Targeting DEFA5 antibodies and assays for diagnosis and treatment of inflammatory bowel disease

Abstract

Disclosed herein are targeted DEFA5 antibodies and assays for the diagnosis and treatment of inflammatory bowel disease. The targeted DEFA5 antibodies have a high degree of specificity for the DEFA5 protein, in particular for the peptide sequences of the P, B and / or M binding sites of the DEFA5 protein. Targeted DEFA5 antibodies can be incorporated into assays for diagnosing and treating ulcerative colitis and Crohn's disease in subjects suffering from inflammatory bowel disease. The assay may be provided in a kit. The targeted DEFA5 antibodies can be used in methods for measuring the level of DEFA5 or DEFA5 expression in a sample collected from a subject, and determining whether the subject has ulcerative colitis or Crohn's disease based on the level of DEFA5 or DEFA5 expression. The treatment may be based on a determination of whether the subject has ulcerative colitis or Crohn's disease.

Description

[0001] This application is a divisional application of the invention application filed on June 20, 2018, with Chinese national application number 201880054065.0, entitled "Targeted DEFA5 antibody and assay method for diagnosing and treating inflammatory bowel disease". Technical Field

[0002] This invention relates to the field of antibody diagnostics. Background Technology

[0003] Inflammatory bowel disease (IBD) is a chronic inflammation of all or part of the digestive tract. Common causes of IBD include ulcerative colitis (“UC”) and Crohn’s disease, also known as Crohn’s colitis (“CD” or “CC”). Ulcerative colitis causes chronic inflammation and ulcers in the innermost lining of the large intestine (i.e., the colon and rectum). Crohn’s disease causes chronic inflammation of the lining of the digestive tract, where the inflammation extends beyond the lining and into the affected tissues. Crohn’s disease can affect the small intestine, the large intestine, or both.

[0004] UC and CD affect an estimated 1.6 million people in the United States alone, with associated annual healthcare costs exceeding $6.3 billion. Although both UC and CD are types of IBD, the differences between UC and CD patients are significant. Currently, clinicians use inaccurate combinations of classifications (including clinical, endoscopic, radiological, and histopathological) to diagnose IBD patients as CD or UC. Nevertheless, differentiating between UC and CD patients among those with IBD remains so challenging that cases of IBD patients who are difficult to classify as UC or CD are classified as unclassified colitis (“IC”). Despite the use of state-of-the-art classification systems applying clinical, endoscopic, radiological, and histological tools, significant subgroups of IBD patients are still misdiagnosed or have their correct diagnosis delayed. In fact, it is estimated that 30% of patients with IBD cannot currently be accurately diagnosed as CD or UC.

[0005] In addition, 15% of colonic IDB cases undergoing ileal pouch-anal anastomosis are initially diagnosed with UC, but the initial diagnosis is subsequently changed to CD based on postoperative follow-up, clinical and histopathological changes, and the development of neoplastic CD within the ileal pouch. Ileal pouch-anal anastomosis is a treatment generally suitable for UC but not for CD, restoring gastrointestinal continuity after surgical resection of the colon and rectum, and involves creating a small intestinal pouch to reconstruct the resected rectum.

[0006] The significance of differentiating between UC and CD cases includes the selection of medical treatment, timing of surgery, prognosis, whether to provide patients with ileal pouch anal anastomosis, and lifestyle expectations. For these reasons, improvements are needed in the diagnosis and subsequent treatment of subjects with IBD. Summary of the Invention

[0007] The expression of DEFA5 proteins (e.g., HD5) and the DEFA5 gene has been found to be useful as biomarkers for determining whether patients with IBD also have UC or CD. Specifically, anti-DEFA5 antibodies have been identified and discovered that exhibit high specificity for binding to DEFA5, unlike their binding to other defensin proteins, which is highly advantageous for identifying DEFA5 as a biomarker in subjects.

[0008] In a first aspect, a method for measuring DEFA5 protein in patients with inflammatory bowel disease (IBD) or at risk of IBD is disclosed. The method includes measuring the level of DEFA5 or DEFA5 expression in a sample from the subject using an anti-DEFA5 antibody.

