Inspection method for immune-related adverse event enteritis

Abstract

Provided herein is a method for inspecting irAE enteritis, comprising a detection step for detecting an antibody that immunologically reacts with a fragment or all of integrin [alpha] v [beta] 6 in a sample as an indicator of ulcerative colitis-like irAE enteritis.

Description

Technical Field

[0001] The present invention relates to a method for examining immune-related adverse event (irAE) enteritis, an examination kit for examining irAE enteritis, and the like. Background Art

[0002] When treating cancer patients with immune checkpoint inhibitors (ICI), as a compensation for releasing the brakes of the immune system, the immune system gets out of control, and as a result, irAE sometimes occurs. There are various types of irAE that occur in various parts of the body, and enteritis (hereinafter referred to as irAE enteritis) is one of them. When the treatment of irAE enteritis is delayed, the patient sometimes dies, which is one of the major side effects of ICI therapy.

[0003] Since irAE enteritis is basically caused by an excessive immune response resulting from the release of immunosuppression by ICI, once the administration of ICI is stopped and treatment with an immunosuppressant such as a steroid is carried out, improvement can be seen. On the other hand, immunosuppression caused by the use of an immunosuppressant is generally contraindicated for infectious enteritis caused by bacteria and the like. Further, in the case where enteritis occurs as a side effect of a combined therapy of ICI and a chemotherapeutic agent, the treatment method is different depending on whether the enteritis is irAE enteritis caused by ICI treatment or enteritis caused by the chemotherapeutic agent. When an immunosuppressant is administered to enteritis caused by a chemotherapeutic agent, not only does the symptom of enteritis not improve, but sometimes the cause symptom of the enteritis deteriorates. Therefore, it is necessary to diagnose whether the enteritis occurring in the current case is irAE that requires the use of an immunosuppressant in order to carry out appropriate treatment. However, since the diagnosis of irAE enteritis is basically an exclusion diagnosis, it is currently difficult to definitely diagnose irAE enteritis. In addition, the treatment effect of ICI for cancer patients itself is brought about by the activation of the immune response, and the administration of an immunosuppressant, which is the opposite, may weaken the treatment effect of ICI, which is also one of the reasons for hesitating to administer an immunosuppressant. On the other hand, in the case where the enteritis is irAE enteritis, when the timing of the administration of the immunosuppressant is delayed, irAE enteritis becomes severe. Therefore, in the clinical field, the development of an examination method for clearly determining whether the cause of this enteritis is irAE is an urgent task.

[0004] Furthermore, it is also known that not all patients with irAE enteritis require treatment with immunosuppressants such as steroids and anti-TNFα antibodies. For example, in the case of relatively mild irAE enteritis without mucosal exfoliation, treatment with an immunosuppressant is not required, and sometimes treatment such as temporarily stopping the administration of ICI is sufficient. Therefore, a method for determining whether it is irAE enteritis that requires an immunosuppressant is also needed.

[0005] As a technique for diagnosing a specific type of enteritis, a diagnostic method using a biomarker as an index is proposed. For example, as an index for ulcerative colitis or primary sclerosing cholangitis, the detection of integrin αvβ6 autoantibodies in a test sample has been reported (Patent Document 1). However, no autoantibody that can be an index for irAE enteritis has been reported so far.

[0006] Prior art documents

[0007] Patent documents

[0008] Patent Document 1: WO2020 / 141608 Summary of the invention

[0009] Problems to be solved by the invention

[0010] An object of the present invention is to develop a method for examining irAE enteritis, particularly a method for determining whether enteritis developed in a cancer patient receiving ICI treatment is irAE enteritis, or a method for determining whether a cancer patient who is to undergo ICI treatment in the future is likely to develop irAE enteritis.

[0011] Means for solving the problems

[0012] In view of the above problems, the present inventors conducted intensive studies and, as a result, surprisingly found that in patients with ICI-administered cancer who developed enteritis and showed a morbid state similar to ulcerative colitis, the blood concentration of autoantibodies against integrin αvβ6 was significantly high, and the symptoms of enteritis improved when an immunosuppressant was administered to the patient. Further, the present inventors surprisingly found that in patients who developed ulcerative colitis-like irAE enteritis, the blood concentration of autoantibodies against integrin αvβ6 was significantly high before receiving ICI treatment. Thus, the present invention has been completed.

[0013] Therefore, the present invention provides the following aspects.

[0014] (1) A method for examining irAE enteritis, comprising:

[0015] A detection step of detecting, as an index for ulcerative colitis-like irAE enteritis, an antibody that immunologically reacts with a fragment or all of integrin αvβ6 in a test sample.

[0016] (2) The method according to (1), wherein the detection step includes a step of using a fragment or all of integrin αvβ6 as an antigen to detect an antibody that immunologically reacts therewith.

[0017] (3) The method according to (1) or (2), wherein the test sample is a blood sample.

[0018] (4)The method according to any one of (1) to (3), wherein the aforementioned index is an index of severe ulcerative colitis-like irAE enteritis.

[0019] (5)A test kit for examining irAE enteritis, which comprises a fragment or all of integrin αvβ6.

[0020] (6)The test kit according to (5), which further comprises an antibody for detection.

[0021] (7)The test kit according to (5) or (6), which further comprises a positive standard solution and / or a negative standard solution.

[0022] (8)The test kit according to any one of (5) to (7), which comprises the aforementioned fragment or all of integrin αvβ6 in a form immobilized on a solid phase.

[0023] (9)The method according to any one of (1) to (4), which is used to determine whether the enteritis that occurs in a patient receiving immune checkpoint inhibitor (ICI) therapy is ulcerative colitis-like irAE enteritis, wherein

[0024] in the aforementioned detection step, the aforementioned sample is a sample derived from a patient receiving ICI therapy, and the antibody titer of the antibody that immunologically reacts with a fragment or all of integrin αvβ6 detected in the aforementioned sample is higher than the antibody titer of the aforementioned antibody detected in a sample from a control source, which is an index for determining that the enteritis that occurs in the aforementioned patient is ulcerative colitis-like irAE enteritis.

[0025] Here, the aforementioned patient is a human or a non-human animal, preferably a human.

[0026] (10)The method according to (9), wherein the antibody titer of the antibody that immunologically reacts with a fragment or all of integrin αvβ6 detected in the aforementioned sample is higher than the antibody titer of the aforementioned antibody detected in a sample from a control source, which is an index for determining that the enteritis that occurs in the aforementioned patient is severe ulcerative colitis-like irAE enteritis.

[0027] (11)The method according to (9) or (10), wherein the aforementioned patient is a patient receiving combined therapy of ICI and a cancer drug therapy other than ICI.

[0028] (12)The method according to any one of (1) to (4), which is used to predict the risk of developing ulcerative colitis-like irAE enteritis in a patient, wherein

[0029] In the foregoing detection step, the foregoing sample is a sample derived from a patient, and the antibody titer of the antibody that immunologically reacts with a fragment or all of integrin αvβ6 detected in the foregoing sample is higher than the antibody titer of the foregoing antibody detected in a sample from a control source, which is an index for determining the risk of developing ulcerative colitis-like irAE enteritis in the foregoing patient.

[0030] Here, the foregoing patient is a human or non-human animal, preferably a human.

[0031] The method according to (13) or (12), wherein the antibody titer of the antibody that immunologically reacts with a fragment or all of integrin αvβ6 detected in the foregoing sample is higher than the antibody titer of the foregoing antibody detected in a sample from a control source, which is an index for determining the risk of developing severe ulcerative colitis-like irAE enteritis in the foregoing patient.

[0032] The method according to (14), (12) or (13), wherein the foregoing patient is a patient before ICI treatment or a patient receiving ICI treatment.

[0033] (15)A method for examining irAE enteritis, comprising:

[0034] A detection step of detecting an antibody that immunologically reacts with a fragment or all of integrin αvβ6 in a sample as an index for enteritis for recommending discontinuation of the administration of ICI.

[0035] The method according to (16) or (15), wherein the foregoing index is an index for enteritis for recommending discontinuation of the administration of ICI and administering one or more immunosuppressants.

[0036] The method according to (17), (15) or (16), wherein the foregoing detection step includes a step of using a fragment or all of integrin αvβ6 as an antigen to detect an antibody that immunologically reacts therewith.

[0037] The method according to any one of (18), (15) to (17), wherein the foregoing sample is a blood sample.

[0038] The method according to any one of (19), (15) to (18), which is used to determine whether the enteritis developed in a patient receiving ICI treatment is enteritis for which it is recommended to discontinue the administration of ICI, wherein

[0039] In the foregoing detection step, the foregoing sample is a sample derived from a patient receiving ICI treatment, and the antibody titer of the antibody that immunologically reacts with a fragment or all of integrin αvβ6 detected in the foregoing sample is higher than the antibody titer of the foregoing antibody detected in a sample from a control source, which is an index for determining that the enteritis developed in the foregoing patient is enteritis for which it is recommended to discontinue the administration of ICI.

[0040] The method described in (20) (19), wherein the antibody titer of the antibody that immunologically reacts with a fragment or the whole of integrin αvβ6 detected in the aforementioned sample is higher than the antibody titer of the aforementioned antibody detected in a sample from a control source, is an index for determining that the enteritis that has developed in the aforementioned patient is enteritis for which it is recommended to discontinue the administration of ICI and administer one or more immunosuppressants.

[0041] The method described in (21) (19) or (20), wherein the aforementioned patient is a patient receiving combined treatment with ICI and a cancer drug therapy other than ICI.

[0042] The method described in any one of (22) (15) to (18), which is used to predict the risk of enteritis for which it is recommended to discontinue the administration of ICI in a patient, wherein

[0043] In the aforementioned detection step, the aforementioned sample is a sample from the patient, and the antibody titer of the antibody that immunologically reacts with a fragment or the whole of integrin αvβ6 detected in the aforementioned sample is higher than the antibody titer of the aforementioned antibody detected in a sample from a control source, which is an index for determining the risk of enteritis for which it is recommended to discontinue the administration of ICI in the aforementioned patient.

[0044] The method described in (23) (22), wherein the antibody titer of the antibody that immunologically reacts with a fragment or the whole of integrin αvβ6 detected in the aforementioned sample is higher than the antibody titer of the aforementioned antibody detected in a sample from a control source, which is an index for determining the risk of enteritis for which it is recommended to discontinue the administration of ICI and administer one or more immunosuppressants in the aforementioned patient.

[0045] The method described in (24) (22) or (23), wherein the aforementioned patient is a patient before ICI treatment or a patient receiving ICI treatment.

[0046] The kit described in any one of (25) (5) to (8), which is used to determine whether the enteritis that has developed in a patient receiving ICI treatment is ulcerative colitis-like irAE enteritis.

[0047] Here, the aforementioned patient is a human or a non-human animal, preferably a human.

[0048] The kit described in (26) (25), wherein the aforementioned patient is a patient receiving combined treatment with ICI and a cancer drug therapy other than ICI.

[0049] The kit described in any one of (27) (5) to (8), which is used to predict the risk of ulcerative colitis-like irAE enteritis in a patient.

[0050] Here, the aforementioned patient is a human or a non-human animal, preferably a human.

[0051] The kit described in (28) (27), wherein the aforementioned patient is a patient before ICI treatment or a patient receiving ICI treatment.

[0052] (29) A method for treating ulcerative colitis-like irAE enteritis, comprising:

[0053] The step of detecting an antibody that immunologically reacts with a fragment or all of integrin αvβ6 in a sample derived from a patient,

[0054] In the case where the antibody titer of the aforementioned antibody detected in the sample derived from the aforementioned patient is higher than the antibody titer of the aforementioned antibody detected in the sample derived from the control, for the aforementioned patient, the step of stopping ICI treatment and / or administering an immunosuppressant.

