people, and has a composition that worsens IBD. On the other hand, the specific flora composition is different in each IBD patient. Therefore, the IgA antibody used in this method uses an antibody obtained from the patient as an antibody that improves the flora in the digestive tract of the IBD patient to be treated.

[0167] Confirmation that the IgA antibody can improve the digestive tract flora of IBD patients can be implemented, for example, by the following methods: analyzing the amount of IBD-related bacteria bound to the IgA; analyzing the binding distribution map of multiple bacteria bound to the IgA antibody; conducting physical, chemical or biological property tests on digestive tract contents or excreta modified by the IgA antibody, such as confirming that the diversity of the flora in the digestive tract contents or excreta is increased, confirming that the diversity of the flora in the intestine is increased by administering the digestive tract contents or excreta, confirming that the short-chain fatty acids in the intestine are increased by administering the digestive tract contents or excreta, confirming that the Enterobacteriaceae present in the intestine are reduced by administering the digestive tract contents or excreta, confirming that the symptoms of IBD are improved by administering the digestive tract contents or excreta, etc.

[0168] In the case of confirming that the IgA antibody can make the digestive tract flora of the IBD patient sound by analyzing the IBD-related bacteria bound to the IgA antibody, for example, it can be implemented as follows: the intestinal bacteria (IBD-related bacteria) known to be associated with the deterioration of IBD in the digestive tract contents or excreta from the IBD patient and the digestive tract contents or excreta from a healthy person are compared with the binding amount of the IgA antibody, and it is confirmed that the IgA antibody is bound to a larger amount of the IBD-related bacteria in the digestive tract contents or excreta from the IBD patient. This confirmation test can be implemented using methods known to those skilled in the art, such as FACS analysis, ELISA analysis, etc. The IBD-related bacteria in the present disclosure are bacteria known to those skilled in the art as intestinal bacteria known to be associated with the deterioration of IBD. Non-limiting examples of the IBD-related bacteria include Enterobacteriaceae.

[0169] When confirming that the IgA antibody can improve the digestive tract flora of IBD patients by analyzing the binding distribution map of multiple bacteria bound to the IgA antibody, the method can be implemented as follows, for example: the digestive tract contents or excreta from the IBD patient are brought into contact with the IgA antibody, and a binding distribution map is prepared based on the types and amounts of bacteria bound to the IgA antibody; the digestive tract contents or excreta from a healthy person are brought into contact with the IgA antibody, and a binding distribution map is prepared based on the types and amounts of bacteria bound to the IgA antibody, and the two binding distribution maps prepared above are compared to confirm that the binding of the IgA antibody to more types of IBD-related bacteria or a larger binding amount to IBD-related bacteria is observed in the binding distribution map prepared from the digestive tract contents or excreta from the IBD patient. In one example, the above binding distribution diagram can be represented by quantitative data based on the IgA index (calculated based on the amount of each bacterium bound to IgA and the amount not bound 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 implemented using methods known to those skilled in the art, such as FACS analysis, ELISA analysis, and analysis using a next-generation sequencer.

[0170] In the case where the IgA antibody is confirmed to make the digestive tract flora of IBD patients healthy by confirming that the flora diversity in the digestive tract contents or excreta treated with the IgA antibody increases, for example, the flora diversity of the digestive tract contents or excreta before treatment with the IgA antibody can be compared with the flora diversity of the digestive tract contents or excreta after treatment with the IgA antibody, and the IgA antibody can be confirmed to make the digestive tract flora of IBD patients healthy by confirming that the flora diversity of the digestive tract contents or excreta after treatment with the IgA antibody increases. This confirmation test can be implemented using methods known to those skilled in the art, such as FACS analysis, ELISA analysis, analysis using a next-generation sequencer, etc.

[0171] In the case where the diversity of the intestinal flora is confirmed to increase by administering the digestive tract contents or excreta treated with the IgA antibody to the subject, and thus confirming that the IgA antibody can make the digestive tract flora of the IBD patient healthy, for example, the diversity of the intestinal flora of the subject before the administration can be compared with the diversity of the intestinal flora after the administration, and the increase in the diversity of the digestive tract contents or excreta after the administration can be confirmed to confirm that the IgA antibody can make the digestive tract flora of the IBD patient healthy. This confirmation test can use the digestive tract contents or excreta of the subject after administration as a sample, and use methods known to those skilled in the art to implement, for example, FACS analysis, ELISA analysis, analysis using a next-generation sequencer, etc. can be used. In this test, IBD patients or IBD model animals can be used as subjects.

[0172] In the case where the increase of short-chain fatty acids in the intestine is confirmed by administering the digestive tract contents or excreta, and thus confirming that the IgA antibody can make the digestive tract flora of the IBD patient healthy, for example, the short-chain fatty acid content of the digestive tract contents or excreta of the subject before the administration can be compared with the short-chain fatty acid content of the digestive tract contents or excreta after the administration, and the IgA antibody can make the digestive tract flora of the IBD patient healthy by confirming that the short-chain fatty acid content of the digestive tract contents or excreta after the administration increases. This confirmation test can be implemented using methods known to those skilled in the art, such as chromatographic analysis. In this test, IBD patients or IBD model animals can be used as subjects.

[0173] In the case where the Enterobacteriaceae present in the intestinal tract is confirmed to be reduced by administering the digestive tract contents or excreta, and thus confirming that the IgA antibody can make the digestive tract flora of the IBD patient sound, for example, the Enterobacteriaceae content of the digestive tract contents or excreta of the subject before the administration can be compared with the Enterobacteriaceae content of the digestive tract contents or excreta after the administration, and the Enterobacteriaceae content of the digestive tract contents or excreta after the administration is confirmed to be reduced to confirm that the IgA antibody can make the digestive tract flora of the IBD patient sound. This confirmation test can be implemented using methods known to those skilled in the art, for example, colony culture tests can be used. In this test, IBD patients or IBD model animals can be used as subjects.

[0174] In the case where the symptoms of IBD are confirmed to be improved by administering the digestive tract contents or excreta, and thus confirming that the IgA antibody can make the digestive tract flora of the IBD patient healthy, for example, the IBD symptoms of the subject before the administration can be compared with the IBD symptoms of the subject after the administration, and the IgA antibody can make the digestive tract flora of the IBD patient healthy by confirming the improvement of the symptoms after the administration. This confirmation test can be implemented using IBD diagnostic indicators known to those skilled in the art. In this test, IBD patients or IBD model animals can be used as subjects.

[0175] The confirmation that the above-mentioned IgA antibodies can improve the digestive tract flora of IBD patients can also be implemented in advance as a method separate from the treatment method disclosed in the present invention, or can be performed as one of the steps constituting the present treatment method, using the digestive tract contents or excrement obtained in step (A1) as step (A1-2).

[0176] The IgA antibody used in the method of the present disclosure may be further bound to other compounds within the range where its binding properties are not lost, and may be fused to a heterologous peptide.

[0177] The IgA antibody used in the method of the present disclosure may have an amino acid sequence derived from the same species as the IBD patient, or may have amino acids derived from a different species such as a closely related xenobiotic. Specifically, for example, examples include those derived from humans, mice, rats, hamsters, rabbits, goats, donkeys, pigs, cattle, horses, chickens, monkeys, chimpanzees, camels, and llamas.

[0178] The IgA antibodies disclosed herein may be those produced by antibody-producing cells derived from B cells such as hybridomas, or nucleic acids encoding the antibodies may be introduced into cells other than the immune system using genetic recombination techniques and the produced antibodies may be used as recombinant antibodies.

[0179] In one embodiment, the IgA antibody of the present disclosure comprises:

[0180] A heavy chain variable region comprising the amino acid sequence of a heavy chain CDR1, the amino acid sequence of a heavy chain CDR2, and the amino acid sequence of a heavy chain CDR3 of the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 7; and

[0181] A light chain variable region comprising the amino acid sequence of the light chain variable region represented by SEQ ID NO:8, comprising the amino acid sequence of the light chain CDR1, the amino acid sequence of the light chain CDR2, and the amino acid sequence of the light chain CDR3.

[0182]

[0183] In one embodiment, the IgA antibody of the present disclosure comprises:

[0184] The heavy chain variable region comprises

[0185] A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1,

[0186] A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and

[0187] A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3; and

[0188] The light chain variable region comprises

[0189] A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4,

[0190] A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5,

[0191] A light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:6.

[0192] Sequence name (sequence number) Amino acid sequence SNK0001_HCDR1 (SEQ ID NO: 1) SYGIS SNK0001_HCDR2 (SEQ ID NO: 2) EIYPRSGNTYYNEKFK SNK0001_HCDR3 (SEQ ID NO: 3) FCARLASS SNK0001_LCDR1 (serial number 4) KASQDINSYLS SNK0001_LCDR2 (serial number 5) RANRLVD SNK0001_LCDR3 (serial number 6) LQYDEFPLT

[0193] In one embodiment, the IgA antibody of the present disclosure comprises:

[0194] A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 7 or a sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto; and

[0195] A light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 8 or a sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0196] In one embodiment, the IgA antibody disclosed herein is a recombinant purified antibody SNK0001AR obtained by determining a nucleic acid sequence encoding the IgM antibody SNK0001M produced by a hybridoma obtained from spleen-derived B cells and producing the IgA antibody using recombinant technology.

[0197] The antibody SNK0001AR has the following amino acid sequence composition.

[0198]

[0199] In one embodiment, the IgA antibody of the present disclosure comprises:

[0200] A heavy chain variable region comprising the amino acid sequence of a heavy chain CDR1, the amino acid sequence of a heavy chain CDR2, and the amino acid sequence of a heavy chain CDR3 of the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 17; and

[0201] A light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of the light chain variable region containing the amino acid sequence represented by SEQ ID NO:18.

[0202]

[0203] In one embodiment, the IgA antibody of the present disclosure comprises:

[0204] The heavy chain variable region comprises

[0205] A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 11,

[0206] A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 12, and

[0207] A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 13; and

[0208] The light chain variable region comprises

[0209] A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 14,

[0210] A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 15,

[0211] A light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:16.

[0212] Sequence name (sequence number) Amino acid sequence SNK0002_HCDR1 (SEQ ID NO: 11) TFGMG SNK0002_HCDR2 (SEQ ID NO: 12) LAHIWWDDDKYYNPAL SNK0002_HCDR3 (SEQ ID NO: 13) YYCARIAG SNK0002_LCDR1 (serial number 14) SASSSVSYMHW SNK0002_LCDR2 (serial number 15) STSNLASG SNK0002_LCDR3 (serial number 16) QRSSYPYTF

[0213] In one embodiment, the IgA antibody of the present disclosure comprises:

[0214] A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 17 or a sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto; and

[0215] A light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 18 or a sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0216] In one embodiment, the IgA antibody disclosed herein is a recombinant purified antibody SNK0002AR obtained by determining the nucleic acid sequence of the IgM antibody SNK0002M produced by a hybridoma obtained from spleen-derived B cells and producing the IgA antibody using recombinant technology.

[0217] The antibody SNK0002AR has the following amino acid sequence composition.

[0218]

[0219] In one embodiment, the IgA antibody of the present disclosure comprises:

[0220] A heavy chain variable region comprising the amino acid sequence of a heavy chain CDR1, the amino acid sequence of a heavy chain CDR2, and the amino acid sequence of a heavy chain CDR3 of the heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 27; and

[0221] A light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of the light chain variable region containing the amino acid sequence represented by SEQ ID NO:28.

[0222]

[0223] In one embodiment, the IgA antibody of the present disclosure comprises:

[0224] The heavy chain variable region comprises

[0225] A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21,

[0226] A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22, and

[0227] A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23; and

[0228] The light chain variable region comprises

[0229] A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 24,

[0230] A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 25,

[0231] A light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:26.

[0232] Sequence name (sequence number) Amino acid sequence SNK0003_HCDR1 (SEQ ID NO: 21) SYNIH SNK0003_HCDR2 (SEQ ID NO: 22) AIYSGNGATSHNQKFK SNK0003_HCDR3 (SEQ ID NO: 23) FCTRVGLR SNK0003_LCDR1 (serial number 24) KASQSVGTSVA SNK0003_LCDR2 (serial number 25) SASYRYS SNK0003_LCDR3 (serial number 26) QQYNNYPYT

[0233] In one embodiment, the IgA antibody of the present disclosure comprises:

[0234] A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 27 or a sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto; and

[0235] A light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 28 or a sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0236] In one embodiment, the IgA antibody disclosed herein is IgA antibody SNK0003A produced by a hybridoma obtained from B cells derived from the intestinal lamina propria. The nucleic acid sequence encoding antibody SNK0003A is determined, and the recombinant purified antibody SNK0003AR produced by applying recombinant technology can also be used in the same manner.

[0237] Antibody SNK0003A has the following amino acid sequence composition.

[0238]

[0239] In one embodiment, the IgA antibody of the present disclosure is a monoclonal antibody that binds to Clostridium difficile bacteria, and is an antibody comprising a heavy chain variable region and a light chain variable region.

[0240] The heavy chain variable region comprises:

[0241] A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31,

[0242] A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32, and

[0243] A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33,

[0244] The light chain variable region comprises:

[0245] A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41,

[0246] A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 42,

[0247] A light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:43.

[0248] In one embodiment, the IgA antibody of the present disclosure comprises:

[0249] A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 37 or a sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto; and

[0250] A light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 38 or a sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0251] In one embodiment, the IgA antibody of the present disclosure is the following antibody:

[0252] and a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 37, and

[0253] Compared to a reference antibody having a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 38,

[0254] At least one amino acid mutation is present in at least one region selected from the group consisting of heavy chain CDR1 to 3, light chain CDR1 to 3, and light chain FR1,

[0255] It binds to the amino acid sequence RQEEHIELIAS (SEQ ID NO: 72) in the SHMT protein of Escherichia coli and the amino acid sequence VLDMMKLEKPE (SEQ ID NO: 73) in the iPGM protein of Clostridium difficile (C. difficile).