[0009] In a second aspect, a method for treating patients with or at risk of IBD is disclosed. The method includes measuring the level of DEFA5 or DEFA5 expression in a sample from a subject using an anti-DEFA5 antibody, and intervening in the patient to treat Crohn's disease. The method may further include comparing the expression or concentration of DEFA5 in the sample to a typical baseline value for a subject without Crohn's disease; and diagnosing Crohn's disease if the expression or concentration of DEFA5 in the sample significantly exceeds the baseline value.

[0010] In a third aspect, a method is provided for diagnosing a subject with or at risk of Crohn's disease (CD), comprising measuring the level of DEFA5 or DEFA5 expression in a sample from the subject using an anti-DEFA5 antibody; comparing the expression or concentration of DEFA5 in the sample to a typical baseline value for a subject without Crohn's disease; and diagnosing Crohn's disease if the expression or concentration of DEFA5 in the sample significantly exceeds the baseline value.

[0011] In a fourth aspect, a method for treating patients with or at risk of ulcerative colitis is disclosed. The method includes measuring DEFA5 in the patient according to the method of the first aspect; and intervening in the patient to treat ulcerative colitis. The method may further include comparing the expression or concentration of DEFA5 in the sample with a typical baseline value for a subject without Crohn's disease; and diagnosing ulcerative colitis if the expression or concentration of DEFA5 in the sample does not significantly exceed the baseline value.

[0012] In a fifth aspect, a kit for measuring DEFA5 in a sample is provided. The kit includes an assay containing an anti-DEFA5 antibody; and a sample container configured to contain samples selected from: fecal samples, blood samples, intestinal tissue samples, and serum samples. The kit can be used for the diagnosis and subsequent treatment of inflammatory bowel disease. The kit contains any anti-DEFA5 antibody disclosed herein as part of an immunoassay. The antibody may be labeled, conjugated, truncated, or otherwise modified to function in the assay, as known in the art. The kit may further include one or more of a sample container and a sampling tool. The container and sampling tool can be configured to collect and store various types of samples, including fecal samples, blood samples, serum samples, rectal lavage samples, and biopsy samples. The sampling tool can be any of a biopsy instrument, a rectal lavage kit, a swab, a blood sampler, and a vacuum blood collection tube.

[0013] In a sixth aspect, a method for diagnosing and treating Crohn's disease in a subject with inflammatory bowel disease is provided. The method includes obtaining a sample from the patient; measuring the concentration of human DEFA5 in the sample using an anti-DEFA5 antibody having a higher affinity for human DEFA5 than for human DEFA1 or human DEFA6; comparing the concentration of DEFA5 in the sample to a typical baseline value for a subject without Crohn's disease; diagnosing Crohn's disease if the concentration of DEFA5 in the sample significantly exceeds the baseline value; and treating the subject with Crohn's disease via a non-surgical intervention.

[0014] The above presents a simplified overview to provide a basic understanding of some aspects of the claimed subject matter. This overview is not an extensive summary. It is not intended to identify key or important elements or to depict the scope of the claimed subject matter. Its sole purpose is to present concepts in a simplified form as a prelude to the more detailed description that follows. Attached Figure Description

[0015] Figure 1 It is a commercially available DEFA5 antibody for HD1 to HD6 specific dot staining.

[0016] Figure 2 A shows the alignment of the primary sequence of DEFA5 with the primary sequences of HD1 and HD6.

[0017] Figure 2 B is a schematic diagram showing the DEFA5 antibody epitope to distinguish pre-DEFA5 from mature protein in the serum of IBD patients.

[0018] Figure 2 C is a sandwich ELISA model used to detect pre-DEFA5 and mature DEFA5 in the serum of IBD patients.

[0019] Figure 3 A shows the initial diagnostic information of 21 subjects diagnosed with ulcerative colitis, undifferentiated colitis, or Crohn's disease.

[0020] Figure 3 B shows that Figure 3 The diagnostic information of 21 subjects was reassessed 9.4 years after A's initial diagnostic information.

[0021] Figure 4 A-4C is a histological staining of DEFA5 tissue samples from patients treated with various therapies.

[0022] Figure 4 D is a quantitative count of staining spots in patients who underwent RPC and IPAA surgery for ulcerative colitis without altering their original diagnosis, compared with those who changed from ulcerative colitis to recurrent Crohn's disease.