[0055] Here, the aforementioned patient is a human or non-human animal that has developed enteritis while receiving ICI treatment, preferably a human.

[0056] (30) The method described in (29), comprising: in the case where the antibody titer of the aforementioned antibody detected in the sample derived from the aforementioned patient is higher than the antibody titer of the aforementioned antibody detected in the sample derived from the control, for the aforementioned patient, the step of stopping ICI treatment and administering an immunosuppressant.

[0057] (31) A method for treating enteritis that develops in a patient receiving combined treatment of ICI and a cancer drug therapy other than ICI, comprising:

[0058] The step of detecting an antibody that immunologically reacts with a fragment or all of integrin αvβ6 in a sample derived from the aforementioned patient,

[0059] In the case where the antibody titer of the aforementioned antibody detected in the sample derived from the aforementioned patient is higher than the antibody titer of the aforementioned antibody detected in the sample derived from the control, for the aforementioned patient, the step of stopping the administration of ICI and / or administering an immunosuppressant, or

[0060] In the case where the antibody titer of the aforementioned antibody detected in the sample derived from the aforementioned patient is the same as or lower than the antibody titer of the aforementioned antibody detected in the sample derived from the control, or in the case where the aforementioned antibody is not detected in the sample derived from the aforementioned patient, for the aforementioned patient, the step of stopping the cancer drug therapy other than ICI.

[0061] Here, the aforementioned patient is a human or non-human animal, preferably a human.

[0062] (32) The method described in (31), comprising: in the case where the antibody titer of the aforementioned antibody detected in the sample derived from the aforementioned patient is higher than the antibody titer of the aforementioned antibody detected in the sample derived from the control, for the aforementioned patient, the step of stopping the administration of ICI and administering an immunosuppressant.

[0063] (33) A method according to any one of (29) to (32), wherein the detection comprises a step of using a fragment or the whole of integrin αvβ6 as an antigen to detect an antibody that immunologically reacts therewith.

[0064] (34) A method according to any one of (29) to (33), wherein the sample is a blood sample.

[0065] (35) A method for evaluating the effect of treatment for ulcerative colitis-like irAE enteritis, comprising:

[0066] a detection step of detecting an antibody that immunologically reacts with a fragment or the whole of integrin αvβ6 in a sample obtained from a patient to whom treatment for ulcerative colitis-like irAE enteritis has been administered.

[0067] (36) A method for diagnosing ulcerative colitis-like irAE enteritis, comprising:

[0068] a step of detecting an antibody that immunologically reacts with a fragment or the whole of integrin αvβ6 in a sample derived from a patient, and

[0069] in the case where the antibody is detected, a step of determining that the patient has ulcerative colitis-like irAE enteritis.

[0070] Here, the patient is a human or non-human animal that has received ICI treatment, preferably a human.

[0071] (37) A method according to (35) or (36), wherein the detection comprises a step of using a fragment or the whole of integrin αvβ6 as an antigen to detect an antibody that immunologically reacts therewith.

[0072] (38) A method according to any one of (35) to (37), wherein the sample is a blood sample.

[0073] (39) A biomarker for diagnosing ulcerative colitis-like irAE enteritis, which comprises an antibody that immunologically reacts with a fragment or the whole of integrin αvβ6.

[0074] (40) A method for screening a candidate substance for a therapeutic agent for ulcerative colitis-like irAE enteritis, comprising:

[0075] an antibody detection step of detecting an antibody that immunologically reacts with a fragment or the whole of integrin αvβ6 in a sample obtained from an animal to which a test substance has been administered, and

[0076] a selection step of selecting the test substance as a candidate substance for a therapeutic agent for ulcerative colitis-like irAE enteritis in the case where the antibody in the sample is reduced by the action of the test substance.

[0077] (41) Method for obtaining an index of ulcerative colitis-like irAE enteritis, comprising:

[0078] A detection step of detecting an antibody in a sample that immunologically reacts with a fragment or the whole of integrin αvβ6.

[0079] (42) The method according to (40) or (41), wherein the aforementioned detection step includes a step of using a fragment or the whole of integrin αvβ6 as an antigen to detect an antibody that immunologically reacts therewith.

[0080] (43) The method according to any one of (40) to (42), wherein the aforementioned sample is a blood sample.

[0081] (44) Use of a fragment or the whole of integrin αvβ6 in the preparation of a test kit or test reagent for examining ulcerative colitis-like irAE enteritis.

[0082] (45) The use according to (44), wherein the aforementioned fragment or the whole of integrin αvβ6 is in a form immobilized on a solid phase.

[0083] (46) A fragment or the whole of integrin αvβ6, which is used for use in the examination of ulcerative colitis-like irAE enteritis.

[0084] (47) The fragment or the whole of integrin αvβ6 according to (46), which is in a form immobilized on a solid phase.

[0085] (48) Method for detecting an antibody in a patient that immunologically reacts with a fragment or the whole of integrin αvβ6, comprising:

[0086] A step of detecting whether the aforementioned antibody is present in a sample derived from the patient.

[0087] Herein, the aforementioned patient is a human or a non-human animal, preferably a human. The aforementioned patient can be an animal suffering from or suspected of suffering from ulcerative colitis-like irAE enteritis.

[0088] (49) The method according to (48), wherein the aforementioned detection is a detection of detecting whether the aforementioned antibody is present in the aforementioned sample by bringing the aforementioned sample into contact with an antigen that is a fragment or the whole of integrin αvβ6 and detecting the binding of the aforementioned antigen to the aforementioned antibody.

[0089] The method described in (50) (49), wherein the detection is to detect the binding of the antibody to the anti-human IgG antibody, anti-human IgA antibody, anti-human IgM antibody, and / or anti-human IgE antibody by contacting the sample with an antigen that is a fragment or the whole of integrin αvβ6, and further contacting with the anti-human IgG antibody, anti-human IgA antibody, anti-human IgM antibody, and / or anti-human IgE antibody, so as to detect whether the antibody is present in the sample.

[0090] Here, the antigen is preferably immobilized on a solid phase.

[0091] The method described in any one of (51) (48) to (50), wherein the sample is a blood sample isolated from the patient. Here, the blood sample is preferably a serum sample, a plasma sample, or whole blood.

[0092] A method for diagnosing ulcerative colitis-like irAE enteritis in a patient, which includes:

[0093] A step of detecting the binding of the antigen to an antibody that immunologically reacts with the antigen by contacting a sample derived from the patient with a solid-phase carrier immobilized with an antigen that is a fragment or the whole of integrin αvβ6, so as to detect whether the antibody is present in the sample, and

[0094] A step of determining that the patient has ulcerative colitis-like irAE enteritis in the case where the presence of the antibody is detected in the sample.

[0095] Here, the patient is a human or non-human animal receiving ICI treatment, preferably a human.

[0096] The method described in (53) (52), wherein the detection is to detect the binding of the antibody to the anti-human IgG antibody, anti-human IgA antibody, anti-human IgM antibody, and / or anti-human IgE antibody by contacting the sample with an antigen that is a fragment or the whole of integrin αvβ6, and further contacting with the anti-human IgG antibody, anti-human IgA antibody, anti-human IgM antibody, and / or anti-human IgE antibody, so as to detect whether the antibody is present in the sample.

[0097] The method described in (54) (52) or (53), wherein the sample is a blood sample isolated from the patient. Here, the blood sample is preferably a serum sample, a plasma sample, or whole blood.

[0098] Advantages of the Invention

[0099] If the inspection method for irAE enteritis of the present invention and the inspection kit therefor are applied, the presence or absence of the onset of ulcerative colitis-like irAE enteritis or the possibility of future onset of ulcerative colitis-like irAE enteritis can be detected with high sensitivity and specificity, and it can be specifically detected by differentiating from enteritis caused by chemotherapeutic agents and other factors other than other ICIs. For example, according to the present invention, it can be determined whether it is ulcerative colitis-like irAE enteritis in which immunosuppressants are also effectively used in enteritis. Therefore, according to the present invention, in the case of the onset of enteritis in patients receiving combined therapy of ICI and cancer drug therapy other than ICI, it is possible to determine which of ICI and cancer drug therapy other than ICI causes the enteritis, or whether the use of immunosuppressants is effective, so that appropriate treatment can be carried out at an early stage.

[0100] Furthermore, according to the present invention, even in irAE enteritis, ulcerative colitis-like irAE enteritis can be specifically detected by differentiating it from other irAE enteritis. In the present invention, even in irAE enteritis, high-level expression of anti-integrin αvβ6 antibody in patients with ulcerative colitis-like irAE enteritis and the effectiveness of the administration of immunosuppressants in these patients are shown. Therefore, according to the present invention, in the case of the occurrence of irAE enteritis in cancer patients receiving ICI treatment, appropriate treatment can be implemented for irAE enteritis. In particular, according to the present invention, ulcerative colitis-like irAE enteritis, which is one of the major irAEs leading to death when active treatment intervention is not carried out in the early stage, can be diagnosed, and thus appropriate treatment can be implemented for the patient.

[0101] Furthermore, in the present invention, using the antibody titer of anti-integrin αvβ6 antibody as an index, ulcerative colitis-like irAE enteritis can be differentiated from non-ulcerative colitis-like irAE enteritis. Ulcerative colitis-like irAE enteritis identified using the antibody titer of anti-integrin αvβ6 antibody as an index is cured by the administration of steroids, immunosuppressants such as anti-TNFα antibody, that is, it must be treated with immunosuppressants. On the other hand, non-ulcerative colitis-like irAE enteritis does not necessarily need to be treated by the administration of immunosuppressants, and there are cases where it is cured by other treatment methods such as simply stopping the administration of ICI and observing the course. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1 shows the concentration of anti-human integrin αvβ6 antibody in the sera of patients with ulcerative colitis-like irAE enteritis (irAE colitis UC like) and patients with non-ulcerative colitis-like irAE enteritis (irAE colitis UC unlike).

[0103] Figure 2 ​​Show the time-course changes in the concentration of anti-human integrin αvβ6 antibody (left vertical axis) and Mayo score (right vertical axis) in the sera of patients with ulcerative colitis-like irAE enteritis who received ICI discontinuation and steroid administration.

[0104] Figure 3 Show endoscopic images of the intestines of patients with ulcerative colitis-like irAE enteritis before ICI discontinuation and steroid administration.

[0105] Figure 4 Show endoscopic images of the intestines of patients with ulcerative colitis-like irAE enteritis after ICI discontinuation and steroid administration.

[0106] Figure 5 Show endoscopic images of the intestines of patients with ulcerative colitis-like irAE enteritis after ICI discontinuation and steroid administration.

[0107] Figure 6 Show the time-course changes in the concentration of anti-human integrin αvβ6 antibody (left vertical axis) and Mayo score (right vertical axis) in the sera of patients with ulcerative colitis-like irAE enteritis who discontinued ICI.

[0108] Figure 7 Show a graph of the time-course changes in the concentration of anti-human integrin αvβ6 antibody in the sera of patients from before the start of ICI treatment to after the onset of ulcerative colitis-like irAE enteritis, and show endoscopic images of the large intestine at the time of onset of ulcerative colitis-like irAE enteritis.

[0109] Figure 8 Show the concentration of anti-human integrin αvβ6 antibody in the sera of patients with irAE enteritis and other irAEs (liver damage, endocrine damage, lung damage, others), cancer patients (without irAE), and healthy subjects.

[0110] Figure 9 Show endoscopic images of the intestines of irAE enteritis patients positive and negative for anti-human integrin αvβ6 autoantibody.