[0256]

[0257] At least one amino acid mutation possessed by the above-mentioned antibody can be determined by using the results of three-dimensional structural analysis of the binding body of an antibody having the amino acid sequence of the above-mentioned reference antibody and Escherichia coli SHMT protein, and an antibody having the amino acid sequence of the reference antibody and iPGM protein of Clostridium difficile (C. difficile), using a technique available to those skilled in the art. The number of amino acid mutations compared to the reference antibody can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.

[0258] Such a reference antibody is not particularly limited as long as it has W27G2_HV (sequence number 37) as a heavy chain variable region and W27G2_LV (sequence number 38) as a light chain variable region. For example, an IgG antibody (W27RS_H000_L000GR) can be used. The antibody W27RS_H000_L000GR has the same heavy chain variable region and light chain variable region as the antibody W27G2 produced by a hybridoma obtained from a B cell derived from the intestinal mucosal lamina propria.

[0259]

[0260] The binding of an antibody having a specific mutation to the amino acid sequence RQEEHIELIAS in the E. coli SHMT protein and the amino acid sequence VLDMMKLEKPE in the iPGM protein of Clostridium difficile (C. difficile) can be confirmed by methods known to those skilled in the art, such as ELISA and immunoblotting.

[0261] In one embodiment, the antibody that binds to Clostridium difficile bacteria of the present disclosure is an antibody comprising a heavy chain variable region and a light chain variable region,

[0262] The heavy chain variable region comprises

[0263] A heavy chain CDR1 containing the amino acid sequence of X1YYIH,

[0264] A heavy chain CDR2 comprising the amino acid sequence of RIDPENX2X3TTYAPKFQ,

[0265] A heavy chain CDR3 comprising the amino acid sequence of YCARSTVL,

[0266] The light chain variable region comprises

[0267] A light chain CDR1 containing the amino acid sequence of RX4SQSIVHTNG,

[0268] A light chain CDR2 containing the amino acid sequence of KLLIYKV,

[0269] A light chain CDR3 comprising the amino acid sequence of GVYYFQGS,

[0270] Light chain FR1 contains

[0271] The amino acid sequence of TPLSLPVSLGDQA, or

[0272] The amino acid sequence of SPASX5SVSLGDRX6,

[0273] X1, X2, and X3 are each independently a neutral polar amino acid or an acidic polar amino acid, X4 is a non-polar amino acid or a neutral polar amino acid, and X5 and X6 are each independently a non-polar amino acid.

[0274] The above-mentioned antibody was designed through three-dimensional structural analysis to have the same binding mode as the W27G2 antibody to the amino acid sequence RQEEHIELIAS in the SHMT protein of Escherichia coli and the amino acid sequence VLDMMKLEKPE in the iPGM protein of Clostridium difficile (C. difficile), and actually exerts the same binding mode and physiological activity as the W27G2 antibody against these bacteria.

[0275] In one embodiment, the IgA antibody of the present disclosure is an antibody that binds to Clostridium difficile bacteria.

[0276] It contains a heavy chain variable region and a light chain variable region.

[0277] The heavy chain variable region comprises

[0278] A heavy chain CDR1 containing the amino acid sequence of X1YYIH,

[0279] A heavy chain CDR2 comprising the amino acid sequence of RIDPENX2X3TTYAPKFQ,

[0280] A heavy chain CDR3 comprising the amino acid sequence of YCARSTVL,

[0281] The light chain variable region comprises

[0282] A light chain CDR1 containing the amino acid sequence of RX4SQSIVHTNG,

[0283] A light chain CDR2 containing the amino acid sequence of KLLIYKV,

[0284] A light chain CDR3 comprising the amino acid sequence of GVYYFQGS,

[0285] Light chain FR1 contains

[0286] The amino acid sequence of TPLSLPVSLGDQA, or

[0287] The amino acid sequence of SPASX5SVSLGDRX6,

[0288] X1, X2, and X3 are each independently a neutral polar amino acid or an acidic polar amino acid, X4 is a non-polar amino acid or a neutral polar amino acid, and X5 and X6 are each independently a non-polar amino acid.

[0289] Furthermore, the antibody is one in which X1 is aspartic acid, X2 is aspartic acid, X3 is glutamic acid, and X4 is alanine.

[0290] In one embodiment, the heavy chain of the IgA antibody of the present disclosure comprises:

[0291] A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31 or 41,

[0292] A heavy chain CDR2 comprising the amino acid sequence represented by any one of SEQ ID NOs: 32 and 42 to 44,

[0293] A heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:33.

[0294] Sequence name (sequence number) Amino acid sequence W27G2_HCDR1 (SEQ ID NO: 31) DYYI W27G2_HCDR2 (SEQ ID NO: 32) RIDPENDETTYAPKFQ W27G2_HCDR3 (SEQ ID NO: 33) YCARSTVL RS_HCDR1_D50N (serial number 41) NYYIH RS_HCDR2_D75N (Serial No. 42) RIDPENNETTYAPKFQ RS_HCDR2_E76Q (Serial No. 43) RIDPENDQTTYAPKFQ RS_HCDR2_D75N_E76Q (Serial No. 44) RIDPENNQTTYAPKFQ

[0295] In one embodiment, the heavy chain of the IgA antibody of the present disclosure comprises:

[0296] A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31,

[0297] A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32,

[0298] A heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:33.

[0299] In one embodiment, the heavy chain of the IgA antibody of the present disclosure comprises:

[0300] A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31,

[0301] A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 42,

[0302] A heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:33.

[0303] In one embodiment, the heavy chain of the IgA antibody of the present disclosure comprises:

[0304] A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31,

[0305] A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 43,

[0306] A heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:33.

[0307] In one embodiment, the heavy chain of the IgA antibody of the present disclosure comprises:

[0308] A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31,

[0309] A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44,

[0310] A heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:33.

[0311] In one embodiment, the heavy chain of the IgA antibody of the present disclosure comprises:

[0312] A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41,

[0313] A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32,

[0314] A heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:33.

[0315] In one embodiment, the heavy chain of the IgA antibody of the present disclosure comprises:

[0316] A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41,

[0317] A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 42,

[0318] A heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:33.

[0319] In one embodiment, the heavy chain of the IgA antibody of the present disclosure comprises:

[0320] A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41,

[0321] A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 43,

[0322] A heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:33.

[0323] In one embodiment, the heavy chain of the IgA antibody of the present disclosure comprises:

[0324] A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41,

[0325] A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44,

[0326] A heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:33.

[0327] In one embodiment, the light chain of the IgA antibody of the present disclosure comprises:

[0328] A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34 or 52,

[0329] A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35,

[0330] A light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:36.

[0331] Sequence name (sequence number) Amino acid sequence W27G2_LCDR1 (serial number 34) RASQSIVHTNG W27G2_LCDR2 (serial number 35) KL W27G2_LCDR3 (serial number 36) GVYYCFQG RS_LCDR1_A44S (serial number 52) RSSQSIVHTNG

[0332] In one embodiment, the light chain of the IgA antibody of the present disclosure comprises:

[0333] A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34,

[0334] A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35,

[0335] A light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:36.

[0336] In one embodiment, the light chain of the IgA antibody of the present disclosure comprises:

[0337] A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52,

[0338] A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35,

[0339] A light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:36.

[0340] In one embodiment, the IgA antibody of the present disclosure comprises a sequence that binds to protein L in the light chain variable region. The antibody molecule or antigen-binding fragment thereof comprising the sequence that binds to protein L can be purified using a protein L column.

[0341] In one embodiment, the IgA antibody comprising a sequence that binds to protein L of the present disclosure comprises a light chain variable region comprising an amino acid sequence of SPASX5SVSLGDRX6, wherein X5 and X6 are each independently a non-polar amino acid.

[0342] In one embodiment, the IgA antibody comprising a sequence binding to protein L of the present disclosure comprises a light chain variable region comprising an amino acid sequence of SPASX5SVSLGDRX6, wherein X5 is leucine or methionine, and X6 is alanine or valine.

[0343] In one embodiment, the IgA antibody comprising a sequence that binds to protein L of the present disclosure comprises a light chain variable region comprising an amino acid sequence represented by one selected from the group consisting of SEQ ID NOs: 53 to 55.

[0344] Sequence name (sequence number) Amino acid sequence RS_LFR1_L001 (serial number 53) SPASLSVSLGDRA RS_LFR1_L002 (serial number 54) SPASLSVSLGDRV RS_LFR1_L003 (serial number 55) SPASMSVSLGDRA RS_LFR1_L000 (serial number 56) TPLSLPVSLGDQA

[0345] In one embodiment, the IgA antibody comprising a sequence that binds to protein L of the present disclosure comprises a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:54.

[0346] This sequence is a sequence in which a mutation has been introduced into the sequence TPLSLPVSLGDQA (SEQ ID NO: 56) in the light chain FR1 region of the W27G2 antibody in order to allow protein L to bind to the antibody molecule.

[0347] In one embodiment, the IgA antibody or antigen-binding fragment thereof comprising a sequence that binds to protein L of the present disclosure comprises a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:55.

[0348] This sequence is a sequence in which a mutation is further introduced into the light chain FR1 region of the light chain variable region containing the amino acid sequence represented by SEQ ID NO: 54 so that protein L binds more firmly to the antibody molecule.

[0349] In one embodiment, the IgA antibody or antigen-binding fragment thereof comprising a sequence that binds to protein L of the present disclosure comprises a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:58.

[0350] This sequence is a sequence in which a mutation is further introduced into the light chain FR1 region of the light chain variable region containing the amino acid sequence represented by SEQ ID NO: 54 so that protein L binds more firmly to the antibody molecule.

[0351] The IgA antibody disclosed herein may have any combination of the above-mentioned heavy chains and light chains, and may or may not contain a sequence that binds to protein L.

[0352] In one embodiment, the IgA antibody or antigen-binding fragment thereof of the present disclosure is an antibody comprising a heavy chain variable region, a light chain variable region, and a light chain FR1,

[0353] The heavy chain variable region comprises:

[0354] A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31 or 41,

[0355] A heavy chain CDR2 comprising the amino acid sequence represented by any one of SEQ ID NOs: 32 and 42 to 44, and

[0356] A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33;

[0357] The light chain variable region comprises:

[0358] A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34 or 52,

[0359] A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and

[0360] A light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36;

[0361] The light chain FR1 contains the amino acid sequence represented by any one of SEQ ID NOs: 53 to 56.

[0362] For example, the IgA antibody of the present disclosure comprises a combination of any one of the following heavy chain variable regions and any one of the following light chain variable regions.

[0363]

[0364]

[0365] Preferably, the IgA antibody of the present disclosure comprises a combination of the following heavy chain variable region and light chain variable region.

[0366] Heavy chain variable region Light chain variable region W27G2_HV (serial number 37) RS_LV001 (serial number 57) W27G2_HV (serial number 37) RS_LV004 (serial number 60) W27G2_HV (serial number 37) RS_LV005 (serial number 61) RS_HV001 (serial number 45) RS_LV005 (serial number 61) RS_HV002 (serial number 46) RS_LV005 (serial number 61) RS_HV003 (serial number 47) RS_LV005 (serial number 61) RS_HV004 (serial number 48) RS_LV005 (serial number 61) RS_HV005 (serial number 49) RS_LV005 (serial number 61) RS_HV006 (serial number 50) RS_LV005 (serial number 61) RS_HV007 (serial number 51) RS_LV001 (serial number 57) RS_HV007 (serial number 51) RS_LV002 (serial number 58) RS_HV007 (serial number 51) RS_LV003 (serial number 59) RS_HV007 (serial number 51) RS_LV004 (serial number 60) RS_HV007 (serial number 51) RS_LV005 (serial number 61)

[0367] In one embodiment, the IgA antibody and related antibodies used as IgA of the present disclosure are as follows.

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374] Antibodies produced by CHO cells after introducing mutations into the heavy chain or light chain of the W27G2 antibody without substantially changing the antigenic specificity are referred to as RS mutant recombinant purified antibodies. These RS mutant recombinant purified antibodies further have mutations for binding to protein L.

[0375] The IgA antibody disclosed herein may have a mutation in the constituent amino acid sequence, such as its heavy chain variable region, light chain variable region, heavy chain CDR1 to 3 or light chain CDR1 to 3, within the range of not losing its antigen binding properties. The mutation may be a substitution, a deletion, an insertion, etc., but is not limited to these. For example, if it is a substitution, a conservative substitution technique may be used. In addition, a three-dimensional structural analysis or the like may be used to analyze the binding mode of the antibody to the antigen in detail, thereby introducing various mutations into the IgA antibody disclosed herein within the range of not losing its antigen binding properties.

[0376] (Nucleic Acids)

[0377] The nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention may be a ribonucleotide or a deoxynucleotide. In addition, the form of the nucleic acid is not particularly limited, and may be a single-stranded form or a double-stranded form. The codons used by the nucleic acid sequence are not particularly limited, and various codons may be appropriately selected for use according to the purpose. For example, it may be appropriately selected according to the type of host cell used during manufacture, taking into account the codon frequency, etc. In one embodiment, the nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention is used to express and manufacture the antibody or antigen-binding fragment thereof of the present invention.

[0378] In the case where the base nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention encodes an antigen-binding fragment, for example, the two domains VL and VH of the Fv fragment can be encoded using separate nucleic acid molecules, or a single nucleic acid can be used using recombinant technology to encode a single protein chain (single-chain Fv (scFv)) in which the VL and VH regions form a monovalent molecule.

[0379] The following is an example of sequence information related to the base nucleic acid encoding the antibody or antigen-binding fragment thereof of the present disclosure.