[0023] Figure 5 A-5I shows histological staining of the typical morphological appearance of Paneth cells (PCs) on parallel sections, including the presence of dense apical eosinophilic granules.

[0024] Figure 6 A-6I shows double histological staining of PC, lysosomes, and DEFA5.

[0025] Figure 7 A shows the quantification of DEFA5 transcript levels in samples with moderate UC and CC.

[0026] Figure 7 B shows a DEFA5 protein blot, which reveals higher DEFA5 levels in moderate and severe CC compared to other IBD disease states.

[0027] Figure 7 C shows the DEFA5 levels in various IBD disease states.

[0028] Figure 7 D-7H shows IHC staining of DEFA5 in colon tissue using formalin-fixed paraffin-embedded thin sections.

[0029] Figure 8 A-8F shows representative H&E staining of colectomed tissue.

[0030] Figure 9 A-9D shows IHC and H&E staining of DEFA5 in adjacent normal and diseased tissues from CC patients (A and B) and UC patients (C and D).

[0031] Figure 10A-10C shows the detection of DEFA5 in the serum of IBD patients and the specificity of available DEFA5 antibodies.

[0032] Figure 11 A and 11B show the distribution of fresh frozen tissue and blood samples collected from IBD and non-IBD patients by sex and race. Detailed Implementation

[0033] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art. It will be further understood that terms, such as those defined in common dictionaries, should be interpreted as having the same meaning as they have in the context of the specification, and should not be interpreted in an idealized or overly formal sense unless explicitly defined herein. For the sake of brevity or clarity, well-known functions or constructions may not be described in detail.

[0034] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive. The singular forms “a,” “an,” and “the” used herein are also intended to include the plural forms unless the context clearly indicates otherwise.

[0035] The term "consistent essentially of..." means that, in addition to the listed elements, the claimed content may contain other elements (steps, structures, ingredients, components, etc.) that will not adversely affect the operability of the claimed content for its intended purpose as described in this disclosure. This term excludes such other elements that adversely affect the operability of the claimed content for its intended purpose as described in this disclosure, even if such other elements might enhance the operability of the claimed content for some other purpose.

[0036] The terms “about” and “approximately” generally refer to the acceptable degree of error or variation in a measured quantity, given the nature or precision of the measurement. A typical exemplary degree of error or variation is within 20% of a given value or range of values, preferably within 10%, and more preferably within 5%. For biological systems, the term “about” refers to the standard deviation of an acceptable error, preferably not exceeding twice the given value. Unless otherwise stated, quantities in this detailed description are approximate, meaning that the terms “about” or “approximately” can be inferred when not explicitly stated.

[0037] As used herein, the terms “individual,” “subject,” or “patient” refer to any animal, including mammals such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, primates, and humans. The term may be specified as male or female or both, or exclude male or female.

[0038] As used in this article, the terms "treatment," "treat," and "treating" refer to a process of action (such as the administration of a compound or pharmaceutical composition) initiated after the onset of clinical manifestations of a disease state or symptom, in order to eliminate or reduce those clinical manifestations. Such treatment is not necessarily absolutely effective.

[0039] The terms “first,” “second,” etc., are used herein to describe various features or elements, but these features or elements should not be limited by these terms. These terms are used only to distinguish one feature or element from another. Therefore, the first feature or element discussed below may be referred to as the second feature or element, and similarly, the second feature or element discussed below may be referred to as the first feature or element, without departing from the teaching of this disclosure.

[0040] DEFA5 is a small, antimicrobial innate immune system protein belonging to the α-defensin family of mammalian defensin peptides. DEFA5 is expressed in various tissues, particularly on mucosal surfaces. It is encoded by the DEFA5 gene. DEFA5 participates in host defense mechanisms and is highly expressed in the secretory granules of Paneth cells in the small intestine (ileum). Like most secretory proteins, DEFA5 is synthesized as pro-DEFA5(1-94), which first undergoes proteolytic processing to become inactive pro-DEFA5(20-94), DEFA5(23-94), and DEFA5(29-94). DEFA5(23-94) and DEFA5(29-94) are found in tissues, with DEFA5(20-94) being the predominant intracellular form. Pro-DEFA5 is then processed into two active or mature forms, DEFA5(56-94) and DEFA5(63-94), with DEFA5(63-94) being the most abundant. These mature forms of DEFA5 are cysteine-rich host defense peptides that exert broad-spectrum antimicrobial activity and contribute to the innate immunity of the human gut. The DEFA5 used in this article may refer only to the mature form of DEFA5.