[0111] Figure 10 Show several of the 6 characteristic endoscopic findings in ulcerative colitis seen in the intestines of irAE enteritis patients positive and negative for anti-human integrin αvβ6 autoantibody. The vertical axis shows the number of ulcerative colitis findings seen. The horizontal axis shows anti-human integrin αvβ6 antibody-positive and -negative samples.

[0112] Figure 11 ​​​​​​​​​The change over time in the concentration of anti-human integrin αvβ6 antibody (left vertical axis) and the Partial Mayo score (right vertical axis) in the serum of patients with ulcerative colitis-like irAE enteritis who received ICI discontinuation and steroid administration is shown. Detailed implementation mode

[0113] <1. Ulcerative colitis-like irAE enteritis>

[0114] IrAE enteritis (immune-related adverse event; irAE) is a general term for enteritis that occurs as an adverse event caused by immune-checkpoint inhibitors (ICI), and the pathological conditions are very diverse. That is, it ranges from microscopic colitis-like cases without endoscopic abnormalities but first proven by biopsy of the intestinal mucosa to inflammatory bowel disease (IBD)-like cases with severe mucosal edema, bleeding, and exfoliation (erosion, ulcer). Among them, there is irAE enteritis similar to ulcerative colitis (UC), one of the IBDs, which is fatal without active treatment intervention. In this specification, such irAE enteritis is referred to as "ulcerative colitis-like irAE enteritis" or "UC-type irAE enteritis".

[0115] The clinical manifestations of UC-type irAE enteritis are similar to those of UC. In typical cases, there are edema, bleeding, erosion, and ulcer of the mucosa continuous from the rectum to the oral side. Pathologically, crypt abscesses are formed by immune cell infiltration and a large amount of immune cell infiltration is seen in the tissue. UC is a disease of unknown etiology, while UC-type irAE enteritis is clearly different in that it is caused by ICI.

[0116] Including UC-type irAE enteritis, regardless of the type of irAE enteritis, the basic cause is an excessive immune response caused by the release of immunosuppression by ICIs. Improvement can be seen by stopping the administration of ICIs and implementing treatment with immunosuppressants such as steroids. Thus, in irAE enteritis, it is recommended to stop the administration of ICIs. In irAE enteritis, it is preferred to further administer immunosuppressants after stopping the administration of ICIs. On the other hand, immunosuppression is contraindicated for infectious enteritis generally caused by bacteria and the like. Therefore, the premise for the use of immunosuppressants is that the enteritis occurring in the present case is irAE. However, since the diagnosis of irAE enteritis is basically an exclusion diagnosis, it is currently difficult to confirm the diagnosis as irAE. In addition, since the therapeutic effect of ICIs itself is the activation of immune response, the administration of immunosuppressants such as steroids may weaken such anti-tumor effects, which is also one of the reasons for hesitating to administer immunosuppressants.

[0117] The steroid treatment response of irAE enteritis is generally good in the microscopic type of irAE enteritis without mucosal damage, but in UC-type irAE enteritis, when the treatment intervention is delayed, it sometimes becomes refractory, and it is not uncommon to require anti-TNF-α antibodies in cases with poor response to high-dose steroid treatment. When compared with the concept of no cure but only "remission" in UC, in cases of UC-type irAE enteritis with a response to treatment with immunosuppressants, it is often cured with a short period of treatment, and in some cases, re-administration of ICIs may be possible. UC-type irAE enteritis that occurs until mucosal exfoliation rarely improves only with the discontinuation of ICIs, and the symptoms tend to worsen over time. Therefore, it is important to diagnose irAE enteritis as quickly as possible without missing the opportunity to administer immunosuppressants.

[0118] ICI has been applied as a single agent to date. However, with the development of cancer treatment, the combination therapy with cancer drug therapies other than ICI has been increasingly carried out as a standard practice. In this case, as a cause of enteritis occurring during treatment, the factor of enteritis caused by cancer drug therapies other than ICI is further added. In particular, fluoropyrimidine anticancer agents represented by 5-FU used in digestive organ cancers are known to frequently cause diarrhea and intestinal mucosal damage. In this case, the diagnosis of irAE enteritis becomes extremely difficult. Clinically, enteritis caused by fluoropyrimidine mostly occurs in the main part of the small intestine, especially centered around the ileocecal region. However, since there are exceptions, it is often difficult to diagnose only by imaging. Since enteritis caused by anticancer agents is caused by direct damage to the intestinal mucosa, as a concerning pathological condition, it is bacterial translocation where the indigenous bacteria present in the intestine cross the mucosal barrier and invade the bloodstream, becoming sepsis of severe infectious diseases. Therefore, blindly using immunosuppressants for enteritis occurring due to the combination of ICI and cancer drug therapies other than ICI promotes such infections and is not recommended. In this case, it is necessary to observe the reaction caused by the discontinuation of the anticancer agent and additional treatment with broad-spectrum antibacterial drugs. On the other hand, when the cause of enteritis is ICI, the timing of immunosuppressant administration is delayed and the condition further deteriorates. Therefore, in the clinical field, it is important to confirm whether the cause of such enteritis is irAE.

[0119] <2. Patients, Samples>

[0120] The "patients" targeted by the present invention are patients who have the possibility of developing irAE enteritis or who have developed irAE enteritis. For example, cancer patients are listed. Cancer patients are animals suffering from cancer. The type of cancer is not particularly limited and can be any type, but preferably cancers that are indications for ICI treatment. For example, malignant melanoma, non-small cell carcinoma, renal cell carcinoma, classical Hodgkin lymphoma, head and neck cancer, gastric cancer, malignant pleural mesothelioma, esophageal cancer, MSI-High colorectal cancer, urothelial cancer, MSI-High solid cancer, esophageal squamous cell carcinoma, Merkel cell carcinoma, small cell carcinoma, breast cancer, hepatocellular carcinoma, uterine cancer, cancer of unknown primary origin, TMB-High cancer, etc. are listed, but are not limited thereto. The type of animal is not particularly limited and can be a human or other non-human mammals, but preferably a human.

[0121] The patient can be a patient receiving ICI therapy or a patient scheduled to receive ICI therapy. In this specification, "a patient receiving ICI therapy" includes a patient currently undergoing ICI therapy, and a patient who is not currently receiving ICI therapy but has received ICI therapy in the past and within several months, such as within about 2 to 6 months, since the last ICI administration. "A patient scheduled to receive ICI therapy" can be any patient as long as they have a scheduled ICI therapy, and can also be a patient who has never received ICI therapy, or a patient who has received ICI therapy in the past and the elapsed time since the last ICI administration, such as more than 1 year.

[0122] The ICI used in the present invention is a substance that inhibits the signal transduction of the immune mechanism by binding to an immune checkpoint molecule or its ligand molecule, thereby relieving the inhibitory signal of immune cells caused by the immune checkpoint molecule, and preferably has the effect of restoring the aggressiveness of immune cells against cancer. The ICI can be appropriately selected according to the type of cancer the patient has. For example, anti-PD-1 antibodies (e.g., nivolumab, pembrolizumab, etc.), anti-PD-L1 antibodies (e.g., avelumab, atezolizumab, durvalumab, etc.), anti-CTLA4 antibodies (e.g., ipilimumab, etc.) are listed, but not limited thereto. Two or more ICIs can be administered to the patient.

[0123] A patient receiving ICI therapy can further be a patient receiving combination therapy of ICI and a cancer drug therapy other than ICI. In this specification, "a patient receiving combination therapy of ICI and a cancer drug therapy other than ICI" includes a patient currently undergoing combination therapy, and a patient who is not currently receiving combination therapy but has received combination therapy in the past and within several months, such as within about 2 to 6 months, since the last ICI administration or a cancer drug therapy other than ICI.

[0124] As the drugs used in the "cancer drug therapy other than ICI" in this specification, for example, cytocidal anti-cancer agents and molecular target drugs can be listed. As the "cytocidal anti-cancer agent", for example, agents that inhibit cell proliferation by inhibiting the synthesis and replication of DNA in cells, and agents that inhibit cell proliferation by acting on microtubules, which are one of the cytoskeletons, and inhibiting cell division can be listed. As specific examples of cytocidal anti-cancer agents, depending on the type of cancer the patient has, for example, fluoropyrimidine anti-cancer agents represented by 5-FU (fluorouracil), platinum anti-cancer agents such as cisplatin, oxaliplatin, and carboplatin, antimetabolite anti-cancer agents such as gemcitabine and pemetrexed, etc. can be listed, but are not limited thereto. In addition, the "molecular target drug" is a drug that targets molecules related to the proliferation, invasion, metastasis, etc. of cancer cells, and has the effect of inhibiting the primary cancer and also inhibiting the metastasis of cancer by inhibiting the proliferation of cancer cells or inhibiting the progression process of cancer. As specific examples of molecular target drugs, antibody preparations such as Avastin, low molecular weight compounds such as lenvatinib, etc. can be listed, but are not limited thereto.

[0125] As the "sample" used in the present invention, body fluids collected from patients are listed. Specifically, in addition to blood samples such as serum, plasma, and whole blood, body fluid samples other than blood such as saliva, cerebrospinal fluid, and urine can also be used. In addition, not limited to body fluids, tissues collected from patients, for example, tissues collected from sites showing the pathological conditions of enteritis to be examined (such as the large intestine and rectum) can also be used as samples. The sample is used in the present invention in a form separated from the patient.

[0126] <3. Integrin αvβ6>

[0127] Integrin is a protein composed of heterodimeric molecules in the native form, and the heterodimer is composed of two subunit chains, an α chain and a β chain. As the α chain, α1-α11, αv, αX, αM, αL, αD, αE, αIIb are known, and as the β chain, β1-β8 are known, and there are multiple subtypes with different combinations of them.

[0128] Integrin exists on the surface of epithelial cells and binds to laminin, fibronectin, etc., which are extracellular matrix proteins on the surface of connective tissues.

[0129] Integrin αvβ6 is composed of a heterodimeric molecule containing αv as the α chain and β6 as the β chain. Integrin αvβ6 is hardly expressed in normal tissues, but is expressed on the surface of epithelial cells during inflammatory stimulation, etc.

[0130] In the present invention, the origin of the fragment or the whole of integrin αvβ6 that immunologically binds to the autoantibody to be detected is not particularly limited, but is preferably the same species as the patient or the test animal, particularly human. The base sequence information of the genes encoding the αv chain and β6 chain of integrin in mammalian species such as human, and the amino acid sequence information of each chain can be obtained from publicly known databases (such as GenBank). In particular, the amino acid sequence of the preproprotein of human integrin αv chain subtype 1 is registered as GenBank Accession Number NP_002201.2 and is shown in SEQ ID NO: 1. The amino acid sequence of the precursor of human integrin β6 chain is registered as GenBank Accession Number NP_000879.2 and is shown in SEQ ID NO: 2. The amino acid sequence information of the αv chain and β6 chain of various mammalian species other than human can also be obtained from publicly known databases (such as GenBank). The αv chain and β6 chain with amino acid sequences formed by further undergoing post-translational modification of one or both of the αv chain and β6 chain registered in the database can form integrin αvβ6. For example, the partial sequence from the 1st to the 30th positions in the amino acid sequence of SEQ ID NO: 1 is a signal peptide sequence, and the amino acid sequence of the mature polypeptide of human integrin αv chain contains the sequence from the 31st to the 1048th positions in the amino acid sequence of SEQ ID NO: 1. Similarly, the partial sequence from the 1st to the 21st positions in the amino acid sequence of SEQ ID NO: 2 is a signal peptide sequence, and the amino acid sequence of the mature polypeptide of human integrin β6 chain contains the sequence from the 22nd to the 788th positions in the amino acid sequence of SEQ ID NO: 2.