[0380] >Sequence number 1: SNK0001_HCDR1

[0381]

[0382] >Sequence number 2:SNK0001_HCDR2

[0383]

[0384] > SEQ ID NO: 3: SNK0001_HCDR3

[0385]

[0386] >Serial number 4: SNK0001_LCDR1

[0387]

[0388] >Serial number 5: SNK0001_LCDR2

[0389]

[0390] >Serial number 6: SNK0001_LCDR3

[0391]

[0392] >Serial number 7: SNK0001_HV

[0393]

[0394] >Serial number 8: SNK0001_LV

[0395]

[0396] (SNK0001AR antibody heavy chain amino acid sequence)

[0397] >Serial number 9: SNK0001AR_H

[0398]

[0399] (SNK0001AR antibody light chain amino acid sequence)

[0400] >Serial number 10: SNK0001AR_L

[0401]

[0402] >SEQ ID 11: SNK0002_HCDR1

[0403]

[0404] >SEQ ID 12: SNK0002_HCDR2

[0405]

[0406] >SEQ ID 13: SNK0002_HCDR3

[0407]

[0408] >Serial number 14: SNK0002_LCDR1

[0409]

[0410] >Serial number 15: SNK0002_LCDR2

[0411]

[0412] >Serial number 16: SNK0002_LCDR3

[0413]

[0414] >Serial number 17: SNK0002_HV

[0415]

[0416] >Serial number 18: SNK0002_LV

[0417]

[0418] (SNK0002AR antibody heavy chain amino acid sequence)

[0419] >Serial number 19: SNK0002AR_H

[0420]

[0421] (sNK0002AR antibody light chain amino acid sequence)

[0422] >Serial number 20: SNK0002AR_L

[0423]

[0424] >SEQ ID 21: SNK0003_HCDR1

[0425]

[0426] >SEQ ID 22: SNK0003_HCDR2

[0427]

[0428] >SEQ ID NO: 23: SNK0003_HCDR3

[0429]

[0430] >Serial number 24: SNK0003_LCDR1

[0431]

[0432] >Serial number 25: SNK0003_LCDR2

[0433]

[0434] >Serial number 26: SNK0003_LCDR3

[0435]

[0436] >Serial number 27: SNK0003_HV

[0437]

[0438] >Serial number 28: SNK0003_LV

[0439]

[0440] (SNK0003A antibody heavy chain amino acid sequence)

[0441] >Serial number 29: SNK0003A_H

[0442]

[0443] (SNK0003A antibody light chain amino acid sequence)

[0444] >Serial number 30: SNK0003A_L

[0445]

[0446] > SEQ ID NO: 31: W27G2_HCDR1

[0447]

[0448] > SEQ ID NO: 32: W27G2_HCDR2

[0449]

[0450] > SEQ ID NO: 33: W27G2_HCDR3

[0451]

[0452] >Serial number 34: W27G2_LCDR1

[0453]

[0454] >Serial number 35: W27G2_LCDR2

[0455]

[0456] >Serial number 36: W27G2_LCDR3

[0457]

[0458] >Serial number 37: W27G2_HV

[0459]

[0460] >Serial number 38: W27G2_LV

[0461]

[0462] (Amino acid sequence of the heavy chain of antibody produced by mouse W27G2 hybridoma)

[0463] >Serial number 39: W27G2_H

[0464]

[0465] (Light chain amino acid sequence of antibody produced by mouse W27G2 hybridoma)

[0466] >Serial number 40: W27G2_L

[0467]

[0468]

[0469] >Serial number 41: RS_HCDR1_D50N

[0470]

[0471] >Sequence number 42: RS_HCDR2_D75N

[0472]

[0473] >Serial number 43: RS_HCDR2_E76Q

[0474]

[0475] >Serial number 44: RS_HCDR2_D75N_E76Q

[0476]

[0477] >Serial number 45: RS_HV001

[0478]

[0479] >Serial number 46: RS_HV002

[0480]

[0481] >Serial number 47: RS_HV003

[0482]

[0483] >Serial number 48: RS_HV004

[0484]

[0485] >Serial number 49: RS_HV005

[0486]

[0487] >Serial number 50: RS_HV006

[0488]

[0489] >Serial number 51: RS_HV007

[0490]

[0491] >Serial number 52: RS_LCDR1_A44S

[0492]

[0493] >Serial number 53: RS_LFR1_L001

[0494]

[0495] >Serial number 54: RS_LFR1_L002

[0496]

[0497] >Serial number 55: RS_LFR1_L003

[0498]

[0499] >Serial number 56: RS_LFR1_L000

[0500]

[0501] >Serial number 57: RS_LV001

[0502]

[0503] >Serial number 58: RS_LV002

[0504]

[0505] >Serial number 59: RS_LV003

[0506]

[0507] >Serial number 60: RS_LV004

[0508]

[0509] >Serial number 61: RS_LV005

[0510]

[0511] > SEQ ID NO: 62: RS_HCDR1_X

[0512]

[0513] > SEQ ID NO: 63: RS_HCDR2_X

[0514]

[0515] >Serial number 64: RS_LCDR1_X

[0516]

[0517] (SNK0001AR antibody heavy chain cDNA sequence)

[0518] > SEQ ID NO: 65: SNK0001AR_H_CDNA

[0519]

[0520] (SNK0001AR antibody light chain cDNA sequence)

[0521] > SEQ ID NO: 66: SNK0001AR_L_CDNA

[0522]

[0523] (SNK0002AR antibody heavy chain cDNA sequence)

[0524] > SEQ ID NO: 67: SNK0002AR_H_CDNA

[0525]

[0526] (SNK0002AR antibody light chain cDNA sequence)

[0527] > SEQ ID NO: 68: SNK0002AR_L_CDNA

[0528]

[0529] (SNK0003A antibody heavy chain cDNA sequence)

[0530] > SEQ ID NO: 69: SNK0003A_H_CDNA

[0531]

[0532] (SNK0003A antibody light chain cDNA sequence)

[0533] > SEQ ID NO: 70: SNK0003A_L_CDNA

[0534]

[0535]

[0536] (cDNA sequence of antibody heavy chain produced by mouse W27G2 hybridoma)

[0537] > SEQ ID NO: 71: W27G_MOUSE_IGA_H_CDNA

[0538]

[0539] (Mouse W27G2 hybridoma produces antibody light chain cDNA sequence)

[0540] > SEQ ID 72: W27G_MOUSE_IGA_L_CDNA

[0541]

[0542]

[0543] (Mouse W27G2 IGG antibody heavy chain cDNA sequence)

[0544] > SEQ ID NO: 73: W27G2_MOUSE_IGG_H_CDNA

[0545]

[0546] (Human chimeric W27G2 IgG antibody heavy chain cDNA sequence)

[0547] > SEQ ID NO: 74: W27G2_HUMAN_CHIMERA_IGG_H_CDNA

[0548]

[0549]

[0550] (Human chimeric W27G2 IgG antibody light chain cDNA sequence)

[0551] > SEQ ID NO: 75: W27G2_HUMAN_CHIMERA_IGG_L_CDNA

[0552]

[0553] (Mouse W27G2 IGG antibody heavy chain amino acid sequence)

[0554] >Serial number 76: W27G2_MOUSE_IGG_H

[0555]

[0556] (Human chimeric W27G2 IgG antibody heavy chain amino acid sequence)

[0557] >Serial number 77: W27G2_HUMAN_CHIMERA_IGG_H

[0558]

[0559] (Human chimeric W27G2 IgG antibody light chain amino acid sequence)

[0560] >Serial number 78:W27G2_HUMAN_CHIMERA_IGG_L

[0561]

[0562] (digestive tract contents or excreta)

[0563] The digestive tract contents or excreta used in the method disclosed herein are not particularly limited, and those skilled in the art can appropriately select the specific digestive tract contents or excreta used in consideration of the purpose and operability. In a preferred embodiment, the digestive tract contents or excreta used in the method disclosed herein are feces. The digestive tract contents or excreta are obtained by methods known in the art, for example, they can be obtained from the digestive tract lumen using an endoscope, or a portion of the excreta can be obtained.

[0564] (Modification of digestive tract contents or excreta based on IgA antibodies)

[0565] In the method disclosed herein, modification of the digestive tract contents or excreta by IgA antibodies includes various treatments including a step of contacting the digestive tract contents or excreta with the IgA antibodies.

[0566] In one way, the digestive tract contents or excreta are modified by contacting the IgA antibody that makes the flora in the digestive tract of the IBD patient sound before administration to the subject. In one way, after the step of contacting the IgA antibody with the digestive tract contents or excreta, the IgA antibody is separated from the digestive tract contents or excreta together with its conjugate. In one way, after contacting the IgA antibody with the digestive tract contents or excreta, the IgA antibody is used without being separated from the digestive tract contents or excreta. In a preferred way, after contacting the IgA antibody with the digestive tract contents or excreta, the bacteria bound to the IgA antibody are excluded from the digestive tract contents or excreta together with the IgA antibody. The digestive tract contents or excreta can be further subjected to other treatments before or after being modified using the IgA antibody. For example, the flora can be purified from the digestive tract contents or excreta, the flora is modified using the IgA antibody, and the obtained product is used as modified digestive tract contents or excreta. In another embodiment, after the digestive tract contents or excreta are modified by using IgA antibodies, the bacterial flora can be purified from the modified products and used as modified digestive tract contents or excreta.

[0567] In one embodiment, the digestive tract contents or excreta are modified by contacting the IgA antibody that makes the flora in the digestive tract of the IBD patient sound after being administered to the subject. For example, the digestive tract contents or excreta after being administered to the subject can be modified by using IgA antibodies by combining the composition comprising the digestive tract contents or excreta with the composition comprising IgA antibodies. The composition comprising the digestive tract contents or excreta and the composition comprising IgA antibodies can be respectively included in different dosing units, or can be included in a single dosing unit. In the case where the composition comprising the digestive tract contents or excreta and the composition comprising IgA antibodies are included in different dosing units, these compositions can be administered according to the purpose with any dosing order, dosing frequency, dosing regimen, and dosage combination. In a preferred embodiment, after the administration of the composition comprising the digestive tract contents or excreta, the administration of the composition comprising IgA antibodies is performed.

[0568] The amount of IgA antibody used in the modification of digestive tract contents or excreta is not particularly limited and can be adjusted by a person skilled in the art in consideration of the purpose, workability, etc. For example, when applied to human excreta, the IgA antibody can be applied in the range of 1 mg to 1 g per 1 g of excreta. The amount of IgA antibody applied can be further adjusted according to the number of bacteria contained in the excreta.

[0569] (Administration method of modified digestive tract contents or excreta)

[0570] In the method disclosed herein, the method of administering the digestive tract contents or excreta modified by IgA antibodies to IBD patients is not particularly limited as long as it is a method that can affect the intestinal flora of the patient. For example, administration is performed by conventional fecal transplantation therapy. The digestive tract contents or excreta modified by IgA antibodies disclosed herein can be administered, for example, by rectal administration using a colon endoscope, suppository, enema, upper endoscope, upper push-in small intestinal endoscope, intubation from the nose or mouth using a nasogastric tube, nasointestinal tube or nasojejunal tube, etc.

[0571] In addition, the digestive tract contents or excreta modified by IgA antibodies of the present invention can also be orally administered in the form of solids such as pills, tablets, suspensions, gels, gel sheets, semisolid preparations, tablets, granules, lozenges, capsules or microcapsules, or in the form of enteral preparations, or in the form of compositions such as liquids, suspensions, gels, gel sheets, semisolid preparations, tablets, granules, lozenges or capsules.

[0572] The composition containing the digestive tract contents or excreta modified by IgA antibodies disclosed herein can be further treated before administration. In addition, the composition containing the digestive tract contents or excreta modified by IgA antibodies disclosed herein can be lyophilized, freeze-dried or frozen, or processed into powder.

[0573] The composition of the present disclosure contains one or more pharmaceutically acceptable carriers in one mode. As the carrier used, a carrier known in the art can be used, which can be appropriately selected according to the dosage form and administration method of the composition. For example, as a carrier, a diluent or excipient, such as a bulking agent, a binder, a wetting agent, a disintegrant, a surfactant, a glidant, a lubricant, etc. can be used. Generally, the carrier can be a solid (including a powder), a liquid, or a combination thereof. Each carrier is preferably compatible with other ingredients in the composition and is acceptable in terms of being harmless to the object. The carrier is biologically acceptable and can be inert. For example, the carrier is mixed in order to enable the composition to maintain the vitality of its biological material before being delivered to the appropriate site.

[0574] When the composition of the present disclosure is made into an oral composition, an inert diluent or an edible carrier may be included. For example, sweeteners such as sucrose or saccharin, peppermint, methyl salicylate, orange flavoring and other flavoring agents may be included. In addition, oral compositions can also be prepared by combining the digestive tract contents or excreta modified by the IgA antibody disclosed herein with food. In one embodiment, the food for administration is cooled, for example, provided in the form of ice cream. In one embodiment, an oral composition containing a flora purified from digestive tract contents or excreta can be provided in the form of a food composition.

[0575] The amount of digestive tract contents or excreta contained in each dosage unit of the composition containing digestive tract contents or excreta modified by IgA antibodies disclosed herein can be set by a person skilled in the art in consideration of the route of administration, dosage form, patient's condition, administration frequency, etc. For example, based on the daily dosage of the active ingredient (modified digestive tract contents or excreta), about 0.001 to 1000 milligrams (mg) per kilogram (kg) of body weight can be used, but it is not limited thereto. For example, it can be used in an amount of 0.01 to 100 mg / kg body weight, 0.1 to 50 mg / kg body weight, 1 to 10 mg / kg body weight, etc.

[0576] 2. Method for treating IBD using digestive tract contents or excreta modified with IgA antibodies The composition used

[0577] In one embodiment, the present disclosure provides a composition for treating IBD, comprising digestive tract contents or excreta modified with an IgA antibody, and a method for producing the composition.

[0578] In one embodiment, the composition for treating IBD of the present disclosure is a composition comprising digestive tract contents or excreta modified with IgA antibodies as described in the above “1. Method for treating IBD using digestive tract contents or excreta modified with IgA antibodies”.