[0041] This article describes a method using an anti-DEFA5 antibody, which is used to detect, measure, and / or treat patients with IBD. The anti-DEFA5 antibody forms a complex with DEFA5, which is relatively stable under physiological conditions. Specific binding is characterized by an equilibrium dissociation constant of at least about 1 x 10⁻⁶. -6M or smaller (e.g., a smaller KD indicates a tighter binding). Methods for determining whether two molecules bind specifically are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. However, anti-DEFA5 antibodies can exhibit cross-reactivity with other antigens, such as DEFA5 molecules from other species. Furthermore, as used herein, multispecific antibodies (e.g., bispecific antibodies) that bind to DEFA5 and one or more other antigens are still considered anti-DEFA5 antibodies. As used herein, "anti-DEFA5 antibody" is an antibody that forms a stable complex with DEFA5 under the expected binding conditions (e.g., physiological conditions).

[0042] Anti-DEFA5 antibodies can bind to DEFA5 at various affinity levels. One implementation of anti-DEFA5 antibodies is a high-affinity anti-DEFA5 antibody. The term "high-affinity" antibody refers to an antibody with at least 10 affinity for DEFA5. -10 M, Preferred 10 -11 M, or even more preferably 10 -12 Antibodies with M-binding affinity, such as those via surface plasmon resonance, such as BIACORE. TM Or measured by solution-affinity ELISA.

[0043] Anti-DEFA5 antibodies can bind to DEFA5 with high affinity (“high-affinity anti-DEFA5 antibodies”). As used herein, “high-affinity anti-DEFA5 antibodies” are antibodies with high binding affinity. “High binding affinity” refers to the strong binding strength of an epitope to a single complementary site (antigen-binding site). Antibodies with high binding affinity bind to the antigen more quickly than antibodies with lower affinity, allowing for higher sensitivity in assays and better retention of binding to the complementary site. The anti-DEFA5 antibodies described herein have a binding affinity to DEFA5 as low as 10. -7 10 -8 10 -9 10 -10 10 -11 Or 10 -12 K D s(M) or any range or subvalue thereof, the term "K" D "Koff" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, or the equilibrium dissociation constant of an interaction between an antibody, antibody-binding fragment, or molecule. Equilibrium dissociation can be calculated by obtaining the dissociation rate constant (koff value) and association rate constant of a specific antibody-antigen interaction. A lower Koff value indicates a lower equilibrium dissociation rate. D The value indicates a higher binding affinity.

[0044] The anti-DEFA5 antibody described herein also exhibits high specificity for DEFA5. "Specificity" refers to the ability to bind to a specific antigen without binding to other antigens. Some embodiments of the anti-DEFA5 antibody show an affinity for DEFA5 exceeding that shown for one or more related proteins; such related proteins may include one or more of DEFA1, DEFA2, DEFA3, DEFA4, and DEFA6. These are related neutrophil defensins found in multiple species. Typical human neutrophil defensin 1 (DEFA1) protein is described in UniProt accession number P59665, and its sequence is provided herein as SEQ ID NO:2. Typical human neutrophil defensin 2 (DEFA2) protein is described in UniProt accession number P59665, and its sequence is provided herein as SEQ ID NO:3. Typical human neutrophil defensin 3 (DEFA3) protein is described in UniProt accession number P59666, and its sequence is provided herein as SEQ ID NO:4. Typical human neutrophil defensin 4 (DEFA4) protein is described in UniProt accession number P12838, and its sequence is provided herein as SEQ ID NO:5. Typical human neutrophil defensin 6 (DEFA6) protein is described in UniProt accession number P12838, and its sequence is provided herein as SEQ ID NO:6. Other embodiments of the high-specificity anti-DEFA5 antibody show a higher affinity for DEFA5 than for DEFA1, DEFA6, or both. In one embodiment, the anti-DEFA5 antibody has high specificity for DEFA5 and does not bind or substantially does not bind to HD1 and HD6 (i.e., has low or no binding affinity for them). The anti-DEFA5 antibody may have an affinity greater than about 10 for HD1 and / or HD6. -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 K D The binding affinity of s(M) or any range or subvalue thereof. The K-axis of anti-DEFA5 antibody against one or both of DEFA1 and DEFA6. D s(M) can be greater than one of the following values: 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -410 -3 10 -2 and 10 -1 The anti-DEFA5 antibody recognizes epitope binding regions having at least 80%, 85%, 90%, 92%, 94%, 96%, or 98% sequence identity with positions 51-94 of SEQ ID NO:1. The anti-DEFA5 antibody can also recognize epitope binding regions having 100% sequence identity with positions 51-94 of SEQ ID NO:1. Some embodiments of the anti-DEFA5 antibody do not recognize epitope binding regions having at least 80%, 85%, 90%, 92%, 94%, 96%, or 98% sequence identity with positions 1-49 of SEQ ID NO:1. Specific embodiments of the anti-DEFA5 antibody do not recognize epitope binding regions having 100% sequence identity with positions 1-49 of SEQ ID NO:1.