[0131] Furthermore, unless otherwise specified, integrin αvβ6 in the present invention is not limited to the natural type containing mature or immature amino acid sequences, and can also be a variant in an equivalent form to the natural type of integrin αvβ6.

[0132] In addition, integrin αvβ6 is not limited to the form in which the α chain and β chain of the natural type or variant containing mature or immature amino acid sequences jointly contain their full lengths (i.e., the whole of integrin αvβ6), and can be in the form of a fragment of integrin αvβ6.

[0133] In addition, the autoantibody to be detected is not limited to an antibody that immunologically reacts with that composed only of the amino acid sequence of the fragment or the whole of integrin αvβ6, and can also be an antibody that immunologically binds to that in which other peptides are attached to each chain of the fragment or the whole of integrin αvβ6 (especially that attached to the C-terminal side).

[0134] As a fragment of integrin αvβ6, one in which at least one of the αv chain and the β6 chain constituting the dimer of integrin is shorter than the mature or immature native form or its variant is exemplified. Specifically, as the αv chain, an αv chain containing a partial sequence from Phe at position 31 to Val at position 992 in the amino acid sequence of the αv chain shown in SEQ ID NO: 1, and / or as the β6 chain, a β6 chain containing a partial sequence from Gly at position 22 to Asn at position 707 in the amino acid sequence of the β6 chain shown in SEQ ID NO: 2 can be exemplified as a fragment of integrin αvβ6. The fragment of integrin αvβ6 preferably forms a dimer, and more preferably has binding activity to extracellular matrix proteins such as laminin and fibronectin. The binding activity of the fragment of integrin αvβ6 to extracellular matrix proteins can be confirmed, for example, by ELISA method.

[0135] That the whole or a fragment of integrin αvβ6 forms a dimer can be confirmed, for example, as follows: when the whole or a fragment of integrin αvβ6 is subjected to SDS-PAGE in the absence of 2-mercaptoethanol, a band corresponding to the molecular weight of the dimer can be detected, and when subjected to SDS-PAGE in the presence of 2-mercaptoethanol, the band corresponding to the molecular weight of the dimer disappears.

[0136] As a commercially available integrin αvβ6, recombinant human integrin αvβ6 (R&D Systems, Minnesota, USA, product number 3817-AV) can be exemplified. This recombinant human integrin αvβ6 is a dimer composed of an αv chain consisting of a partial sequence from Phe at position 31 to Val at position 992 in the amino acid sequence of the αv chain shown in SEQ ID NO: 1, an additional linker sequence and an acidic tail sequence at its C-terminus, and a β6 chain consisting of a partial sequence from Gly at position 22 to Asn at position 707 in the amino acid sequence of the β6 chain shown in SEQ ID NO: 2, an additional linker sequence and a basic tail sequence at its C-terminus.

[0137] As a more specific embodiment of the αv chain constituting a fragment or the whole of integrin αvβ6, polypeptides selected from the following are exemplified:

[0138] (I) A polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 or a partial sequence from Phe at position 31 to Thr at position 1048 in the amino acid sequence shown in SEQ ID NO: 1,

[0139] (II) A polypeptide containing a partial sequence of the amino acid sequence shown in SEQ ID NO: 1 and functionally equivalent to the polypeptide of (I),

[0140] (III) A polypeptide containing an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or a partial sequence thereof and functionally equivalent to the polypeptide of (I), and

[0141] (IV) A polypeptide that is functionally equivalent to the polypeptide of (I), which comprises an amino acid sequence having one or more amino acids substituted, deleted, and / or added in the amino acid sequence shown in SEQ ID NO: 1 or a partial sequence thereof.

[0142] The polypeptides of the foregoing (I) to (IV) may be polypeptides that further comprise at least one, preferably an additional amino acid sequence added to the N-terminal side and the C-terminal side, in the amino acid sequence or partial sequence defined in the foregoing (I) to (IV).

[0143] In the foregoing (II), (III), and (IV), the polypeptide that is functionally equivalent to the polypeptide of (I) may form a dimer with the integrin β6 chain (particularly preferably a polypeptide chain consisting of the amino acid sequence shown in SEQ ID NO: 2, a polypeptide chain consisting of a partial sequence from Gly at position 22 to Cys at position 788 in the amino acid sequence shown in SEQ ID NO: 2, or a polypeptide chain consisting of a partial sequence from Gly at position 22 to Asn at position 707 in the amino acid sequence shown in SEQ ID NO: 2), and the formed dimer may be exemplified as a polypeptide having the ability to bind to an extracellular matrix protein that can bind to natural integrin αvβ6 such as laminin or fibronectin or commercially available integrin αvβ6.

[0144] As the partial sequence in the foregoing (II), a partial sequence from Phe at position 31 to Val at position 992 in the amino acid sequence shown in SEQ ID NO: 1 is exemplified. As the partial sequences in the foregoing (III) and (IV), a partial sequence from Phe at position 31 to Thr at position 1048 in the amino acid sequence shown in SEQ ID NO: 1 or a partial sequence from Phe at position 31 to Val at position 992 in the amino acid sequence shown in SEQ ID NO: 1 is exemplified.

[0145] The sequence identity in the foregoing (III) is preferably 90% or more, more preferably 95% or more, further preferably 96% or more, particularly preferably 97% or more, most preferably 98% or more or 99% or more.

[0146] In the foregoing (IV), "one or more" is, for example, 1 to 100, preferably 1 to 50, preferably 1 to 30, preferably 1 to 20, preferably 1 to 15, preferably 1 to 10, preferably 1 to 5, preferably 1 to 4, preferably 1 to 3, preferably 1 to 2, preferably 1.

[0147] As a more specific embodiment of the β6 chain constituting a fragment or all of integrin αvβ6, polypeptides selected from the following are exemplified:

[0148] (V) A polypeptide that comprises the amino acid sequence shown in SEQ ID NO: 2 or a partial sequence from Gly at position 22 to Cys at position 788 in the amino acid sequence shown in SEQ ID NO: 2

[0149] (VI) A partial sequence containing the amino acid sequence shown in SEQ ID NO: 2, a polypeptide functionally equivalent to the polypeptide of (V),

[0150] (VII) A polypeptide containing an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence shown in SEQ ID NO: 2 or a partial sequence thereof, a polypeptide functionally equivalent to the polypeptide of (V), and

[0151] (VIII) A polypeptide containing an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in the amino acid sequence shown in SEQ ID NO: 2 or a partial sequence thereof, a polypeptide functionally equivalent to the polypeptide of (V).

[0152] The polypeptides of the foregoing (V) to (VIII) may be polypeptides that further contain at least one on the N-terminal side and the C-terminal side, preferably an amino acid sequence in which other amino acid sequences are added to the C-terminal side, in the amino acid sequences or partial sequences defined in the foregoing (V) to (VIII).

[0153] In the foregoing (VI), (VII), and (VIII), the polypeptide functionally equivalent to the polypeptide of (V) may form a dimer with the integrin αv chain (particularly preferably a polypeptide chain consisting of the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain consisting of a partial sequence from Phe at position 31 to Thr at position 1048 in the amino acid sequence shown in SEQ ID NO: 1, or a polypeptide chain consisting of a partial sequence from Phe at position 31 to Val at position 992 in the amino acid sequence shown in SEQ ID NO: 1), and the formed dimer may be exemplified as a polypeptide having the ability to bind to an extracellular matrix protein that can bind to natural integrin αvβ6 such as laminin or fibronectin or commercially available integrin αvβ6.

[0154] As the partial sequence in the foregoing (VI), a partial sequence from Gly at position 22 to Asn at position 707 in the amino acid sequence shown in SEQ ID NO: 2 is exemplified. As the partial sequences in the foregoing (VII) and (VIII), a partial sequence from Gly at position 22 to Cys at position 788 in the amino acid sequence shown in SEQ ID NO: 2 or a partial sequence from Gly at position 22 to Asn at position 707 in the amino acid sequence shown in SEQ ID NO: 2 is exemplified.

[0155] The sequence identity in the foregoing (VII) is preferably 90% or more, more preferably 95% or more, still more preferably 96% or more, particularly preferably 97% or more, and most preferably 98% or more or 99% or more.

[0156] In the foregoing (VIII), "one or more" means, for example, 1 to 100, preferably 1 to 50, preferably 1 to 30, preferably 1 to 20, preferably 1 to 15, preferably 1 to 10, preferably 1 to 5, preferably 1 to 4, preferably 1 to 3, preferably 1 to 2, preferably 1.

[0157] In the foregoing (III) and (VII), the sequence identity of the amino acid sequence can be determined using methods and sequence analysis software well-known to those skilled in the art. Examples of sequence analysis software include, but are not limited to, the blastp program of the BLAST algorithm and the fasta program of the FASTA algorithm.

[0158] <4. Detection method for irAE enteritis>

[0159] The first detection method of the present invention

[0160] relates to a detection method for irAE enteritis, which includes:

[0161] a detection step of detecting an antibody that immunologically reacts with a fragment or all of integrin αvβ6 in a sample as an index for ulcerative colitis-like irAE enteritis.

[0162] The second detection method of the present invention

[0163] relates to a detection method for irAE enteritis, which is used to determine whether the enteritis that develops in a patient receiving ICI treatment is ulcerative colitis-like irAE enteritis, and includes:

[0164] a detection step of detecting an antibody that immunologically reacts with a fragment or all of integrin αvβ6 in a sample derived from the aforementioned patient as an index for ulcerative colitis-like irAE enteritis,

[0165] In the foregoing detection step, the antibody titer of the antibody that immunologically reacts with a fragment or all of integrin αvβ6 detected in the aforementioned sample is higher than the antibody titer of the aforementioned antibody detected in a sample from a control source, which is an index for determining that the enteritis that develops in the aforementioned patient is ulcerative colitis-like irAE enteritis.

[0166] The third detection method of the present invention

[0167] relates to a detection method for irAE enteritis, which is used to predict the risk of developing ulcerative colitis-like irAE enteritis in a patient, and includes:

[0168] a detection step of detecting an antibody that immunologically reacts with a fragment or all of integrin αvβ6 in a sample derived from the aforementioned patient as an index for ulcerative colitis-like irAE enteritis,

[0169] In the foregoing detection step, the antibody titer of the antibody that immunologically reacts with the fragment or the whole of integrin αvβ6 detected in the foregoing sample is higher than the antibody titer of the foregoing antibody detected in the sample from the control source, which is an index for determining the risk of developing ulcerative colitis-like irAE enteritis in the foregoing patient.

[0170] In this specification, hereinafter, the "fragment or the whole of integrin αvβ6" will be collectively referred to as "integrin αvβ6". In addition, the first, second, and third inspection methods of the present invention will be collectively referred to as the "inspection methods of the present invention".

[0171] The first and second inspection methods of the present invention are completed based on the following surprising findings: the presence of an antibody (autoantibody) against integrin αvβ6 in a sample obtained from a patient is an index that the patient has ulcerative colitis-like irAE enteritis.

[0172] The first and third inspection methods of the present invention are completed based on the following surprising findings: the presence of an antibody (autoantibody) against integrin αvβ6 in a sample obtained from a patient is an index that the patient has a risk of developing ulcerative colitis-like irAE enteritis. In the present invention, it is surprisingly shown that patients with ulcerative colitis-like irAE enteritis express an antibody (autoantibody) against integrin αvβ6 before receiving ICI treatment.

[0173] In the first, second, and third inspection methods of the present invention, the irAE enteritis can be "enteritis for which it is recommended to stop the administration of ICI", and preferably can be "ulcerative colitis-like enteritis for which it is recommended to stop the administration of ICI".