[0579] The digestive tract contents or excreta contained in the composition for treating IBD disclosed herein are the substances described in (digestive tract contents or excreta) of the above-mentioned "1. Method for treating IBD using digestive tract contents or excreta modified with IgA antibodies". The digestive tract contents or excreta are modified with IgA antibodies that can make the digestive tract flora of IBD patients healthy according to the above-mentioned (IgA antibodies) and (Modification of digestive tract contents or excreta based on IgA antibodies).

[0580] In one embodiment, the digestive tract contents or excreta modified with IgA antibodies are contained in the composition in the form of isolated and purified bacterial flora.

[0581] In one embodiment, the composition for treating IBD of the present disclosure is a composition containing IgA antibodies described in the above “1. Method for treating IBD using digestive tract contents or excreta modified with IgA antibodies”, which modifies digestive tract contents or excreta after administration.

[0582] The composition for treating IBD of the present disclosure is mixed with a carrier in a manner having properties suitable for the various administration routes and administration amounts described in the above-mentioned "1. Method for treating IBD using digestive tract contents or excreta modified with IgA antibodies" (method for administering modified digestive tract contents or excreta and composition containing the same) or is directly prepared as a preparation without being mixed with a carrier.

[0583] 3. Method for obtaining IgA antibodies that improve the flora in the digestive tract of IBD patients

[0584] In one embodiment, the present disclosure provides a method for screening for IgA antibodies that improve the flora in the digestive tract of IBD patients.

[0585] In one embodiment, the screening method of the present disclosure includes the following steps.

[0586] (B1) Steps for preparing digestive tract contents or excreta of IBD patients,

[0587] (B2) a step of modifying the above-mentioned digestive tract contents or excreta using the candidate IgA antibody, and

[0588] (B3) A step of confirming that the modified digestive tract contents or excreta can improve the bacterial flora in the digestive tract.

[0589] In the method disclosed herein, the step of (B1) preparing the digestive tract contents or excreta of an IBD patient is prepared according to the description of (digestive tract contents or excreta) in the above-mentioned "1. Method for treating IBD using digestive tract contents or excreta modified with IgA antibodies".

[0590] In the method disclosed herein, the candidate IgA antibody used in the step (B2) of modifying the above-mentioned digestive tract contents or excreta using the candidate IgA antibody may have an amino acid sequence derived from the same species as the IBD patient, or may have amino acids derived from a different species such as a closely related xenobiotic. Specifically, for example, examples include human, mouse, rat, hamster, rabbit, goat, donkey, pig, cow, horse, chicken, monkey, chimpanzee, camel, llama, etc.

[0591] Candidate IgA antibodies can be further combined with other compounds and fused to foreign peptides.

[0592] In the method disclosed herein, the step (B2) of modifying the above-mentioned digestive tract contents or excreta using a candidate IgA antibody is modified according to the description of the above-mentioned "1. Method for treating IBD using digestive tract contents or excreta modified with IgA antibodies" (Modification of digestive tract contents or excreta based on IgA antibodies).

[0593] In the method disclosed herein, the step (B3) of confirming that the modified digestive tract contents or excreta can make the gastrointestinal flora healthy is carried out by various methods described in the method for confirming that the IgA antibody in (IgA antibody) of the above-mentioned "1. Method for treating IBD using digestive tract contents or excreta modified with IgA antibodies" can make the gastrointestinal flora healthy.

[0594] The present disclosure provides, in one embodiment, a method for producing an IgA antibody, comprising the steps of the above-mentioned screening method.

[0595] 4. Method for treating the digestive tract contents or excreta of IBD patients in vitro using IgA antibodies

[0596] In one embodiment, the present disclosure provides a method for modifying the digestive tract contents or excreta of an IBD patient using an IgA antibody.

[0597] In one embodiment, the method of the present disclosure includes the following steps.

[0598] (C1) Steps for preparing digestive tract contents or excreta of IBD patients,

[0599] (C2) A step of contacting the digestive tract contents or excreta with IgA antibodies that improve the bacterial flora in the digestive tract in vitro.

[0600] The step (C1) of preparing the digestive tract contents or excreta of an IBD patient in the method of the present disclosure is performed according to the description in (digestive tract contents or excreta) of the above-mentioned "1. Method for treating IBD using digestive tract contents or excreta modified with IgA antibodies".

[0601] The IgA antibody used in the step of contacting the above-mentioned digestive contents or excreta with the IgA antibody that makes the digestive tract flora healthy in vitro in the method (C2) of the present invention is an antibody that can make the digestive tract flora healthy in IBD patients, etc., by various methods described in the above-mentioned "1. Method for treating IBD using digestive tract contents or excreta modified with IgA antibodies" (IgA antibody) to confirm that the IgA antibody can make the digestive tract flora healthy in IBD patients.

[0602] The above-mentioned contact between the digestive tract contents or excreta and the IgA antibody is typically carried out by mixing the two. The digestive tract contents or excreta may be diluted with a diluent before mixing, or may not be diluted.

[0603] The digestive tract contents or excreta of an IBD patient modified with an IgA antibody obtained by the method of the present disclosure can be administered to an IBD patient as an active ingredient of a drug for improving IBD.

[0604] 5. IgA antibodies for modifying the digestive tract contents or excreta of IBD patients in vitro Composition

[0605] The present disclosure provides, in one embodiment, a composition containing IgA antibodies for treating the digestive tract contents or excreta of an IBD patient in vitro, and a method for producing the same.

[0606] The IgA antibody contained in the composition of the present disclosure is an antibody that has been confirmed to be able to improve the digestive tract flora of IBD patients by various methods described in the above-mentioned "1. Method for treating IBD using digestive tract contents or excreta modified with IgA antibodies" (IgA antibodies) and the like.

[0607] In one embodiment, the composition of the present invention is prepared together with one or more pharmaceutically acceptable carriers in various shapes suitable for modifying the digestive tract contents or excreta of IBD patients. For example, the preparation is prepared in the form of a solution, a suspension, a gel, a capsule or a microcapsule. As a carrier, a diluent or an excipient, such as a bulking agent, a binder, a wetting agent, a disintegrant, a surfactant, a glidant, a lubricant, etc. can be used.

[0608] The amount of IgA antibody contained in the composition of the present disclosure is not particularly limited and can be adjusted by a person skilled in the art in consideration of the purpose, operability, etc. For example, when applied to human excreta, the IgA antibody can be formulated in a range of 1 mg to 1 g per 1 g of excreta. The amount of IgA antibody used can be further adjusted according to the number of bacteria contained in the excreta.

[0609] 6. Testing of subjects with drugs containing IgA antibodies for the purpose of diagnosing the presence or risk of IBD Test Methods

[0610] The present disclosure provides a method for testing a subject using the IgA antibody of the present disclosure that improves the flora in the digestive tract in order to diagnose whether the subject suffers from IBD or is at risk of developing IBD.

[0611] In one embodiment, the method of the present disclosure includes the following steps.

[0612] (D1) a step of preparing IgA antibodies that improve the flora in the digestive tract of IBD patients;

[0613] (D2) contacting the digestive tract contents or excreta of the subject with the IgA antibody; and

[0614] (D3) A step of identifying bacteria that bind to the above-mentioned IgA antibody.

[0615] Furthermore, when the IgA antibody shows binding to IBD-associated bacteria, the subject is treated as suffering from IBD or having a risk of developing IBD.

[0616] The IgA antibody used in this test is an IgA antibody that has been confirmed in IBD patients to be able to make the flora in the digestive tract of IBD patients healthy. The fact that the IgA antibody can make the flora in the digestive tract healthy can be confirmed, for example, by the same method as described in the method for confirming that the IgA antibody can make the flora in the digestive tract of IBD patients healthy in the above-mentioned (method for treating IBD using digestive tract contents or excreta modified with IgA antibodies).

[0617] The step of contacting the above-mentioned digestive tract contents or excreta with IgA antibodies in (D2) of the present disclosure is typically performed by mixing the two. The digestive tract contents or excreta may be diluted with a diluent or not diluted before mixing. The amount of IgA antibodies used can be appropriately set by a person skilled in the art according to the characteristics of the sample, the expected sensitivity, etc. For example, in the case of application to human excreta, IgA antibodies can be applied in the range of 1 mg to 1 g per 1 g of excreta. The amount of IgA antibodies applied can be further adjusted according to the number of bacteria contained in the excreta.

[0618] The step (D3) of identifying bacteria bound to the above-mentioned IgA antibody of the present disclosure is performed, for example, by comparing the amount of binding of the IgA antibody to intestinal bacteria known to worsen IBD in the digestive tract contents or excreta from the patient and the digestive tract contents or excreta from a healthy person. In another embodiment, the analysis step can be performed, for example, as follows: contacting the digestive tract contents or excreta from the patient with the IgA antibody, and making a binding distribution map based on the type and amount of bacteria bound to the IgA antibody; contacting the digestive tract contents or excreta from a healthy person with the IgA antibody, and making a binding distribution map based on the type and amount of bacteria bound to the IgA antibody, and comparing the above two binding distribution maps. In one example, the binding distribution profile can be represented by quantitative data based on the IgA index (calculated based on the amount of each bacterium bound to IgA and the amount not bound to IgA) (Andrew L. Kau, et al Sci Transl Med 7, 2015, Hirosuke Sugahara, et al., Frontiers in Microbiology 8, 1757, 2017).

[0619] For example, the IgA index is calculated according to the following formula.

[0620] IgA + 分类单元丰度 =IgA + 分类单元丰度 ×IgA binding capacity / 100

[0621] IgA - 分类单元丰度 =IgA - 分类单元丰度 ×(1-IgA binding capacity / 100)

[0622]

[0623] In the method disclosed herein for testing a subject for the treatment of IBD, a variety of IgA antibodies can be used. In one embodiment, in different IBD patients, a variety of IgA antibodies that have been confirmed to be able to make the flora in the digestive tract healthy are used to implement the test disclosed herein, and in the case where one or more of them show specific binding to IBD-related bacteria, the above-mentioned subject is regarded as suffering from IBD, or has an IBD risk of onset for treatment. In the case where multiple IgAs among the multiple IgA antibodies tested show specific binding to IBD-related bacteria, it is more accurately inferred that the subject suffers from IBD or has an IBD risk of onset. For example, in the case where more than one, more than two, more than three, more than four, or more than five IgAs that can make the flora in the digestive tract of an IBD patient healthy are combined with IBD-related bacteria, the subject is regarded as suffering from IBD, or has an IBD risk of onset for treatment.

[0624] Whether a subject suffers from IBD or has a risk of developing IBD can be expressed by qualitative or quantitative indicators. For example, it can be quantified by an indicator showing the amount or specificity of binding of IgA antibodies to IBD-related bacteria, or qualitatively expressed by distinguishing the quantitative data.

[0625] When a subject is treated as suffering from IBD or having a risk of developing IBD, the treatment includes all treatments related to maintaining or improving the health of the subject. For example, it includes: classifying the patient as a patient suffering from IBD or a person at risk of developing IBD in an information processing system; adding a label; providing guidance for improving the life of the patient as a patient suffering from IBD or a person at risk of developing IBD; treating and preventing IBD; etc., but is not limited to these.

[0626] In the case of treating a subject for the purpose of treating IBD disclosed herein, the treatment method is not particularly limited, and known IBD treatment methods can be used. In one embodiment, the IBD treatment method comprises administering to the subject a digestive tract content or excreta modified with an IgA antibody that has been shown to bind to IBD-related bacteria in the test.

[0627] 7. Drugs containing IgA antibodies for testing subjects to determine whether IBD treatment is necessary

[0628] In one embodiment, the present disclosure provides a drug for diagnosing the presence or absence of IBD or the risk of developing IBD, comprising an IgA antibody that improves the flora in the digestive tract of an IBD patient.

[0629] The diagnostic drug disclosed in the present invention is a drug used in "6. Method of conducting a test on a subject using a diagnostic drug containing an IgA antibody for the treatment of IBD", and contains an IgA antibody having the properties described in the method.

[0630] In one embodiment, the composition of the present invention is prepared together with one or more pharmaceutically acceptable carriers in various shapes suitable for testing the digestive tract contents or excreta of IBD patients. For example, the preparation is prepared in the form of a solution, a suspension, a gel, a capsule or a microcapsule. As a carrier, a diluent or an excipient, such as a bulking agent, a binder, a wetting agent, a disintegrant, a surfactant, a glidant, a lubricant, etc. can be used.

[0631] The amount of IgA antibody contained in the composition of the present disclosure is not particularly limited and can be adjusted by a person skilled in the art in consideration of the purpose, operability, etc. For example, when applied to human excreta, the preparation can be prepared in a manner that the IgA antibody is applied in a range of 1 mg to 1 g per 1 g of excreta. The amount of IgA antibody applied can be further adjusted according to the number of bacteria contained in the excreta.

[0632] 8. Use of drugs containing substances in the digestive tract or excreta in patients treated with IBD drugs containing substances in the digestive tract or excreta Methods for testing diagnostic drugs for IgA antibodies

[0633] In one embodiment, the present disclosure provides a method for conducting a test using a diagnostic drug containing IgA antibodies in a patient treated with an IBD therapeutic drug containing digestive tract contents or excreta.

[0634] In one embodiment, the method of the present disclosure includes the following steps.

[0635] (E1) a step of obtaining digestive tract contents or excreta of a patient who is currently receiving, plans to receive, or has received an IBD therapeutic drug containing digestive tract contents or excreta;

[0636] (E2) contacting the digestive tract contents or excreta with IgA antibodies; and

[0637] (E3) A step of analyzing bacteria bound to the above-mentioned IgA antibody.

[0638] Furthermore, when the above analysis results show that the bacterial flora in the digestive tract has become healthy, the treatment with the above IBD therapeutic drug is continued.

[0639] In the method disclosed herein, the patient to be tested is a patient who is currently being administered, is scheduled to be administered, or has been administered in the past an IBD therapeutic drug containing digestive tract contents or excreta.