[0174] The sample in the inspection method of the present invention is a sample from a patient. In the second inspection method of the present invention, a sample from a patient who has received ICI treatment and developed enteritis is used. In the third inspection method of the present invention, a sample from a patient before receiving ICI treatment or a patient who has received ICI treatment and has not developed enteritis is used. In the inspection method of the present invention, a patient receiving ICI treatment can be a patient who is further receiving combined treatment of ICI and a cancer drug therapy other than ICI.

[0175] "Developing enteritis" in this specification means showing symptoms of enteritis. As symptoms of enteritis, for example, diarrhea, an increase in the number of bowel movements, abdominal pain, mucus in the stool, bloody stool, etc. are listed.

[0176] In the first or second inspection method of the present invention, a sample of a patient showing a morbid state similar to ulcerative colitis can be applied. In this case, by the inspection method of the present invention, it can be confirmed whether the morbid state similar to ulcerative colitis is irAE. In the present specification, "morbid state similar to ulcerative colitis" refers to a state showing mucosal findings similar to ulcerative colitis. For example, a state in which the mucosa continuous from the rectum to the oral side is edematous, bleeding, erosive, or ulcerous. Such a morbid state can be confirmed by endoscopy, for example. In addition, the formation of crypt abscesses in the large intestine tissue is also included in the "morbid state similar to ulcerative colitis". Crypt abscesses can be collected from the diseased tissue and confirmed by microscopy, for example.

[0177] The detection step in the inspection method of the present invention is a step of detecting an antibody immunologically reactive with integrin αvβ6 in a sample. In the present specification, "detection" is the following concept: in addition to confirming the presence of an antibody immunologically reactive with integrin αvβ6 in a sample, it also includes measuring, that is, "quantifying" the amount (antibody titer) of the antibody contained in the sample.

[0178] In the present specification, the "antibody immunologically reactive with integrin αvβ6" includes an antibody specifically binding to integrin αvβ6, and an antibody specifically binding to integrin αvβ6 and inhibiting the binding of integrin αvβ6 to extracellular matrix proteins such as laminin and fibronectin.

[0179] The detection step in the inspection method of the present invention may be any method capable of quantitatively or qualitatively detecting the aforementioned antibody in a sample, and there is no particular limitation on the specific method. For example, immunological methods can be applied. For example, antibody enzyme methods (Enzyme-Linked ImmunoSorbent Assay; ELISA method), immunoprecipitation methods (IPP method), immunoblotting methods (IB method), latex agglutination methods, immunochromatography methods, indirect fluorescent antibody methods (IF method), radioimmunoassay methods (RIA method), etc. can be listed. The ELISA method capable of processing multiple samples is particularly preferred. Specifically, in the ELISA method, by immobilizing integrin αvβ6, which is the antigen of the aforementioned antibody, on a solid phase, bringing the sample into contact with the antigen immobilized on the solid phase, and applying a detection antibody labeled with an enzyme to detect the immune complex of the antigen and the aforementioned antibody that may be contained in the sample, the aforementioned antibody can be detected. Methods for suppressing non-specific reactions accompanying the implementation of the ELISA method, labeling substances that can be used during detection, measuring instruments, etc. are well known in the art and are not particularly limited. As the solid phase for immobilizing the antigen, a solid phase of any shape such as a plate, bead, or tube can be applied.

[0180] In immunological methods such as ELISA, steps of contacting a sample that may contain the aforementioned antibody with the aforementioned antigen, necessary washing steps, necessary labeling steps, and necessary detection steps can be carried out in an appropriate buffer. Preferably, it is carried out in a buffer containing one or more metal ions selected from calcium ions, magnesium ions, manganese ions, sodium, lithium, etc. The aforementioned metal ions are preferably divalent metal ions such as calcium ions, magnesium ions, and manganese ions, and particularly preferably one or two of calcium ions, magnesium ions, and manganese ions. The concentration of the aforementioned metal ions in the buffer is not particularly limited, and examples of the total concentration can be 0.02 mM to 200 mM, preferably 0.2 mM to 20 mM, and particularly preferably 0.4 mM to 10 mM. In a buffer containing these metal ions, it is preferred due to the improved detection sensitivity.

[0181] In the detection step, the amount of the antibody that immunologically reacts with integrin αvβ6, for example, can be determined as the measured value of the labeling amount of the detection antibody that binds to the immune complex of the aforementioned antibody and the aforementioned antigen. In addition, a standard curve can be prepared using a positive standard solution containing a known concentration of the aforementioned antibody, and the amount of the aforementioned antibody in the test sample can be calculated based on the measured value of the labeling amount by applying the standard curve.

[0182] In the detection step, the determination of whether there is an antibody that immunologically reacts with integrin αvβ6 (positive / negative determination) can be carried out by comparing the measured value of the aforementioned antibody in the sample obtained from the patient with the measured value of the aforementioned antibody in the sample obtained from the control. Here, the measured value of the aforementioned antibody in the sample obtained from the control can be a value measured simultaneously with the measurement of the aforementioned antibody in the sample obtained from the patient, or a value measured in advance. In the case where the measured value of the aforementioned antibody in the sample obtained from the patient is greater than the measured value of the aforementioned antibody in the sample obtained from the control, preferably, in the case of being significantly greater, it can be judged as positive.

[0183] In addition, in the detection step, the antibody titer of the aforementioned antibody in the control-derived sample compared with the antibody titer of the aforementioned antibody in the patient-derived sample can be a value measured simultaneously with the measurement of the aforementioned antibody in the patient-derived sample, or a value measured in advance.

[0184] The "control" in this specification can be an animal of the same or different species as the patient, preferably an animal of the same species as the patient. In addition, the "control" is an animal that does not have ulcerative colitis-like irAE enteritis. For example, animals that do not show the morbid state of ulcerative colitis-like, animals that have not received ICI treatment, or animals that do not have cancer are listed. Here, animals that do not have ulcerative colitis-like irAE enteritis include animals that have never had ulcerative colitis-like irAE enteritis in the past, and animals that had ulcerative colitis-like irAE enteritis in the past but are currently cured. For example, animals that have been self-cured for several years or more, preferably ten years or more. Animals that have not received ICI treatment include animals that have never received ICI treatment in the past, and animals that received ICI treatment in the past but are not currently receiving such treatment. For example, animals that have not received ICI administration for several years or more, preferably ten years or more since the last ICI administration. Animals that do not have cancer include animals that have never had cancer in the past, and animals that had cancer in the past but are currently cured. For example, animals that have been self-cured for several years or more, preferably ten years or more. Preferably, the control is an animal that does not show the morbid state of ulcerative colitis-like and does not have cancer, more preferably a healthy animal that does not show any morbid state and has not been diagnosed as having or suspected of having any disease, and even more preferably a healthy individual.

[0185] The isotype of the antibody that immunologically reacts with integrin αvβ6 and is detected in the detection step of the detection method of the present invention is not particularly limited and can be isotypes such as IgG antibody, IgA antibody, IgM antibody, IgE antibody, etc., and IgG antibody or IgA antibody is particularly preferred. As the antibody that immunologically reacts with integrin αvβ6, it is particularly preferred to detect IgG or IgA. The subclass in the IgG antibody is not particularly limited. For example, IgG1 antibody, IgG2 antibody, IgG3 antibody, or IgG4 antibody can be detected. As the IgG antibody that immunologically reacts with integrin αvβ6, it is more preferred to detect IgG1 antibody, IgG2 antibody, or IgG4 antibody, and most preferably to detect IgG1 antibody.

[0186] According to the inspection method of the present invention, since it is possible to inspect the presence or absence of ulcerative colitis-like irAE enteritis in a patient, or the risk of onset of ulcerative colitis-like irAE enteritis in a patient, or the presence or absence of "enteritis for which it is recommended to stop the administration of ICI" in a patient, or the risk of onset of "enteritis for which it is recommended to stop the administration of ICI" in a patient, based on the inspection results, the treatment policy of the patient can be determined. For example, based on the inspection results of the second inspection method of the present invention, in the case where it is determined that the enteritis in which the patient has developed is ulcerative colitis-like irAE enteritis or "enteritis for which it is recommended to stop the administration of ICI", it is preferable to stop the administration of ICI to the patient and / or administer an immunosuppressant. For example, based on the inspection results of the third inspection method of the present invention, in the case where it is determined that the patient has a risk of onset of ulcerative colitis-like irAE enteritis or a risk of onset of "enteritis for which it is recommended to stop the administration of ICI", it is preferable to perform a treatment other than ICI treatment on the patient before ICI treatment, stop the administration of ICI to the patient during ICI treatment, and / or administer an immunosuppressant.

[0187] The cut-off value for the above determination based on the inspection results of the second or third inspection method of the present invention can be appropriately determined by those skilled in the art by methods known in the art. For example, a value obtained by adding three times the standard deviation (SD) to the average value of the inspection results (antibody titers) of the control group can be used as the cut-off value.

[0188] The inspection method according to the present invention can particularly detect severe ulcerative colitis-like irAE enteritis. Here, "severe" can be, for example, enteritis with a grade of 3 or higher based on the CTCAE (Common Terminology Criteria for Adverse Events) version 5.0 published by the Cancer Therapy Evaluation Program (CTEP) of the National Cancer Institute (NCI) in November 2017 in the United States, or enteritis with a total score of 11 or more for 4 items of the Mayo score, or enteritis with a total score of 8 or more for 3 items (Partial Mayo score) of the Mayo score. The "grade" in CTCAE indicates the severity of adverse events. Grade 1 is defined as "mild", grade 2 is defined as "moderate", grade 3 is defined as "severe", grade 4 is defined as "life-threatening", and grade 5 is defined as "death". The Mayo score is an established evaluation tool used in clinical trials to measure the severity of ulcerative colitis. It evaluates the total score of 4 items: the number of bowel movements checked (normal number: 0 points, 1 - 2 times more than normal per day: 1 point, 3 - 4 times more than normal per day: 2 points, 5 times more than normal per day: 3 points), bloody stools (no bloody stools: 0 points, small amount of blood attached in less than half of the bowel movements: 1 point, obvious blood mixed in most of the bowel movements: 2 points, mostly blood: 3 points), mucosal findings (normal or non-active findings: 0 points, mild: 1 point, moderate: 2 points, severe: 3 points), and the overall evaluation by a doctor (normal: 0 points, mild: 1 point, moderate: 2 points, severe: 3 points). A Mayo score with a total score of 3 - 5 points is evaluated as "mild", 6 - 10 points as "moderate", and 11 - 12 points as "severe". In addition, in the case of the Partial Mayo score, it is evaluated by the total score of 3 items: the number of bowel movements checked, bloody stools, and the overall evaluation by a doctor. A Partial Mayo score with a total score of 2 - 4 points is evaluated as "mild", 5 - 7 points as "moderate", and 8 - 9 points as "severe". According to the present invention, it is found that there are many severe cases of enteritis among anti-integrin αvβ6 antibody-positive cases.

[0189] <5. Inspection Kit or Inspection Reagent for irAE Enteritis>

[0190] The inspection kit or inspection reagent of the present invention first relates to an inspection kit or inspection reagent for inspecting irAE enteritis, which contains a fragment or all of integrin αvβ6.

[0191] The second inspection kit or inspection reagent of the present invention relates to an inspection kit or inspection reagent for determining whether the enteritis that develops in a patient receiving ICI treatment is ulcerative colitis-like irAE enteritis, and contains a fragment or all of integrin αvβ6.

[0192] The third inspection kit or inspection reagent of the present invention relates to an inspection kit or inspection reagent for predicting the risk of developing ulcerative colitis-like irAE enteritis in a patient receiving ICI treatment, and contains a fragment or all of integrin αvβ6.

[0193] In this specification, hereinafter, the first, second, and third inspection kits or inspection reagents of the present invention described above are collectively referred to as the "inspection kit or inspection reagent of the present invention".