[0640] The IBD therapeutic drug containing digestive tract contents or excreta disclosed in the present invention is preferably an IBD therapeutic drug containing digestive tract contents or excreta modified with IgA antibodies used in this test. In one embodiment, the digestive tract contents or excreta contained in the IBD therapeutic drug disclosed in the present invention are digestive tract contents or excreta obtained from the patient himself. The IgA antibody used in this test is preferably an IgA antibody that has been confirmed to be able to make the digestive tract flora of the patient under examination healthy.

[0641] The step of contacting the above-mentioned digestive tract contents or excreta with IgA antibodies in (D2) of the present disclosure is typically performed by mixing the two. The digestive tract contents or excreta may be diluted with a diluent or not diluted before mixing. The amount of IgA antibodies used can be appropriately set by a person skilled in the art according to the characteristics of the sample, the expected sensitivity, etc. For example, in the case of application to human excreta, IgA antibodies can be applied in the range of 1 mg to 1 g per 1 g of excreta. The amount of IgA antibodies applied can be further adjusted according to the number of bacteria contained in the excreta.

[0642] The step (D3) of analyzing bacteria bound to the above-mentioned IgA antibody of the present disclosure is performed, for example, by comparing the amount of binding of the IgA antibody to intestinal bacteria known to worsen IBD in the digestive tract contents or excreta from the patient and the digestive tract contents or excreta from a healthy person. In another embodiment, the analysis step can be performed, for example, as follows: contacting the digestive tract contents or excreta from the patient with the IgA antibody, and making a binding distribution map based on the type and amount of bacteria bound to the IgA antibody; contacting the digestive tract contents or excreta from a healthy person with the IgA antibody, and making a binding distribution map based on the type and amount of bacteria bound to the IgA antibody, and comparing the above two binding distribution maps.

[0643] In the method disclosed herein, the analysis result of the bacteria bound to the IgA antibody shows that the flora in the digestive tract has become healthy, for example, when the amount of the IgA antibody bound to intestinal bacteria known to worsen IBD in the digestive tract contents or excreta from the patient is compared with that in the digestive tract contents or excreta from a healthy person, no significant difference is observed between the two. In another embodiment, the analysis result of the bacteria bound to the IgA antibody shows that the flora in the digestive tract has become healthy, for example, refers to contacting the digestive tract contents or excreta from the patient with the IgA antibody, and making a binding distribution map based on the type and amount of bacteria bound to the IgA antibody; contacting the digestive tract contents or excreta from a healthy person with the IgA antibody, and making a binding distribution map based on the type and amount of bacteria bound to the IgA antibody, and when comparing the above two binding distribution maps, no significant difference is observed in the distribution map of IBD-related bacteria.

[0644] 9. For testing patients treated with IBD drugs containing digestive tract contents or excreta Diagnostic drugs containing IgA antibodies

[0645] In one embodiment, the present disclosure provides a diagnostic drug containing IgA antibodies for testing patients treated with an IBD therapeutic drug containing digestive tract contents or excreta.

[0646] The diagnostic drug disclosed in the present invention is a drug used in "8. Method for testing a patient treated with an IBD therapeutic drug containing digestive tract contents or excreta using a diagnostic drug containing IgA antibodies", and contains an IgA antibody having the properties described in the method.

[0647] In one embodiment, the composition of the present invention is prepared together with one or more pharmaceutically acceptable carriers in various shapes suitable for testing the digestive tract contents or excreta of IBD patients. For example, the preparation is prepared in the form of a solution, a suspension, a gel, a capsule or a microcapsule. As a carrier, a diluent or an excipient, such as a bulking agent, a binder, a wetting agent, a disintegrant, a surfactant, a glidant, a lubricant, etc. can be used.

[0648] The amount of IgA antibody contained in the composition of the present disclosure is not particularly limited and can be adjusted by a person skilled in the art in consideration of the purpose, operability, etc. For example, when applied to human excreta, the preparation can be prepared in a manner that the IgA antibody is applied in a range of 1 mg to 1 g per 1 g of excreta. The amount of IgA antibody applied can be further adjusted according to the number of bacteria contained in the excreta.

[0649] In one embodiment, the present disclosure of "1. Method for treating IBD using digestive tract contents or excreta modified with IgA antibodies", "6. Method for testing a subject using a diagnostic drug containing IgA antibodies for the treatment of IBD" and "8. Method for testing a diagnostic drug containing IgA antibodies in a patient treated with an IBD therapeutic drug containing digestive tract contents or excreta" and the compositions used in these methods can be used in combination with part or all of them. For example, a patient who is determined to be an IBD treatment target by "6. Method for testing a subject using a diagnostic drug containing IgA antibodies for the treatment of IBD" can be treated by "1. Method for treating IBD using digestive tract contents or excreta modified with IgA antibodies", and the course of his treatment can be diagnosed by "8. Method for testing a diagnostic drug containing IgA antibodies in a patient treated with an IBD therapeutic drug containing digestive tract contents or excreta". The IgA antibodies used in these methods may be the same or different.

[0650] Example

[0651] The present invention will be further described in detail below by way of examples. However, these examples are merely illustrative and do not limit the present disclosure.

[0652] Example 1: Binding properties of endogenous IgA antibodies

[0653] (Methods and Materials)

[0654] Bacterial extraction from human fecal samples

[0655] Human stool samples from patients with inflammatory bowel disease (n=12, 7 patients with ulcerative colitis and 5 patients with Crohn's disease, P1-P12) and healthy subjects (n=12, C1-C12) were provided by Shiga University of Medical Science and Kanazawa University.

[0656] Healthy people were volunteers whose comprehensive physical examination results showed no abnormal values. The stool samples were immediately put into anaerobic bags after collection, stored frozen (-80°C), and sent to the University of Tokyo. In addition, a human stool sample from a patient with ulcerative colitis (P13) was provided by the Department of Gastroenterology at the University of Tokyo and used in the experiment on the same day.

[0657] The samples provided were prepared into bacterial solution and packaged in an anaerobic chamber (80% N2, 10% H2, 10% CO2, Coy laboratory products) by the following method, and stored at -80°C for subsequent experiments. All reagents used in the anaerobic chamber were sterilized by passing through a 0.22μm filter and allowed to stand overnight in the anaerobic chamber for anaerobic oxidation. 30ml of PBS was added to the frozen fecal sample, dissolved in the anaerobic chamber, further suspended, and centrifuged at 50g and 4°C for 15 minutes. The weight of the sample provided by the University of Tokyo was measured, 9 times the amount of PBS was added and suspended, and centrifuged at 50g and 4°C for 15 minutes. The supernatant was recovered in another 50ml tube, and after sufficient suspension, 1ml was dispensed into 1.5ml tubes per tube and stored at -80°C as a bacterial freezing storage solution for human fecal samples.

[0658] Determination of bacterial counts in frozen stock solutions of human fecal samples

[0659] The frozen bacterial stock solution of the human fecal sample was thawed in an anaerobic chamber. Take 10μl of the frozen bacterial stock solution and dilute it to 1,000 times with PBS. The bacterial count was determined according to the operating procedures of the cell viability detection kit (BD). In this study, all bacteria contained in the human fecal sample were used as the object, so propidium iodide (PI) for detecting dead bacteria was not used, but only thiazole orange (TO) for staining nucleic acids to detect bacteria was used for bacterial count determination. PBS, liquid counting microspheres (BD), and thiazole orange (final concentration 42nM, BD) were added to the bacterial dilution solution and reacted on ice for 10 minutes. The bacterial count determination solution was analyzed by flow cytometry using a SONY SA3800 analyzer (SONY). The bacterial count concentration of the bacterial dilution solution was calculated based on the number of counting microspheres detected in the analysis.

[0660] ·16S rRNA gene amplicon sequencing analysis of bacterial DNA

[0661] For bacterial DNA, a polymerase chain reaction (PCR) targeting the V3-V4 region of the variable region of the 16S rRNA gene was performed. Primers for the next generation sequencer using Miseq were prepared, and in order to distinguish the samples to be analyzed, they were used after adding an index sequence (see below). After the PCR reaction, 10× loading buffer (TAKARA) was added and agarose electrophoresis was performed. The gel used to run the PCR sample used a 1.5% agarose gel (VWR Life Sciences) to which LED StainG (LABTAS+) was added. A 100 bp DNA ladder marker (TAKARA) was used as a size marker for DNA. Electrophoresis was performed at 100 V for 30 minutes. After running, a PCR product of about 500 bp was cut out, and the PCR product was purified using the Fast Gene gel extraction kit (Nippon Genetics). The purified PCR product was electrophoresed again to confirm whether purification was achieved. Sequencing analysis was performed using the Mi-seq kit V3 (Illumina) and commissioned to TAK-Circulator Co., Ltd. The sequence data obtained by sequencing analysis were used for bacterial flora analysis using Qiime2 software (ver.2020-2).

[0662] The primer sequences used for 16S rRNA gene amplicon sequencing analysis are shown below.

[0663]

[0664] The sequence indicated by XXX is the index sequence, and the gray one indicates the sequence targeting the template 16S rRNA.

[0665] ·Calculation method of IgA index

[0666] The higher the value of the IgA index, the stronger the binding of the bacteria to the IgA antibody. On the other hand, the lower the value, the weaker the binding of the bacteria to the IgA antibody. The IgA index of each person is calculated taking into account that the binding ratio of the IgA antibody to the total flora of each person is different. The IgA binding ability is the ratio of bacteria that bind to the IgA antibody relative to the total flora. This value is used to calculate the ratio of bacteria bound to IgA antibodies or bacteria not bound by antibodies for each person (IgA + 分类单元丰度(taxon abundance) , IgA - 分类单元丰度 ), and then the IgA index was calculated (Andrew L.Kau, et al Sci TranslMed 7, 2015, Hirosuke Sugahara, et al., Frontiers in Microbiology 8, 1757, 2017).

[0667] IgA + 分类单元丰度 =IgA + 分类单元丰度 ×IgA binding capacity / 100

[0668] IgA - 分类单元丰度 =IgA - 分类单元丰度 ×(1-IgA binding capacity / 100)

[0669]

[0670] Specifically, the relative abundance of IgA-binding bacteria obtained by 16S rRNA analysis was multiplied by the IgA binding capacity (the ratio of IgA antibody-binding bacteria to the total bacterial population) to obtain the taxon abundance of IgA antibody-binding bacteria. On the other hand, for antibody-unbound bacteria, the relative abundance was multiplied by 1-IgA binding capacity (the number obtained by subtracting the ratio of IgA antibody-binding bacteria from 100%) to obtain the taxon abundance of IgA antibody-unbound bacteria. The IgA index was calculated using the respective taxon abundances.

[0671] ·Separation of RS_H000_L001 IgA antibody-bound and non-bound bacteria from human fecal samples using magnetic cell sorter (MACS) and cell sorter

[0672] The operation was performed in an anaerobic chamber. After the bacterial count was determined, the bacteria in the human fecal sample were added to PBS to prepare 6×10 7 / ml. Then centrifuge at 8,000g, 4℃ for 5 minutes and remove the supernatant. Add 100μl of PBS containing 20% ​​normal rat serum (Wako) to the bacteria after removing the supernatant, and react on ice for 20 minutes. This inhibits the nonspecific binding of the antibodies used. Add FACS buffer to the bacterial solution, centrifuge at 8,000g, 4℃ for 5 minutes, and remove the supernatant. Relative to 6×10 6 15 μg of biotinylated RS_H000_L001 IgA antibody was added to the antibody reaction solution and reacted on ice for 20 minutes. FACS buffer was added to the antibody reaction solution and centrifuged at 8,000 g and 4°C for 5 minutes, and the supernatant was removed. 6 15 μl of microbeads-streptavidin (Milteny) was added to each bacterial cell and reacted on ice for 20 minutes. The bacterial separation operation was performed according to the operating procedures of the MACS column (LS column, Milteny) kit. The bacteria adsorbed to the MACS column were recovered as RS_H000_L001 IgA antibody-bound bacteria, and the bacteria that directly passed through the column were recovered as RS_H000_L001 IgA antibody-unbound bacteria. For RS_H000_L001 IgA antibody-bound bacteria, the RS_H000_L001 IgA antibody-bound bacteria were concentrated using the MACS column again. The recovered RS_H000_L001 IgA antibody-bound bacteria and unbound bacteria were centrifuged at 8,000g and 4°C for 5 minutes, and the supernatant was removed. FACS buffer, TO (final concentration 42nM, BD) and phycoerythrin (PE)-anti-mouse IgA antibody (final concentration 5μg / ml, Biolegend) were added to the RS_H000_L001 IgA antibody-bound bacteria and unbound bacteria recovered by MACS, and the reaction was allowed to proceed on ice in the dark for 10 minutes. After that, RS_H000_L001 IgA antibody-bound bacteria and unbound bacteria were separated by cell sorting using a SONY cell sorter SH800 (SONY). The bacteria of each fraction recovered by cell sorting were analyzed again using a SONY cell sorter SH800, and it was confirmed that the target fraction was recovered at a ratio of more than 90%.

[0673] Separation of human endogenous IgA antibody-bound and non-bound bacteria from human fecal samples using magnetic cell sorter (MACS) and cell sorter

[0674] Bacteria from human fecal samples were thawed in an anaerobic chamber. All reagents used in the anaerobic chamber were sterilized by passing through a 0.22 μm filter and allowed to stand overnight in the anaerobic chamber for anaerobic oxidation. After the bacterial count was determined, PBS was added to prepare 6×10 7 / ml. Then centrifuge at 8,000g, 4℃ for 5 minutes and remove the supernatant. Add 100μl of PBS containing 20% ​​normal rat serum (Wako) to the bacteria after removing the supernatant, and react on ice for 20 minutes. This will inhibit the nonspecific binding of the antibodies used. Add FACS buffer to the bacterial solution, centrifuge at 8,000g, 4℃ for 5 minutes, and remove the supernatant. Relative to 6×10 6 15 μl of anti-IgA-PE human antibody (Milteny) was added to each cell and reacted on ice for 20 minutes. FACS buffer was added to the antibody reaction solution, centrifuged at 8,000 g and 4°C for 5 minutes, and the supernatant was removed. 6 15 μl of anti-PE microbeads (Milteny) were added to each number of bacteria and reacted on ice for 20 minutes. The bacterial separation operation was carried out according to the operating procedures of the MACS column (LS column, Milteny) kit. The bacteria adsorbed on the MACS column were recovered as human endogenous IgA antibody-bound bacteria, and the bacteria directly passing through the column were recovered as human endogenous IgA antibody-unbound bacteria. For human endogenous IgA antibody-bound bacteria, the human endogenous IgA antibody-bound bacteria were concentrated again using the MACS column. The recovered human endogenous IgA antibody-bound bacteria and unbound bacteria were centrifuged at 8,000g and 4°C for 5 minutes, and the supernatant was removed. FACS buffer and TO (final concentration 42nM, BD) were added to the human endogenous IgA antibody-bound bacteria and unbound bacteria recovered by MACS, and reacted on ice in the dark for 10 minutes. Then, human endogenous IgA antibody-bound bacteria and unbound bacteria were separated by cell sorting using a SONY cell sorter SH800 (SONY). The bacteria in each fraction recovered by cell sorting were analyzed again using a Sony cell sorter SH800, and it was confirmed that the target fraction was recovered at a ratio of 90% or more.

[0675] (result)

[0676] Endogenous polyclonal IgA antibody-binding bacteria were collected from healthy individuals or IBD patients, and the binding properties to various intestinal bacteria contained in feces collected from healthy individuals or IBD patients were tested.

[0677] The results, such as Figure 1As shown, endogenous polyclonal IgA of healthy people showed a median IgA index of >0 for Enterobacteriaceae, Erysipelotrichaceae, Gemellaceae, and Bacteroidaceae, confirming that it binds to these bacteria. In particular, it showed high binding to Enterobacteriaceae. This bacterium is said to be related to the deterioration of the intestinal environment such as enteritis, suggesting that endogenous IgA of healthy people binds to these bacteria that worsen IBD and inhibits their activity, thereby exerting a biological defense function. On the other hand, the endogenous polyclonal IgA of IBD showed a median of IgA index>0 for Lactobacillaceae, Veillonellaceae, Enterococcus, Enterobacteriaceae, Actinomycetaceae and Bacteroidaceae, and was confirmed to bind to these bacteria. The endogenous polyclonal IgA of IBD patients showed the highest specificity for Lactobacillaceae, which is a fermentative bacterium generally considered to be unrelated to the deterioration of the intestinal environment. From this perspective, it showed a significantly different binding property from the endogenous polyclonal IgA of healthy people, which has attracted attention. This result suggests that in IBD patients, the biological defense mechanism based on endogenous polyclonal IgA does not function soundly.

[0678] Example 2: IgA from IBD patients has reduced binding ability to IBD-associated bacteria

[0679] (Methods and Materials)

[0680] Preparation of human IgA antibodies from stool samples

[0681] Add an appropriate amount of PBS to a stool sample from a healthy person or patient (frozen at -80°C after collection), and dissolve and suspend at room temperature. All operations are performed under aerobic conditions. Centrifuge the suspended solution at 2,000g and 4°C for 10 minutes, and recover the supernatant. Further centrifuge the supernatant at 8,000g and 4°C for 10 minutes, and recover the supernatant. Repeat this operation once more. After that, add the same volume of saturated ammonium sulfate while stirring the supernatant on ice, and then let it stand at 4°C overnight. The next day, centrifuge at 10,000g and 4°C for 10 minutes to obtain an ammonium sulfate precipitate. Add 50% saturated ammonium sulfate solution to it and suspend it, and wash it by centrifugation at 10,000g and 4°C for 10 minutes. The subsequent precipitate is dissolved in PBS, further dialyzed with PBS, and packaged as an IgA antibody solution from human feces and stored frozen at -20°C.

[0682] Determination of human IgA antibody titers from stool samples

[0683] The IgA antibody concentration in the IgA antibody solution extracted from the fecal sample was measured by conventional sandwich ELISA. Goat anti-human IgA INLB (final concentration 2 μg / ml, Southern Biotech) was used as the anti-human IgA antibody coated on the ELISA plate (C96 MaxiSorp Nunc Immuno plate (Thermo Fisher)). The ELISA plate with the anti-human IgA antibody immobilized was washed 3 times with PBS, and 150 μl of PBS supplemented with 1% bovine serum albumin (BSA, Wako) was added to each well, and it was left to stand at 4°C overnight for blocking. The control IgA antibody used a human IgA isotype control (Genway, 1 mg / ml), and a serial dilution of the control IgA antibody and the human endogenous IgA antibody solution extracted from healthy people or patients was prepared in a 96-well plate. Dilution was performed using PBS supplemented with 1% BSA, with gradient dilutions of 1x, 1 / 3x, 1 / 10x, 1 / 30x, 1 / 100x, 1 / 300x, 1 / 1,000x, and 1 / 3,000x. After blocking, the PBS supplemented with 1% BSA on the ELISA plate was removed, and 50 μl of the gradient diluted antibody was added to each well, and reacted at room temperature for 1 hour. Next, the ELISA plate was washed three times with PBS supplemented with 0.05% Tween 20 (Chem Cruz), and then alkaline phosphatase-conjugated goat anti-human IgA (final concentration 0.5 μg / ml, Southern Biotech) as a secondary antibody was added and reacted at room temperature for 1 hour. The substrate reaction solution used for color development was used by dissolving magnesium chloride (final concentration 2 mM) and phosphatase substrate (Sigma) in sodium bicarbonate (final concentration 6.5 mM) and sodium carbonate (final concentration 18.5 mM) solutions. After the secondary antibody reaction, the plate was washed again with PBS supplemented with 0.05% Tween 20, and 50 μl of substrate reaction solution was added to each well. The color was developed by shielding with aluminum foil, and the absorbance (OD405 nm) was measured using Tristar2 LB942 (BERTHOLD TECHNOLOGIES). The IgA antibody concentration of each sample was calculated by comparing the reaction curve with the control IgA antibody.

[0684] · Determination of the binding capacity of human IgA antibodies to bacteria (based on ELISA method)

[0685] Each bacterium was cultured aerobically or anaerobically overnight under optimal conditions. Then, PBS was added and centrifuged at 8,000 g and 4°C for 5 minutes, and the supernatant was removed. After the bacterial count of each bacterium was determined, the ELISA plate (C96 MaxiSorp Nunc Immuno plate (Thermo Fisher)) was used at a concentration of 1.0 × 10 7The cells were suspended in 50 mM sodium carbonate solution at 50 μl / well and added to an ELISA plate at 50 μl / well and allowed to stand overnight at 4°C to immobilize the bacteria.

[0686] The ELISA plate was washed using a plate washer (Vaccu-Pette / 96-well multi-well plate pipette, Sigma-Aldrich). The bacteria-immobilized ELISA plate was washed three times with PBS, and 150 μl of PBS supplemented with 1% bovine serum albumin (BSA, Wako) was added to each well, and the plate was left to stand overnight at 4°C for blocking. Serial dilutions of human endogenous IgA antibody samples (adjusted to a concentration of 30 μg / ml) extracted from healthy individuals or patients were prepared in a 96-well plate. PBS supplemented with 1% BSA was used for dilution, and gradient dilutions were performed to 1x, 1 / 3x, 1 / 10x, 1 / 30x, 1 / 100x, 1 / 300x, 1 / 1,000x, and 1 / 3,000x. After blocking, the PBS supplemented with 1% BSA was removed from the ELISA plate, and 50 μl of gradient diluted antibodies were added to each well, and the reaction was carried out at room temperature for 1 hour. Then, the plate was washed three times with PBS supplemented with 0.05% Tween 20 (Chem Cruz), and then alkaline phosphatase-conjugated goat anti-human IgA (final concentration 0.5μg / ml, Southern Biotech) was added as a secondary antibody and reacted at room temperature for 1 hour. The substrate reaction solution used for color development is a solution of magnesium chloride (final concentration 2mM) and phosphatase substrate (Sigma) dissolved in sodium bicarbonate (final concentration 6.5mM) and sodium carbonate (final concentration 18.5mM). After the secondary antibody reaction, the plate was washed again with PBS supplemented with 0.05% Tween 20, and 50μl of the substrate reaction solution was added to each well, and color development was performed in the dark. The absorbance (OD405nm) was measured using Tristar2 LB942 (BERTHOLD TECHNOLOGIES). After confirming that each sample has developed color in the order of serial dilution, the absorbance (405nm) value of each sample when the IgA antibody concentration is 10mg / ml was compared. Figure 2 The horizontal axis represents the median. The P value was determined by a two-sided Student's T test.

[0687] (result)

[0688] The results of measuring the binding capacity of human IgA antibodies to bacteria by ELISA showed that there was no difference in the binding capacity of IgA antibodies to probiotics (beneficial bacteria) such as Bifidobacterium bifidum between healthy people and patients, but the response of IgA antibodies to bacteria belonging to the Proteobacteria such as Escherichia coli (E. coli) and Pseudomonas fulva was significantly reduced in IBD patients ( Figure 2 ).

[0689] Example 3: IgA antibodies with high binding affinity to IBD-related bacteria

[0690] (Methods and Materials)

[0691] Bacterial extraction from human fecal samples

[0692] The samples provided in the same manner as in Example 1 were prepared into bacterial liquids in an anaerobic chamber (80% N2, 10% H2, 10% CO2, products of Coy laboratories) using the following method and were packaged and stored at -80°C for subsequent experiments. All reagents used in the anaerobic chamber were sterilized through a 0.22 μm filter and allowed to stand overnight in the anaerobic chamber for anaerobic oxidation. 30 ml of PBS was added to the frozen fecal sample, dissolved in the anaerobic chamber, further suspended, and centrifuged at 50 g and 4°C for 15 minutes. The weight of the sample provided by the University of Tokyo was measured, 9 times the amount of PBS was added for suspension, and centrifuged at 50 g and 4°C for 15 minutes. The supernatant was recovered in another 50 ml tube, and after sufficient suspension, 1 ml was dispensed into 1.5 ml tubes per tube and stored at -80°C as a bacterial freezing storage solution for human fecal samples.

[0693] Determination of bacterial counts in frozen stock solutions of human fecal samples

[0694] The frozen bacterial stock solution of the human fecal sample was thawed in an anaerobic chamber in the same manner as in Example 1. Take 10 μl of the frozen bacterial stock solution and dilute it to 1,000 times with PBS. The bacterial count was determined according to the operating procedures of the cell viability detection kit (BD). In this study, all bacteria contained in the human fecal sample were taken as the object, so propidium iodide (PI) for detecting dead bacteria was not used, but only thiazole orange (TO) for staining nucleic acids to detect bacteria was used to determine the bacterial count. PBS, liquid counting microspheres (BD), and thiazole orange (final concentration 42nM, BD) were added to the bacterial dilution and reacted on ice for 10 minutes. The bacterial count determination solution was analyzed by flow cytometry using a SONY SA3800 analyzer (SONY). The bacterial count concentration of the bacterial dilution was calculated based on the number of counting microspheres detected in the analysis.

[0695] Human endogenous IgA antibody binds bacteria ( Figure 3 ) Analysis of the ratio of bacteria to human fecal samples

[0696] The operation was performed in an anaerobic chamber. After the bacterial count was determined, bacteria from human feces were added to PBS to prepare 6×10 7 / ml. Then centrifuge at 8,000g, 4℃ for 5 minutes and remove the supernatant. Add 100μl of PBS containing 20% ​​normal rat serum (Wako) to the bacteria after removing the supernatant, and react on ice for 30 minutes. This inhibits the nonspecific binding of antibodies. Add FACS buffer to the bacterial solution, centrifuge at 8,000g, 4℃ for 5 minutes, and remove the supernatant. Relative to 6×10 6 15 μl of anti-IgA-PE human antibody (Milteny) was added to each number of bacteria and reacted on ice for 20 minutes. FACS buffer was added to the antibody reaction solution, centrifuged at 8,000g, 4°C for 5 minutes, and the supernatant was removed. FACS buffer and thiazole orange (final concentration 42nM, BD) were added to the bacteria and reacted on ice for 10 minutes. The proportion of all endogenous IgA antibody-bound bacteria was then analyzed using a SONY cell sorter SH800 (SONY).

[0697] ·RS_H000_L001 IgA antibody binding bacteria ( Figure 4 )、RS_H007_L004 antibody-binding bacteria ( Figure 5 )、SNK0003A antibody-binding bacteria ( Figure 6 )、SNK0001A antibody-binding bacteria ( Figure 7 )、SNK0002A antibody-binding bacteria ( Figure 8 ) Analysis of the ratio of bacteria to human fecal samples

[0698] The operation was performed in an anaerobic chamber. After the bacterial count was determined, the bacteria in the human fecal sample were added to PBS to prepare 6×10 7 / ml. Then centrifuge at 8,000g and 4°C for 5 minutes and remove the supernatant. Add 100μl of PBS containing 20% ​​normal rat serum (Wako) to the bacteria after removing the supernatant, and react on ice for 30 minutes. This inhibits the nonspecific binding of antibodies. The fluorescence activated cell sorter (FACS) buffer for antibody staining is PBS with 10% fetal bovine serum (FBS, Nichirei) and EDTA (final concentration 5μM, Nacalai Tesque) added, and sterilized by passing through a 0.22μm filter. Add FACS buffer to the bacterial solution, centrifuge at 8,000g and 4°C for 5 minutes, and remove the supernatant. Relative to 6×10 6Add 15 μg of each biotinylated antibody to each number of bacteria and react on ice for 20 minutes. Add FACS buffer to the antibody reaction solution, centrifuge at 8,000g and 4°C for 5 minutes, and remove the supernatant. Add 20 μl of PE / Cyanine 7 (Cyanine7) labeled streptavidin (final concentration 10 μg / ml, BioLegend) to the bacteria and react on ice in the dark for 20 minutes. Add FACS buffer to the reaction solution, centrifuge at 8,000g and 4°C for 5 minutes, and remove the supernatant. Add FACS buffer and thiazole orange (final concentration 42 nM, BD) to the bacteria and react on ice for 10 minutes. Then use the SONY cell sorter SH800 (SONY) to analyze the proportion of bacteria bound to each antibody.