[0194] In the first, second, and third inspection kits or inspection reagents of the present invention, the irAE enteritis can be "enteritis for which it is recommended to stop the administration of ICI", and preferably can be "ulcerative colitis-like enteritis for which it is recommended to stop the administration of ICI".

[0195] The inspection kit or inspection reagent of the present invention can be used in the inspection method of the present invention.

[0196] As needed, the inspection kit of the present invention can contain reagents required for immunological assays, such as buffers, one or more metal salts selected from calcium salts, magnesium salts, manganese salts, sodium salts, lithium salts, etc.

[0197] The inspection reagent of the present invention can be a liquid or solid composition, and contains integrin αvβ6 and a carrier such as a solvent and an excipient that are permitted for formulation purposes.

[0198] In the inspection kit or inspection reagent of the present invention, the form of integrin αvβ6 is not particularly limited, and can be in a solution state, a dry state, or a form immobilized on a solid phase. In the case where integrin αvβ6 is in a dry state, a buffer or solvent that is in a solution state before use can be included in the inspection kit or inspection reagent of the present invention.

[0199] An inspection kit or inspection reagent containing integrin αvβ6 in a form fixed to a solid phase can be applied in the ELISA method. As the form of the solid phase, it can be any form such as a plate, beads, a tube, etc. In addition to the aforementioned solid phase, the inspection kit for application in the ELISA method may further contain a positive standard solution, a negative standard solution, a blocking solution, a washing solution, a sample diluent, a detection antibody, a substrate solution, etc. The detection antibody can be labeled with a detectable label such as an enzyme. In the positive standard solution, in addition to the serum diluent of a patient with ulcerative colitis-like irAE enteritis containing autoantibodies against integrin αvβ6 itself, a solution containing an antibody against integrin αvβ6 is added. As the antibody added to this solution, an antibody of the same isotype as the autoantibody to be measured, such as IgG antibody, IgA antibody, IgM antibody, IgE antibody, etc., is preferably used. The negative standard solution uses a serum diluent of an animal (such as a human) not suffering from ulcerative colitis-like irAE enteritis, the serum of a patient with other intestinal diseases that are medically distinguishable from ulcerative colitis-like irAE enteritis, the serum of a healthy person (control serum), and its diluent, etc.

[0200] <6. Method for treating ulcerative colitis-like irAE enteritis>

[0201] The first treatment method of the present invention

[0202] relates to a method for treating ulcerative colitis-like irAE enteritis, which includes:

[0203] the step of detecting an antibody that immunologically reacts with a fragment or the whole of integrin αvβ6 in a sample derived from a patient who has received ICI treatment and developed enteritis,

[0204] In the case where the antibody titer of the aforementioned antibody detected in the sample derived from the aforementioned patient is higher than the antibody titer of the aforementioned antibody detected in the sample derived from the control source, for the aforementioned patient, the step of stopping ICI treatment and / or administering an immunosuppressant.

[0205] The second treatment method of the present invention

[0206] relates to a method for treating enteritis that develops in a patient receiving combined treatment of ICI and a cancer drug therapy other than ICI, which includes:

[0207] the step of detecting an antibody that immunologically reacts with a fragment or the whole of integrin αvβ6 in the sample derived from the aforementioned patient,

[0208] In the case where the antibody titer of the aforementioned antibody detected in the sample derived from the aforementioned patient is higher than the antibody titer of the aforementioned antibody detected in the sample derived from the control source, for the aforementioned patient, the step of stopping the administration of ICI and / or administering an immunosuppressant, or

[0209] In a case where the antibody titer of the aforementioned antibody detected in the aforementioned patient-derived sample is the same or lower than the antibody titer of the aforementioned antibody detected in the control-derived sample, or in a case where the aforementioned antibody is not detected in the aforementioned patient-derived sample, the step of stopping cancer drug therapy other than ICI for the aforementioned patient.

[0210] In the present specification, hereinafter, the first and second treatment methods of the present invention described above are collectively referred to as "the treatment method of the present invention".

[0211] In the first treatment method of the present invention, ulcerative colitis-like irAE enteritis may be "ulcerative colitis-like enteritis for which discontinuation of ICI administration is recommended".

[0212] In the treatment method of the present invention, in a case where the antibody titer of the aforementioned antibody detected in the patient-derived sample is higher than the antibody titer of the aforementioned antibody detected in the control-derived sample, it is preferable to stop ICI treatment for the patient and administer an immunosuppressant.

[0213] As the "immunosuppressant" used in the treatment method of the present invention, as long as it has the effect of suppressing the inflammatory reaction accompanying excessive immune response occurring in the body, there is no particular limitation. For example, steroids (e.g., prednisolone, Rinderon, dexamethasone, Saxizon, Medrol, etc.), anti-TNFα antibodies (e.g., infliximab, adalimumab, golimumab, etc.), tacrolimus, cyclosporine, mycophenolate mofetil, etc. may be mentioned, and two or more immunosuppressants may also be used.

[0214] As described above, the antibody that immunologically reacts with integrin αvβ6 in the sample is useful as an index for ulcerative colitis-like irAE enteritis. Therefore, in a case where the antibody titer of the aforementioned antibody detected in the patient-derived sample is higher than the antibody titer of the aforementioned antibody detected in the control-derived sample, treatment of irAE, that is, discontinuation of ICI and / or administration of an immunosuppressant, is carried out. In a case where the aforementioned antibody is not detected in the patient-derived sample, or in a case where the antibody titer of the aforementioned antibody detected in the patient-derived sample is the same or lower than the antibody titer of the aforementioned antibody detected in the control-derived sample, treatment for general enteritis (i.e., enteritis that is not irAE) is carried out. Treatment for general enteritis, for example, includes administration of a gastrointestinal regulator, fasting as needed, or administration of an antibiotic in a case where bacterial infection is suspected, etc., and can be appropriately determined by those skilled in the art based on the symptoms of enteritis or the course of treatment. For example, in a case where no recovery is seen, after discontinuing ICI, a low dose of an immunosuppressant may also be administered empirically.

[0215] In the treatment method of the present invention, the step of detecting the aforementioned antibody, the method of comparing the antibody titer, etc. are the same as those described in the inspection method regarding the present invention.

[0216] <7. Method for evaluating the effect of treating ulcerative colitis-like irAE enteritis>

[0217] The method for evaluating the treatment effect of the present invention, firstly,

[0218] relates to a method for evaluating the effect of treating ulcerative colitis-like irAE enteritis, which includes:

[0219] a detection step of detecting an antibody that immunologically reacts with a fragment or the whole of integrin αvβ6 in a sample obtained from a patient who has undergone treatment for ulcerative colitis-like irAE enteritis.

[0220] As described above, the antibody in the sample that immunologically reacts with integrin αvβ6 is useful as an index for ulcerative colitis-like irAE enteritis. Therefore, the amount of the aforementioned antibody in the sample obtained from a patient who has undergone treatment for ulcerative colitis-like irAE enteritis decreases in the case where the treatment is effective, and does not change or increases from before the start of the treatment in the case where the treatment does not fully exert its effect. Therefore, by detecting the aforementioned antibody in the sample obtained from a patient who has undergone treatment for ulcerative colitis-like irAE enteritis, the effect of the treatment can be evaluated.

[0221] Compare the quantitative result of the aforementioned antibody in the sample obtained from a patient who has undergone treatment for ulcerative colitis-like irAE enteritis in the detection step of the method for evaluating the treatment effect of the present invention with, for example, the quantitative result of the aforementioned antibody in the sample obtained from the same individual before the start of the aforementioned treatment. In the case where the former is smaller than the latter, it can be evaluated that the treatment is effective. On the other hand, in the case where the former is the same as or larger than the latter, it can be evaluated that the treatment is ineffective or insufficient. Based on this evaluation result, it is possible to judge whether to continue the treatment, change the treatment policy, discontinue the treatment, etc.

[0222] In addition, compare the quantitative result of the aforementioned antibody in the sample obtained from a patient who has undergone treatment for ulcerative colitis-like irAE enteritis in the detection step of the method for evaluating the treatment effect of the present invention with, for example, the quantitative result of the aforementioned antibody in the sample obtained from a healthy individual of the same animal species. In the case where the former is the same as or smaller than the latter, it can be evaluated that the treatment is effective. On the other hand, in the case where the former is larger than the latter, it can be evaluated that the treatment is ineffective or insufficient. Based on this evaluation result, it is possible to judge whether to continue the treatment, change the treatment policy, discontinue the treatment, etc.

[0223] In the evaluation method of the present invention, the detection step of the aforementioned antibody, etc. is the same as that described in the inspection method of the present invention.

[0224] In addition, in the evaluation method of the present invention, ulcerative colitis-like irAE enteritis may be "ulcerative colitis-like enteritis for which the administration of ICI is recommended to be stopped".

[0225] <8. Method for screening candidate substances for therapeutic agents for ulcerative colitis-like irAE enteritis>

[0226] The first screening method of the present invention,

[0227] relates to a method for screening candidate substances for therapeutic agents for ulcerative colitis-like irAE enteritis, which includes:

[0228] an antibody detection step of detecting an antibody that immunologically reacts with a fragment or all of integrin αvβ6 in a sample obtained from an animal to which a test substance has been administered, and

[0229] a selection step of selecting the test substance as a candidate substance for a therapeutic agent for ulcerative colitis when the antibody in the sample is reduced by the action of the test substance.

[0230] Here, the above-mentioned animal is an animal suffering from ulcerative colitis-like irAE enteritis, preferably a mammal, more preferably a non-human mammal.

[0231] As described above, an antibody that immunologically reacts with a fragment or all of integrin αvβ6 in the sample is useful as an index for ulcerative colitis-like irAE enteritis. Therefore, when the antibody in the sample is reduced by the action of the test substance, the test substance can be selected as a candidate substance for a therapeutic agent for ulcerative colitis-like irAE enteritis.

[0232] In the screening method of the present invention, the antibody detection step and the like are the same as those described in the inspection method of the present invention.

[0233] In the screening method of the present invention, the test substance is a test substance that may be a candidate for a new drug, and there is no particular limitation.

[0234] In the selection step of the screening method of the present invention, the quantitative result of the antibody in the sample obtained from the animal to which the test substance has been administered in the antibody detection step is compared with, for example, the quantitative result of the antibody in the sample obtained from the same individual before the test substance is administered. When the former is smaller than the latter, it is determined that the antibody in the sample is reduced by the action of the test substance, and the test substance can be selected as a candidate substance for a therapeutic agent for ulcerative colitis-like irAE enteritis.

[0235] In addition, in the selection step of the screening method of the present invention, the quantitative result of the aforementioned antibody in the sample obtained from the animal to which the test substance has been applied in the antibody detection step is compared with, for example, the quantitative result of the aforementioned antibody in the sample obtained from a healthy individual of the same animal species. In the case where the former is equal to or smaller than the latter, it is determined that the test substance can be selected as a candidate substance for a therapeutic agent for ulcerative colitis-like irAE enteritis by reducing the aforementioned antibody in the aforementioned sample by the action of the aforementioned test substance.

[0236] In addition, in the screening method of the present invention, ulcerative colitis-like irAE enteritis may be "ulcerative colitis-like enteritis for which it is recommended to discontinue the administration of ICI".

[0237] Examples

[0238] Hereinafter, the present invention will be described in further detail with reference to application examples, but the present invention is not limited to these examples.