[0699] Analysis of the ratio of RS_H000_L001 antibody-binding bacteria to bacteria in human fecal samples

[0700] The operation was performed in an anaerobic chamber. After the bacterial count was determined, the bacteria in the human fecal sample were added to PBS to prepare 6×10 7 / ml. Then centrifuge at 8,000g and 4°C for 5 minutes and remove the supernatant. Add 100μl of PBS containing 20% ​​normal rat serum (Wako) to the bacteria after removing the supernatant, and react on ice for 30 minutes. This inhibits the nonspecific binding of antibodies. The fluorescence activated cell sorter (FACS) buffer for antibody staining is PBS with 10% fetal bovine serum (FBS, Nichirei) and EDTA (final concentration 5μM, Nacalai Tesque) added, and sterilized by passing through a 0.22μm filter. Add FACS buffer to the bacterial solution, centrifuge at 8,000g and 4°C for 5 minutes, and remove the supernatant. Relative to 6×10 6 15 μg of biotinylated RS_H000_L001 antibody was added to each bacterial cell and reacted on ice for 20 minutes. FACS buffer was added to the antibody reaction solution, centrifuged at 8,000g and 4°C for 5 minutes, and the supernatant was removed. 20 μl of PE / cyanine 7-labeled streptavidin (final concentration 10 μg / ml, BioLegend) was added to the bacteria and reacted on ice in the dark for 20 minutes. FACS buffer was added to the reaction solution, centrifuged at 8,000g and 4°C for 5 minutes, and the supernatant was removed. FACS buffer and thiazole orange (final concentration 42 nM, BD) were added to the bacteria and reacted on ice for 10 minutes. The proportion of RS_H000_L001 antibody-bound bacteria was then analyzed using a Sony cell sorter SH800 (SONY).

[0701] ·Gemella morbillorum proliferation inhibition test

[0702] The operation was performed in an anaerobic chamber. A culture medium prepared by adding sheep defibrinated blood (Japan Bioserum, final concentration 5%) to tryptone soy broth (BD) was used for the culture of Gemella morbillorum (G. morbillorum, ATCC27824 strain). G. morbillorum was revived from a glycerol stock solution and cultured in 10 ml of culture medium at 37°C overnight. 450 μl of culture medium was added to 50 μl of bacterial solution, and the mixture was centrifuged at 8,000 g at room temperature for 5 minutes, and the supernatant was removed. 500 μl of culture medium was added, and after the bacterial count was determined, the bacterial solution was added to the culture medium to prepare 2×10 3 / μl. Add 25μl of PBS or RS_H000_L001 IgA antibody (1mg / ml) to 5μl of bacterial solution and react at 37℃ for 1 hour. After the reaction, add 30μl of culture medium and culture at 37℃ for 8 hours. After the culture, inoculate the bacterial solution in agar medium, culture at 37℃ overnight, and measure the number of colonies.

[0703] (result)

[0704] As a result of studying IgA antibodies with high binding affinity to IBD-related bacteria in the feces of IBD patients, it was found that RS_H000_L001 antibody and its mutants (RS_H007_L004), SNK0003A, SNK0001A and SNK0002A had high binding affinity to IBD-related bacteria ( Figures 3 to 8 ). Figure 3 The results of FACS analysis of the ratio of all bacteria bound to endogenous IgA antibodies (number of bound bacteria / total number of bacteria in stool samples) in bacteria obtained from feces of healthy people or IBD patients (Crohn's disease: CD, ulcerative colitis: UC) are shown. Regarding the ratio of all bacteria bound to endogenous IgA antibodies, no significant difference in the ratio of binding to antibody samples was observed between bacteria from healthy people's feces and bacteria from IBD patients' feces ( Figure 3 On the other hand, when RS_H000_L001 and its mutants (RS_H007_L004), SNK0003A, SNK0001A, and SNK0002A were used as antibody samples, the binding ratio of bacteria from healthy human feces was low, while bacteria from IBD patient feces showed significantly higher binding ratios to these antibody samples ( Figures 4 to 8 ).

[0705] The results of confirming the binding properties of RS_H000_L001 IgA antibody to bacteria in feces obtained from healthy individuals or IBD patients showed that no bacteria that showed specific binding were found for bacteria in feces from healthy individuals. In contrast, for bacteria collected from feces from IBD patients, binding was shown to Enterococcus and Enterobacteriaceae, Gemellaceae, and Veillonellaceae, which are known to cause enteritis. Fig. 9 ).

[0706] Gemellaceae is one of the bacteria that IgA antibodies from healthy people bind to in feces from healthy people ( Figure 1 ), RS_H000_L001 antibody showed a strong proliferation inhibition effect on Gemella morbillorum in an in vitro proliferation inhibition test ( Fig.10 ). These results suggest that IgA antibodies with high binding affinity to IBD-related bacteria can improve IBD disease by inhibiting the proliferation of IBD-related bacteria in the digestive tract of IBD patients and improving intestinal flora.

[0707] Example 4: Feces treated with IgA antibodies that have high binding capacity to IBD-related bacteria and the Therapeutic effect of oral administration of IgA antibodies on IBD

[0708] (Methods and Materials)

[0709] Mice for human fecal sample transplantation

[0710] Activation-induced cytidine deaminase (AID) knockout mice with endogenous IgA deficiency were used. Mice were raised in sterile vinyl isolators. Male mice aged 5-19 weeks were used. Mice were euthanized by cervical dislocation when samples were collected after the experiment.

[0711] Bacterial transplantation of mice by oral administration of intestinal bacteria from IBD patient samples (P10 and P13)

[0712] The same operation as in Example 3 was carried out in an anaerobic chamber. After the bacterial count was determined, PBS was added to the bacterial suspension to prepare 3×10 8 pcs / tube. Then centrifuge at 8,000g, 4°C for 5 minutes and remove the supernatant. Add 100μl of sterile PBS and orally administer to mice. The number and interval of oral administration of bacterial solution are different in P10 and P13. The schematic diagram and details of each experiment are shown in Fig.11 and Fig.15 middle.

[0713] · Removal of RS_H000_L001 antibody-binding bacteria from IBD patient samples (P10) using a column

[0714] The operation was performed in an anaerobic chamber. After the bacterial count was determined, PBS was added to the bacteria in the human feces sample to prepare 3×10 8 / tube. Then centrifuge at 8,000g, 4℃ for 5 minutes and remove the supernatant. Add 100μl of PBS containing 20% ​​normal rat serum (Wako) to the bacteria after removing the supernatant, react on ice for 30 minutes to block nonspecific binding. Add FACS buffer to the bacterial solution, centrifuge at 8,000g, 4℃ for 5 minutes, and remove the supernatant. Relative to 6×10 6 15 μg of biotinylated RS_H000_L001 IgA antibody was added to each bacterial count and reacted on ice for 40 minutes. FACS buffer was added to the antibody reaction solution, centrifuged at 8,000g, 4°C for 5 minutes, and the supernatant was removed. Veritas' magnetic bead separation (MACS) kit was used to remove RS_H000_L001 antibody-bound bacteria. Myone C1-streptavidin magnetic beads (Veritas) were used to remove RS_H000_L001 IgA antibody-bound bacteria in P10 fecal samples. 15 μl of magnetic beads prepared according to the Veritas magnetic bead washing protocol were added to 15 μg of biotinylated RS_H000_L001 IgA antibody and reacted on ice for 30 minutes. Bacterial isolation was performed according to the Veritas MACS bacterial isolation protocol. The bacteria adsorbed to the magnet were recovered as RS_H000_L001 IgA antibody-bound bacteria, and the bacteria recovered as flow-through were recovered as RS_H000_L001 antibody-unbound bacteria, and suspended in 500 μl of FACS buffer. 5 μl of RS_H000_L001 antibody-unbound bacteria were recovered, and FACS buffer, TO (final concentration 42 nM, BD) and phycoerythrin (PE)-anti-mouse IgA antibody (final concentration 5 μg / ml, Biolegend) were added, and reacted on ice in the dark for 10 minutes. Then, FACS analysis using a SONY cell sorter SH800 (SONY) confirmed that only unbound bacteria were recovered at a ratio of more than 90%. The remaining RS_H000_L001 antibody-unbound bacteria were centrifuged at 8,000g and 4°C for 5 minutes, and the supernatant was removed. 100 μl was prepared with PBS and orally administered to mice.

[0715] · RS_H000_L001 Oral administration of IgA antibody

[0716] Starting from the last day of bacterial solution administration, RS_H000_L001 antibody was orally administered to mice once a day via gastric feeding tube for 7 days (P10) or 8 days (P13). The single dose of RS_H000_L001 antibody was prepared in PBS to 200 μl at 100 μg / mouse.

[0717] Dextran sulfate sodium (DSS)-induced enteritis model mice

[0718] From the day after the last day of RS_H000_L001 antibody administration, 4% dextran sulfate sodium (DSS (MP Biomedicals)) was added to the water bottle and orally administered to mice to induce enteritis. DSS was added to sterile water to a final concentration of 4%, and the mice were administered with free drinking water for 7 days. The body weight and fecal score of the mice were measured daily.

[0719] Freezing and staining of colon tissue

[0720] On the 7th day after DSS administration, mice were euthanized by cervical dislocation, and the large intestine was removed by autopsy. The contents of the large intestine tissue were washed, embedded in an optimal cutting temperature (OCT) compound (Sakura), frozen with liquid nitrogen, and stored at -80°C.

[0721] Tissue sections with a thickness of 6 μm were prepared from the colon tissue of mice cryopreserved using OCT embedding medium. The tissue sections were fixed with acetone for 15 minutes, stained with hematoxylin-eosin and alcian blue, and observed under an optical microscope. Hematoxylin-eosin staining used Gill III modified hematoxylin solution (Merck) and eosin alcohol solution and acid extract (Wako). Alcian blue staining used Alcian blue solution, pH 2.5 (Wako) and nuclear fast red solution (Muto Chemical).

[0722] Method for extracting bacterial DNA from mouse feces

[0723] Mouse fecal samples were collected before (day 0) and after administration of RS_H000_L001 IgA antibody. The weight of the mouse fecal sample was measured and 9 times the amount of sterilized PBS was added. The sample was suspended using a vortex shaker and centrifuged at 50g and 4°C for 15 minutes. The supernatant was collected in a 1.5 ml tube and centrifuged at 8,000g and 4°C for 5 minutes to remove the supernatant. The DNA lysis buffer for DNA extraction was prepared by adding Tris-HCl (final concentration 50mM, Nacalai Tesque), NaCl (final concentration 300mM, Nacalai Tesque), EDTA (final concentration 1mM, Nacalai Tesque), and 0.5% SDS (Nacalai Tesque) to ultrapure water. After adding 500 μl of DNA lysis buffer to the bacteria and suspending, transfer to a sterilized 2.0 ml tube (TM-625S, TOMY) with glass beads (GB-01, 0.1Φ, TOMY), and use a bead cell disruptor (MS-100, TOMY) at 3,500 rpm for 1 minute. Add protein kinase K (final concentration 0.5 mg / ml, Nacalai Tesque) to the bead-disrupted bacterial solution and vortex oscillate, and let stand at 55°C overnight to completely dissolve the bacteria. Return from 55°C to room temperature, add 500 μl of phenol: chloroform: isoamyl alcohol = 24:25:1 (Nacalai Tesque) to the bacterial lysate, mix using a vortex oscillator, centrifuge at 13,000 rpm, 4°C for 5 minutes, and recover the upper layer into another 1.5 ml tube. Add chloroform (Nacalai Tesque) in an amount equal to the amount of recovered liquid, mix using a vortex oscillator, and centrifuge at 13,000 rpm and 4°C for 5 minutes. Recover the upper layer again in another 1.5 ml tube, add isopropanol (Nacalai Tesque) and glycogen (final concentration 0.04 mg / ml, Nacalai Tesque) in an amount equal to the amount recovered, and mix by inversion. The DNA solution after inversion and mixing was allowed to stand at -20°C for 20 minutes, then centrifuged at 13,000 rpm and 4°C for 10 minutes, and the supernatant was removed. Add 500 μl of 70% ethanol to the precipitated DNA, centrifuge at 13,000 rpm and 4°C for 5 minutes, and remove the supernatant. After air-drying the precipitated DNA, add ultrapure distilled water (Invitrogen) to dissolve the DNA. The concentration of DNA was measured using Bio Drop DUO (Biochrome).

[0724] (result)

[0725] Total flora obtained from the feces of a specific IBD patient (P10) or RS_H000_L001IgA antibody-unbound bacteria after removing bacteria that bind to RS_H000_L001 IgA antibody showing high binding affinity to IBD-related bacteria were orally administered to IBD model mice multiple times. Analysis of RS_H000_L001 IgA antibody-bound bacteria in the flora of the IBD patient (P10) revealed a high IgA index for Enterobacteriaceae and the like and a low IgA index for Bifidobacteriaceae and the like, and oral administration of the bacteria was performed on some of these mice while RS_H000_L001 IgA antibody was orally administered. That is, the test groups were (1) total flora / antibody non-administration group, (2) total flora / RS_H000_L001 IgA antibody oral administration group, (3) RS_H000_L001 IgA antibody non-binding bacteria / antibody non-administration group, and (4) RS_H000_L001 IgA antibody non-binding bacteria / RS_H000_L001 IgA antibody oral administration group ( Fig.11 ).

[0726] As a result, in the (1) total flora / antibody non-administration group, body weight loss, which is considered to be associated with the onset of DSS-induced enteritis, was observed after oral administration of bacteria. In contrast, in the (2) total flora / RS_H000_L001 IgA antibody oral administration group, body weight loss was significantly suppressed ( Fig.12 ).

[0727] On the other hand, in the (3) RS_H000_L001 IgA antibody unbound bacteria / antibody unadministered group, almost no weight loss, which is believed to be associated with the onset of DSS-induced enteritis, was observed after oral administration of bacteria, suggesting that the enteritis-causing effect was reduced by modifying the fecal flora of IBD patients with RS_H000_L001 IgA antibodies. In addition, in the (4) RS_H000_L001 IgA antibody unbound bacteria / RS_H000_L001 IgA antibody oral administration group, which was combined with oral administration of RS_H000_L001 IgA antibodies, the weight change was almost the same as that of the (3) RS_H000_L001 IgA antibody unbound bacteria / antibody unadministered group ( Fig.13 ).

[0728] In addition, the diversity of the intestinal flora of the treated mice was analyzed. The results showed that the (2) total flora / RS_H000_L001 IgA antibody oral administration group, (3) RS_H000_L001 IgA antibody unbound bacteria / antibody unbound group, and (4) RS_H000_L001 IgA antibody unbound bacteria / RS_H000_L001 IgA antibody oral administration group showed higher diversity than (1) total flora / antibody untreated group ( Fig.14 ).

[0729] These results indicate that, when autologous fecal transplantation (microbiota transplantation) is performed on IBD patients, by combining oral administration of IgA antibodies with high binding affinity to IBD-related bacteria, or by modifying the microbiota contained in autologous feces using IgA antibodies with high binding affinity to IBD-related bacteria, the intestinal microbiota can be improved in IBD patients, thereby improving IBD disease symptoms.

[0730] The same experiment was conducted using the fecal flora from an IBD patient (P13) different from the above IBD patient (P10). Fig.15 As shown, the purpose of this study was to test the antibody effect of the total flora and RS_H000_L001 IgA antibody in more detail.

[0731] As a result of FACS analysis, it was observed that about 70% of the fecal flora from IBD patients (P13) bound to the RS_H000_L001 antibody ( Fig.16 ).

[0732] The results of observing the changes in body weight after oral administration of bacteria in the (1) total flora / antibody non-administration group and (2) total flora / RS_H000_L001 IgA antibody oral administration group showed that, for the flora from the feces of IBD patients (P13), almost no changes in body weight were observed in the (1) total flora / antibody non-administration group, and the same was true for the (2) total flora / RS_H000_L001 IgA antibody oral administration group ( Fig.17 ).

[0733] In addition, the diversity of intestinal flora after treatment was analyzed ( Figures 18 to 21 ), and the Shannon index showed almost no change ( Fig.18 On the other hand, the results of confirming the relative abundance ratios of various bacteria showed that the relative abundance ratios of Fusobacteriaceae and Bacteroidaceae, which are thought to be associated with IBD, decreased in both the (1) total flora / antibody non-administration group and the (2) total flora / RS_H000_L001 IgA antibody oral administration group ( Fig.19 , Fig.21 ), whereas an increase in the relative abundance of Lachnospiraceae was observed ( Fig. 20 ).

[0734] Further observation of the colon tissue sections after treatment revealed that abnormal tissue and cell structures were observed in the (1) total flora / antibody non-administration group, whereas normal tissue and cell structures were observed in the (2) total flora / RS_H000_L001 IgA antibody oral administration group ( Fig. 22 ).

[0735] These results also verify that when autologous fecal transplantation (microbiota transplantation) is performed on IBD patients, the intestinal microbiota and IBD disease symptoms can be improved in IBD patients by combining oral administration of IgA antibodies with high binding force for IBD-related bacteria. In addition, in a mouse administration test using microbiota from the feces of IBD patients (P10), without the use of IgA antibodies, a significant weight loss was observed, which is believed to be associated with the onset of DSS-induced enteritis. In contrast, in a mouse administration test using microbiota from the feces of IBD patients (P13), no significant weight loss was observed, suggesting that the microbiota morphology involved in the onset of IBD patients is different in each IBD patient. In the present invention, a more effective IBD improvement effect can be obtained by using IgA antibodies that make the microbiota in the digestive tract of each patient sound.

[0736] Example 5: Other IgA antibodies with high binding affinity to IBD-related bacteria

[0737] The same method as in Example 3 was used to test other IgA antibodies that have high binding affinity to IBD-related bacteria.

[0738] The binding ratios of bacteria from the feces of healthy people and those of bacteria from the feces of IBD patients that bound to IgA antibody clones SNK0004 and SNK0005 isolated from the intestinal mucosa of mice were also compared.

[0739] It should be noted that the amino acid sequences of the heavy chain and light chain of SNK0004 and SNK0005 are shown below.

[0740]

[0741]

[0742] The results are shown in Fig.23 and 24Regarding the SNK0004 antibody, the binding ratio of bacteria from IBD patient feces that bound to the antibody was higher than the binding ratio of bacteria from healthy human feces that bound to the antibody. On the other hand, regarding SNK0005, the binding ratio of bacteria from healthy human feces was not significantly different from the binding ratio of bacteria from IBD patient feces.

[0743] On the other hand, the binding characteristics of each bacterial species were confirmed for bacteria from IBD patient feces that these antibodies bound to. The results showed that both SNK0004 and SNK0005 showed specific binding to Enterobacteriaceae, but showed different binding characteristics to other bacteria ( Fig.25 and Fig.26 ).

[0744] For the SNK0001, SNK0002, and SNK0003 antibodies used in Example 3, bacteria from IBD patient feces that specifically bind to them were also confirmed ( Figure 27 to Figure 29 ) These results show that, in antibodies with a higher binding ratio to bacteria from IBD patient feces than to bacteria from healthy human feces, the bacteria to which each exhibited specific binding were different.

[0745] The results of comparison of the ratios of bacteria from IBD patient feces that bind to RS_H000_L001, SNK0001, SNK0002, SNK0003, SNK0004, and SNK0005 antibodies are shown in Fig.30 The results showed that these antibodies all bind to Enterobacteriaceae, and therefore are believed to have an effect on improving the intestinal flora. On the other hand, the bacterial species that bind to each antibody are different.

[0746] Regarding IBD patients, it is believed that IBD-related bacteria showing abnormal abundance ratios are different in each patient. From this result, it can be seen that different types of IgA antibodies can be used to test the status of bacteria showing abnormal abundance ratios in these IBD patients; and based on the test results, IgA antibodies that are more suitable for the patient can be used as a drug for improving the flora in the digestive tract and an IBD therapeutic drug.

[0747] Industrial Applicability

[0748] The present disclosure provides: a method for obtaining digestive tract contents or excreta with improved digestive tract flora by treating the digestive tract contents or excreta with IgA antibodies in a therapeutic method of administering digestive tract contents or excreta for the treatment of IBD; and a method for obtaining IgA antibodies related to the method; and pharmaceutical compositions used in these methods, which have extremely high industrial utilization value.

Claims

1. A pharmaceutical composition for treating inflammatory bowel disease (IBD), comprising digestive tract contents or excreta treated with IgA antibodies that improve the flora in the digestive tract.

2. A pharmaceutical composition for treating IBD, which is used in combination with a composition containing digestive tract contents or excreta, and contains IgA antibodies that improve the flora in the digestive tract.

3. The pharmaceutical composition according to claim 1 or 2, wherein The IgA antibody that improves the flora in the digestive tract has one or more of the following characteristics: (1) when the IgA antibody comes into contact with digestive tract contents or excreta from an IBD patient or digestive tract contents or excreta from a healthy person, the IgA antibody binds to more IBD-associated bacteria when the IgA antibody comes into contact with digestive tract contents or excreta from an IBD patient; (2) contacting the digestive tract contents or excreta from an IBD patient with the IgA antibody, and preparing a first binding profile based on the types and amounts of bacteria bound to the IgA antibody; The IgA antibody is brought into contact with digestive tract contents or excreta from a healthy person, and a second binding profile is prepared based on the types and amounts of bacteria bound to the IgA antibody, and when the first binding profile is compared with the second binding profile, binding of the IgA to a greater number of types of IBD-related bacteria or a greater amount of binding to IBD-related bacteria is observed in the first binding profile; (3) When the bacterial diversity of the digestive tract contents or excreta from IBD patients before treatment with IgA antibodies is compared with the bacterial diversity of the digestive tract contents or excreta after treatment with IgA antibodies, the bacterial diversity of the digestive tract contents or excreta after treatment with IgA antibodies increases; (4) when digestive tract contents or excreta modified with the IgA antibody are administered to a patient, and when the bacterial diversity in the digestive tract of the patient before administration is compared with the bacterial diversity in the digestive tract contents or excreta after administration, the bacterial diversity in the digestive tract contents or excreta after administration increases; (5) when the digestive tract contents or excreta modified with the IgA antibody are administered to a patient, and when the short-chain fatty acid content in the digestive tract of the patient before administration is compared with the short-chain fatty acid content in the digestive tract contents or excreta after administration, the short-chain fatty acid content after administration increases; (6) Gastrointestinal contents or excreta modified with the IgA antibody are administered to a patient, and the Enterobacteriaceae content in the digestive tract contents or excreta of the subject before administration is compared with the Enterobacteriaceae content in the digestive tract contents or excreta after administration, whereby the Enterobacteriaceae content in the digestive tract contents or excreta after administration is reduced.

4. The pharmaceutical composition according to claim 1 or 2, wherein The digestive tract contents or excrement are from a subject to which the composition is administered.

5. A method for screening IgA antibodies that improve the flora in the digestive tract, comprising the following steps: (B1) Steps for preparing digestive tract contents or excreta of an IBD patient; (B2) a step of modifying the digestive tract contents or excreta using the candidate IgA antibody; (B3) A step of confirming that the modified digestive tract contents or excreta can improve the bacterial flora in the digestive tract.

6. The method of claim 5, wherein: The step (B3) of confirming that the modified digestive tract contents or excreta can make the flora in the digestive tract healthy is carried out by any of the following methods: (1) when the IgA antibody is brought into contact with digestive tract contents or excreta from an IBD patient or digestive tract contents or excreta from a healthy person, it is confirmed that the IgA antibody binds to more IBD-associated bacteria when brought into contact with digestive tract contents or excreta from an IBD patient; (2) contacting the digestive tract contents or excreta from an IBD patient with the IgA antibody, and preparing a first binding distribution map based on the types and amounts of bacteria bound to the IgA antibody; The IgA antibody is brought into contact with digestive tract contents or excreta from a healthy person, and a second binding profile is prepared based on the types and amounts of bacteria bound to the IgA antibody, and when the first binding profile is compared with the second binding profile, it is confirmed that the IgA antibody binds to more types of IBD-related bacteria or in a greater amount to IBD-related bacteria in the first binding profile; (3) When the bacterial diversity of the digestive tract contents or excreta from IBD patients before treatment with IgA antibodies was compared with the bacterial diversity of the digestive tract contents or excreta after treatment with IgA antibodies, it was confirmed that the bacterial diversity of the digestive tract contents or excreta after treatment with IgA antibodies increased; (4) administering digestive tract contents or excreta modified with the IgA antibody to a patient, and comparing the bacterial diversity in the patient's digestive tract before administration with the bacterial diversity in the digestive tract contents or excreta after administration, confirming that the bacterial diversity in the digestive tract contents or excreta after administration increases; (5) administering to a patient digestive tract contents or excreta modified with the IgA antibody, and comparing the short-chain fatty acid content in the digestive tract of the patient before administration with the short-chain fatty acid content in the digestive tract contents or excreta after administration, the short-chain fatty acid content after administration increases; (6) Gastrointestinal contents or excreta modified with the IgA antibody are administered to a patient, and the Enterobacteriaceae content in the digestive tract contents or excreta of the subject before administration is compared with the Enterobacteriaceae content in the digestive tract contents or excreta after administration, whereby the Enterobacteriaceae content in the digestive tract contents or excreta after administration is reduced.

7. A method for treating digestive tract contents or excreta of an IBD patient, comprising the following steps: (C1) Steps for preparing digestive tract contents or excreta from an IBD patient; (C2) a step of contacting the digestive tract contents or excreta with IgA that improves the bacterial flora in the digestive tract in vitro.

8. A composition for modifying the flora of digestive tract contents or excreta in vitro, comprising IgA that improves the flora in the digestive tract.

9. A method for conducting an experiment to diagnose whether a subject suffers from IBD or is at risk of developing IBD, comprising the following steps: (D1) a step of preparing IgA antibodies that improve the flora in the digestive tract of IBD patients; (D2) contacting the digestive tract contents or excreta of the subject with the IgA antibody; as well as (D3) a step of identifying bacteria that bind to the IgA antibody, In the case where the IgA antibody shows binding to IBD-associated bacteria, the subject is considered to be suffering from IBD, or at risk of developing IBD and is treated.

10. A drug for diagnosing the presence or absence of IBD or the risk of developing IBD, comprising an IgA antibody that improves the flora in the digestive tract of an IBD patient.

11. A method for testing the therapeutic effect of an IBD therapeutic drug, comprising the following steps: (E1) a step of obtaining digestive tract contents or excreta of a patient who is currently receiving, plans to receive, or has received an IBD therapeutic drug containing digestive tract contents or excreta; (E2) a step of contacting the digestive tract contents or excreta with IgA antibodies; as well as (E3) a step of analyzing bacteria bound to the IgA antibody, When the analysis results show that the bacterial flora in the digestive tract has become healthy, the treatment with the IBD therapeutic drug is continued.

12. The method of claim 11, wherein: The digestive tract contents or excreta are modified by the IgA.

13. A diagnostic drug containing IgA antibodies for use in testing the therapeutic effect of an IBD therapeutic drug on a patient who is currently being administered, is scheduled to be administered, or has been administered an IBD therapeutic drug containing digestive tract contents or excreta.

14. The diagnostic drug according to claim 13, wherein The digestive tract contents or excreta are modified by the IgA.

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