[0239] <Example 1: Diagnosis of ulcerative colitis-like irAE enteritis based on anti-integrin αvβ6 antibody - 1>

[0240] As an antigen, human integrin αvβ6 (3817-AV, R&D system) was immobilized on a solid phase, and it was confirmed by ELISA whether the sera of patients with ulcerative colitis-like irAE enteritis and patients without ulcerative colitis-like irAE enteritis contained antibodies that bind to the aforementioned antigen. In this ELISA method, autoantibodies with human IgG as the detection target were used, and in order to detect the antibodies in the patient sera that bind to the antigen immobilized on the solid phase, a rabbit anti-human IgG polyclonal antibody labeled with HRP (horseradish peroxidase) was used as the detection antibody.

[0241] 1. Serum specimens

[0242] Serum specimens were obtained from 9 patients diagnosed with ulcerative colitis-like irAE enteritis by endoscopic examination. As a control, serum specimens were obtained from 13 patients diagnosed with non-ulcerative colitis-like irAE enteritis patients by endoscopic examination.

[0243] 2. Procedure

[0244] In the following experiments, unless otherwise specified, ELISA Starter Accessory Kit (E101, Bethyl Laboratories) was used. ELISA Coating Buffer, ELISA Wash Solution, ELISA Blocking Buffer, and Conjugate Diluent were all prepared according to the instructions of the aforementioned kit. However, in ELISA Wash Solution, ELISA Blocking Buffer, and Conjugate Diluent, CaCl 2 and MgCl 2 were added at a final concentration of 1 mM each. All steps were carried out at room temperature unless otherwise specified.

[0245] 2.1. Coating with Antigen

[0246] (1) The above antigen was diluted in the ELISA Coating Buffer of the aforementioned kit to form a 2 μg / ml concentration solution. 100 μl of this solution was added to each well of the microplate of the aforementioned kit.

[0247] (2) The above microplate was incubated at 4°C for 60 minutes.

[0248] (3) After incubation, the solution was aspirated and removed from each well.

[0249] (4) Each well was washed with the ELISA Wash Solution of the aforementioned kit. Specifically, the ELISA Wash Solution was filled in each well, and then the washing operation of aspirating and removing the ELISA Wash Solution was carried out 3 times.

[0250] 2.2. Blocking

[0251] (1) 200 μl of the ELISA Blocking Buffer of the aforementioned kit was added to each well.

[0252] (2) Incubate for 30 minutes.

[0253] (3) After incubation, after removing the ELISA Blocking Buffer, each well was washed 3 times.

[0254] 2.3. Test Specimen

[0255] (1) The serum specimens of ulcerative colitis-like irAE enteritis patients (n = 9) or non-ulcerative colitis-like irAE enteritis patients (n = 13) were diluted at a ratio of 1:100 with the Conjugate Diluent of the aforementioned kit.

[0256] (2) Add 100 μl of the serum diluent obtained by the aforementioned dilution to each well of the blocked microplate.

[0257] (3) Incubate for 60 minutes.

[0258] (4) After incubation, remove the aforementioned serum diluent and wash each well 5 times.

[0259] 2.4. Antibody for HRP Conjugate Detection

[0260] (1) Dilute the detection antibody conjugated with HRP (abcam6759, rabbit anti-human IgG H&L(HRP)) at a ratio of 1:50,000 with the conjugate diluent of the aforementioned kit.

[0261] (2) Add 100 μl of the detection antibody diluent obtained by the aforementioned dilution to each well of the microplate in contact with the serum.

[0262] (3) Incubate for 60 minutes.

[0263] (4) After incubation, remove the aforementioned detection antibody diluent and wash each well 5 times.

[0264] 2.5. Enzyme Substrate Reaction

[0265] (1) Prepare the TMB (3,3’,5,5’-tetramethylbenzidine) solution according to the recommended conditions of the manufacturer.

[0266] (2) Add 100 μl of the aforementioned TMB solution to each well of the microplate in contact with the detection antibody.

[0267] (3) Incubate for 7 to 8 minutes.

[0268] (4) To stop the oxidation of TMB, add 100 μl of 0.18 M H 2 SO 4 to each well.

[0269] (5) Use an enzyme-linked immunosorbent assay reader to measure the color development caused by the product of the aforementioned oxidation reaction at a wavelength of 450 nm.

[0270] 3. Results and Discussion

[0271] The results are shown in Figure 1 . Figure 1The absorbance at 450 nm (OD value) proportional to the amount of detection antibody on the solid phase is shown on the vertical axis. "UC-like irAE colitis" represents the serum sample of a patient with ulcerative colitis-like irAE enteritis, and "UC-unlike irAE colitis" represents the serum sample of a patient with non-ulcerative colitis-like irAE enteritis (control). The cut-off value indicated by the dotted line is the value of the average absorbance of the control serum sample plus 3 times the standard deviation (SD). As a control, the serum sample of a healthy person was used.

[0272] The anti-human integrin αvβ6 antibody was positive in 6 out of 9 cases of the serum samples of patients with ulcerative colitis-like irAE enteritis, and the sensitivity was 66.6%. On the other hand, it was negative in all 13 cases of the serum samples of patients with non-ulcerative colitis-like irAE enteritis, and the specificity was 100% ( Figure 1 ). The sensitivity and specificity were calculated using the following formulas.

[0273] Sensitivity (%) = [Number of patients diagnosed as positive for ulcerative colitis-like irAE enteritis based on antibody titer / Total number of patients with ulcerative colitis-like irAE enteritis examined] × 100

[0274] Specificity (%) = [Number of patients diagnosed as negative for ulcerative colitis-like irAE enteritis based on antibody titer / Total number of patients with non-ulcerative colitis-like irAE enteritis examined] × 100

[0275] This result confirmed that an increase in the serum concentration of the autoantibody against human integrin αvβ6 is a diagnostic indicator for ulcerative colitis-like irAE enteritis, and ulcerative colitis-like irAE enteritis can be diagnosed based on the blood concentration of the autoantibody against human integrin αvβ6.

[0276] <Example 2: Correlation between anti-integrin αvβ6 antibody titer and clinical image - 1>

[0277] In patients diagnosed with ulcerative colitis-like irAE enteritis, the serum concentration of the anti-integrin αvβ6 antibody was measured over time and the intestinal clinical images were taken, and it was confirmed that the anti-integrin αvβ6 antibody titer was correlated with the clinical image.

[0278] The patient was a 57-year-old female with peritoneal cancer. From October 12, 2017 to January 23, 2018, she received ICI treatment administered through atezolizumab. On February 27, 2018, ulcerative colitis-like irAE enteritis occurred. After the onset, discontinuing atezolizumab did not improve the condition, and additional treatment with prednisone 30 mg / day was started orally since May 29, 2018. The administered dose was gradually reduced, and the administration ended on October 1, 2018. Serum samples were obtained from the patient on May 7, 2018, September 10, 2018, October 7, 2019, December 7, 2020, and December 13, 2021. The anti-integrin αvβ6 antibody titer in the samples was measured by ELISA as described in Example 1. In addition, lower gastrointestinal endoscopy was performed on April 27, 2018, September 7, 2018, and October 7, 2019. Further, based on the findings three days before and after May 7, 2018, September 10, 2018, October 7, 2019, December 7, 2020, and December 13, 2021, the patient was evaluated using the Mayo score. The examination items of the Mayo score are the number of bowel movements, bloody stools, mucosal findings, and overall evaluation by the physician, a total of four items (Table 1). The mucosal findings by endoscopy were evaluated using the data of one day, and the remaining three items were evaluated based on the data of three days. The Mayo score is the sum of the scores of the above four items, and the higher the score, the more severe the symptoms.

[0279] [Table 1]

[0280]

[0281] The results are shown in Figures 2 - 5 . Figure 2 Shows the time-course changes in the antibody titer in the patient's serum sample and the Mayo score, which is an evaluation tool for the severity of ulcerative colitis. The left vertical axis shows the absorbance at 450 nm proportional to the amount of detection antibody on the solid phase, and the right vertical axis shows the Mayo score. The horizontal axis shows the date. Figure 2 The cut-off value of the antibody titer shown by the dotted line in Figure 3 、 Figure 4 and Figure 5 are lower gastrointestinal endoscopy photos taken on April 27, 2018, September 7, 2018, and October 7, 2019, respectively.

[0282] The antibody titer of the anti-integrin αvβ6 antibody in the patient's serum sample was 1.86 on May 7, 2018, 0.74 on September 10, 2018, and 0.18 on October 7, 2019. As per Figure 2It is known that the antibody titer is high at the onset of ulcerative colitis-like irAE enteritis, but the antibody titer decreases with ICI discontinuation and steroid treatment. In addition, as described in Figure 3 , Figure 4 , and Figure 5 It is known that the colon endoscopy images at the time of high antibody titer show inflammatory findings such as redness, erosion, and ulcers from the ascending colon through the sigmoid colon and rectum. However, as the antibody titer decreases, the inflammatory findings improve, and when the antibody titer is below the cut-off value, almost no inflammatory findings are seen. In addition, the Mayo Score also improves as the antibody titer decreases. Thus, it is shown that the antibody titer of anti-integrin αvβ6 antibody is correlated with the clinical picture. Therefore, the amount of the antibody titer of anti-integrin αvβ6 antibody can be used as an index for the degree of development of ulcerative colitis-like irAE enteritis and the severity of ulcerative colitis-like irAE enteritis. In addition, it is known that by observing the time-course change of the antibody titer of anti-integrin αvβ6 antibody, it can also be used in the evaluation of the treatment effect of ulcerative colitis-like irAE enteritis.

[0283] <Example 3: Correlation between anti-integrin αvβ6 antibody titer and clinical picture - 2>

[0284] The case was an 82-year-old male patient with non-small cell carcinoma who received ICI treatment with pembrolizumab since October 2021, and developed ulcerative colitis-like irAE enteritis on February 28, 2022. The symptoms improved spontaneously only by discontinuing pembrolizumab on the same day. Serum samples were obtained from the patient on April 11, 2022, April 25, 2022, and July 21, 2022, and the anti-integrin αvβ6 antibody titer in the samples was measured by ELISA as described in Example 1. In addition, lower gastrointestinal endoscopy was performed on March 30, 2022. Further, based on the findings for the three days before and after April 11, 2022, April 25, 2022, and July 21, 2022, the patient was evaluated using the Partial Mayo score. The Partial Mayo score is evaluated by three items: the number of bowel movements, bloody stools, and overall evaluation by the physician, excluding the endoscopic findings (mucosal findings) (Table 1).

[0285] The results are shown in Figure 6 . Figure 6 The figure in shows the time-course changes of the antibody titer in the patient's serum sample and the Partial Mayo score. The left vertical axis shows the absorbance at 450 nm proportional to the amount of the detection antibody on the solid phase, and the right vertical axis shows the Partial Mayo score. The horizontal axis shows the date. Figure 6 The cut-off value of the antibody titer indicated by the dotted line in is the average of the measured values of the healthy population + 3SD.

[0286] As described according to Figure 6 It is known that endoscopic images at the onset of irAE enteritis show inflammatory findings in the rectum, and the antibody titer is also a high value of 0.85. However, the antibody titer decreases as the symptoms improve thereafter. In addition, endoscopic examination cannot be performed after the first time, but the Partial Mayo score based on endoscopic findings shows a tendency to improve.

[0287] <Example 4: Anti-integrin αvβ6 antibody titer before the onset of ulcerative colitis-like irAE enteritis>

[0288] In cancer patients, the antibody titers of anti-integrin αvβ6 antibody in blood were measured before the start of ICI treatment, after the start of ICI treatment, and after the onset of ulcerative colitis-like irAE enteritis. The antibody titers were measured by the same method as in Example 1.

[0289] The patient was a 69-year-old female with malignant melanoma. Resection surgery was performed in May 2018, but recurrence was seen in December 2019, and reoperation was performed in January 2020. ICI treatment was started in March 2020. Nivolumab was administered in March 2020, and nivolumab and ipilimumab were administered on June 8, 2020. On June 15, 2020, the onset of ulcerative colitis-like irAE enteritis was confirmed by endoscopic examination. Serum samples were obtained from the patient on February 17, 2020 before the start of ICI treatment, on June 1, 2020 after the start of ICI treatment and before the onset of irAE, and on June 29, July 13, and July 20, 2020 after the onset of irAE, and the antibody titers of anti-integrin αvβ6 antibody were measured.

[0290] The results are shown in Figure 7 . Figure 7 The figure in shows the time-course change of the antibody titer in the patient's serum sample. The vertical axis shows the absorbance at 450 nm proportional to the amount of the detection antibody on the solid phase, and the horizontal axis shows the date. The points in the figure respectively show the dates of February 17, 2020, June 1, 2020, June 29, 2020, July 13, 2020, and July 20, 2020, which are the dates of serum sample collection. Figure 7 The endoscopic examination image in is the one at the time of confirmation of the onset of ulcerative colitis-like irAE enteritis (June 15, 2020). Figure 7 The cut-off value of the antibody titer indicated by the dotted line in is the average of the measured values of the healthy population + 3SD.

[0291] As described according to Figure 7It is known that patients who will develop ulcerative colitis-like irAE enteritis have high antibody titers of anti-integrin αvβ6 antibody even before the start of ICI treatment. Therefore, anti-integrin αvβ6 antibody is useful as an indicator of the risk of developing ulcerative colitis-like irAE enteritis in the future at the time before the start of ICI treatment or before the onset of enteritis.

[0292] <Example 5: Diagnosis of ulcerative colitis-like irAE enteritis based on anti-integrin αvβ6 antibody - 2>

[0293] (1)Antibody test

[0294] As an antigen, human integrin αvβ6 (3817-AV, R&D system) was immobilized on a solid phase, and it was confirmed by the same ELISA method as in Example 1 whether the serum of irAE enteritis patients contained antibodies that bind to the aforementioned antigen. As a control, serum samples from other irAE patients (liver damage, endocrine damage, lung damage, others), cancer patients without irAE, and healthy individuals were used for the same test.

[0295] The results are shown in Figure 8 . Figure 8 The vertical axis in shows the absorbance at 450 nm proportional to the amount of detection antibody on the solid phase. The cut-off value indicated by the dotted line is the value of the average of the absorbance of the healthy individual serum sample plus three times the standard deviation (SD).

[0296] Anti-human integrin αvβ6 antibody was positive in 8 out of 26 serum samples of irAE enteritis patients, and the sensitivity was 30.8%. On the other hand, 154 out of 157 control serum samples were negative, and the specificity was 98.1% ( Figure 8 ). The sensitivity and specificity were calculated using the following formulas.

[0297] Sensitivity (%) = [Number of patients determined to be positive for irAE enteritis based on antibody titer / Total number of irAE enteritis patients examined] × 100

[0298] Specificity (%) = [Number of patients determined to be negative for irAE enteritis based on antibody titer / Total number of non-irAE enteritis patients examined] × 100

[0299] (2)Endoscopic image examination - 1

[0300] Furthermore, the endoscopic images of irAE enteritis patients were verified. As a result, in antibody-positive cases, findings similar to ulcerative colitis, such as multiple diffuse / continuous redness, granular mucosa, and disappearance of the vascular perspective image, were observed. On the other hand, in antibody-negative cases, these findings were absent ( Figure 9 ).

[0301] (3)Endoscopic image examination - 2

[0302] Next, for a more objective evaluation, the antibody titer was evaluated in a blinded state by two IBD specialist doctors to determine which of the following six characteristic items of ulcerative colitis were met by each case, and the average of the two evaluations was calculated.

[0303] 1. Diffuse / continuous erythema

[0304] 2. Continuous lesions starting from the rectum

[0305] 3. Granular mucosa

[0306] 4. Disappearance of the vascular pattern

[0307] 5. Bleed easily

[0308] 6. Ulcers

[0309] The results are shown in Figure 10 . In all 8 antibody-positive cases, more than 3 characteristic ulcerative colitis findings were seen. On the other hand, in the antibody-negative cases, only 4 out of 18 cases had more than 3 characteristic ulcerative colitis findings. Therefore, there was a significant difference between the antibody-positive and negative cases, and multiple characteristic findings were present in ulcerative colitis.

[0310] These results confirm that an increase in the serum concentration of autoantibodies against human integrin αvβ6 is a diagnostic marker for ulcerative colitis-like irAE enteritis, and ulcerative colitis-like irAE enteritis can be diagnosed based on the blood concentration of autoantibodies against human integrin αvβ6.

[0311] <Example 6: Correlation between anti-integrin αvβ6 antibody titer and clinical picture - 3>

[0312] The case was a 69-year-old female with renal cancer who received ICI treatment with pembrolizumab starting from March 18, 2019, and developed ulcerative colitis-like irAE enteritis on November 1, 2020. The antibody titer decreased after ICI discontinuation and steroid treatment. Serum samples were obtained from the patient on December 16, 2020, and September 15, 2022, and the anti-integrin αvβ6 antibody titer in the samples was measured by ELISA as described in Example 1. The patient was evaluated using the Partial Mayo score. The Partial Mayo score is evaluated based on three items: the number of bowel movements, bloody stools, and overall evaluation by the physician (Table 1), excluding endoscopic findings.

[0313] The results are shown in Figure 11 . Figure 11The figure shows the time-course changes in the antibody titer and the Partial Mayo score in the patient serum sample. The left vertical axis shows the absorbance at 450 nm proportional to the amount of detection antibody on the solid phase, and the right vertical axis shows the Partial Mayo score. The horizontal axis shows the date. Figure 11 The cut-off value of the antibody titer represented by the dashed line in [reference] is the average of the measured values of the healthy population + 3SD.

[0314] As shown according to Figure 11 It is known that in the endoscopic image at the onset of irAE enteritis, inflammatory findings are seen in the rectum, and the antibody titer is also a high value of 1.08. However, the antibody titer decreases as the symptoms improve thereafter. The Partial Mayo score also shows a decreasing trend, similar to the antibody titer.

[0315] <Example 7: Evaluation of the severity of irAE colitis>

[0316] The clinical characteristics of 26 irAE enteritis patients (8 antibody-positive and 18 antibody-negative) who underwent antibody examination in Example 5 were compared. These irAE enteritis patients are cancer patients treated with immune checkpoint inhibitors (ICI). After the onset of enteritis, ICI administration was stopped, and they received any one of conservative adjunctive treatments (only ICI withdrawal), steroid administration, or steroid and anti-TNFα antibody (infliximab) administration. Regarding the severity of adverse events including enteritis, it was evaluated based on the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 published by the Cancer Therapy Evaluation Program (CTEP) of the National Cancer Institute (NCI) in the United States in November 2017. The Japanese translation "CTCAEv5.0 - JCOG" of CTCAE by the Japan Clinical Oncology Group (JCOG) is shown in Table 2. The results are summarized in Table 3.

[0317] [Table 2]

[0318]

[0319] [Table 3]

[0320]

[0321] As can be seen from Table 3, multiple severe enteritis cases (enteritis with a CTCAE grade of 3 or higher) were seen in the antibody-positive cases, 6 out of 8 cases (75%) in the positive cases and 1 out of 18 cases (5.6%) in the negative cases.

[0322] In addition, regarding the treatment method, only in the antibody-positive cases, among 4 out of 8 cases that did not improve with steroid treatment alone, infliximab was additionally administered. All of these 4 cases had severe colitis with a disease grade of 3 or higher, and the symptoms of colitis improved with this additional administration. In the antibody-negative cases, infliximab was not administered to any of the 18 cases. That is, in the antibody-negative cases, since the symptoms of colitis improved with single-dose steroid administration or conservative adjunctive treatment, additional administration of infliximab was not required.

[0323] Based on the above, in the case of anti-integrin αvβ6 antibody being positive in irAE colitis, it is inclined to judge that the colitis is severe and difficult as a treatment method. It is shown that measuring this antibody may be useful in the treatment management of irAE enteritis.

Claims

1. A method for detecting irAE enteritis, which comprises: A detection step of detecting, in a sample, an antibody that immunologically reacts with a fragment or the whole of integrin αvβ6 as an index of ulcerative colitis-like irAE enteritis.

2. The method according to claim 1, wherein, The detection step includes a process of using a fragment or the whole of integrin αvβ6 as an antigen to detect an antibody that immunologically reacts therewith.

3. The method according to claim 1 or 2, wherein, The sample is a blood sample.

4. The method according to any one of claims 1 to 3, wherein, The index is an index of severe ulcerative colitis-like irAE enteritis.

5. A detection kit for detecting irAE enteritis, which comprises a fragment or the whole of integrin αvβ6.

6. The detection kit according to claim 5, which further comprises a detection antibody.

7. The detection kit according to claim 5 or 6, which further comprises a positive standard solution and / or a negative standard solution.

8. The detection kit according to any one of claims 5 to 7, wherein, The fragment or the whole of integrin αvβ6 is included in a form immobilized on a solid phase.

9. The method according to any one of claims 1 to 4, which is used to determine whether the enteritis that develops in a patient receiving immune checkpoint inhibitor (ICI) treatment is ulcerative colitis-like irAE enteritis, wherein, In the detection step, the sample is a sample from a patient receiving ICI treatment, and the antibody titer of the antibody that immunologically reacts with a fragment or the whole of integrin αvβ6 detected in the sample is higher than the antibody titer of the antibody detected in a control-source sample, which is an index for determining that the enteritis that develops in the patient is ulcerative colitis-like irAE enteritis.

10. The method according to claim 9, wherein, The patient is a patient receiving combined treatment of ICI and a cancer drug therapy other than ICI.

11. The method according to any one of claims 1 to 4, which is used to predict the risk of developing ulcerative colitis-like irAE enteritis in a patient, wherein, In the detection step, the sample is a sample from a patient, and the antibody titer of the antibody that immunologically reacts with a fragment or the whole of integrin αvβ6 detected in the sample is higher than the antibody titer of the antibody detected in a control-source sample, which is an index for determining that the patient has a risk of developing ulcerative colitis-like irAE enteritis.

12. The method according to claim 11, wherein, The patient is a patient before ICI treatment or a patient receiving ICI treatment.

13. A method for detecting irAE enteritis, which comprises: A detection step of detecting, in a sample, an antibody that immunologically reacts with a fragment or the whole of integrin αvβ6 as an index of enteritis for which it is recommended to stop the administration of ICI.

14. The method according to claim 13, wherein, The index is an index of enteritis for which it is recommended to stop the administration of ICI and to administer one or more immunosuppressants.

15. The method according to claim 13 or 14, which is used to determine whether the enteritis that occurs in a patient receiving ICI treatment is the enteritis for which the administration of ICI is recommended to be stopped, wherein, in the detection step, the sample is a sample from a patient receiving ICI treatment, and the antibody titer of the antibody that immunologically reacts with a fragment or all of integrin αvβ6 detected in the sample is higher than the antibody titer of the antibody detected in a sample from a control source, which is an index for determining that the enteritis that occurs in the patient is the enteritis for which the administration of ICI is recommended to be stopped.

16. The method according to claim 13 or 14, which is used to predict the risk of enteritis for which the administration of ICI is recommended to be stopped in a patient, wherein, in the detection step, the sample is a sample from a patient, and the antibody titer of the antibody that immunologically reacts with a fragment or all of integrin αvβ6 detected in the sample is higher than the antibody titer of the antibody detected in a sample from a control source, which is an index for determining the risk of enteritis for which the administration of ICI is recommended to be stopped in the patient.

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