[0045] Examples of commercially available anti-DEFA5 antibodies include: anti-α5 defensin antibody [EPR14309(B)] from ABCAM, Cambridge, United Kingdom; anti-α5 defensin antibody (ab167591) from ABCAM, Cambridge, United Kingdom; anti-α5 defensin antibody [8C8] from ABCAM, Cambridge, United Kingdom (catalog number ab90802); and anti-α5 defensin antibody from THERMO FISHER SCIENTIFIC. INC., Waltham, MA: Defensin 5 monoclonal antibody (8C8) (Catalog No. MA1-46026); Anti-α-defensin-5 (DEFA5) antibody, clone 8C8, from MILLIPORESIGMA, Burlington, MA (Catalog No. MABF31); Defensin 5 antibody LS-C50934 (Catalog No. LS-C50934-100), from LSBIO, Seattle, WA; Defensin α5 antibody (8C8) from NOVUSBIOLOGICALS, Littleton, CO (Catalog No. NB110-60002 / NB110-60002SS); Defensin α5 antibody (8C8) from NOVUSBIOLOGICALS, Littleton, CO (Catalog No. NBP1-84282); Defensin α5 antibody from BIORBYT, Cambridge, United Kingdom (Catalog No. orb156565); From BIOSS Defensive alpha 5 antibody from INC., Woburn, MA (catalog number bs-4313R); defensive alpha 5 antibody [N1C3] from GENETEX, INC., Irvine, CA (catalog number GTX116079); anti-DEFA5 antibody from ATLASANTIBODIES, Bromma, Sweden (HPA015775); DEFA5 antibody from R&D SYSTEMS, Minneapolis, MN (catalog number 972207.111 or CSL 1450400); and alpha-defensive 5 antibody from SANTA CRUZ BIOTECHNOLOGY, INC., Dallas, TX (catalog number 53997), etc.

[0046] In one embodiment, the anti-DEFA5 antibody is an α-defensin 5 antibody (catalog number 53997) from Santa Cruz Biotechnology, Inc., Dallas, TX. Surprisingly, the α-defensin 5 antibody (catalog number 53997) has been found to be particularly advantageous for measuring DEFA5 and DEFA5 expression levels in samples. Specifically, the α-defensin 5 antibody (catalog number 53997) exhibits high affinity and high specificity for DEFA5, including high affinity and high specificity for HD5 and low to zero affinity and specificity for other defensins (e.g., HD1-HD4 or HD6). The anti-DEFA5 antibody may be a κ light chain polypeptide subunit. In some embodiments, the anti-DEFA5 antibody is a mammalian antibody, such as a human antibody or a canine antibody.

[0047] A method for diagnosing ulcerative colitis or Crohn's disease in a subject is disclosed. The subject may have IBD. The method includes measuring the level of α-defensin 5 (“DEFA5”) or DEFA5 expression in a sample from the subject using an anti-DEFA5 antibody, and diagnosing the subject with Crohn's disease if the level or expression of DEFA5 indicates Crohn's disease, or diagnosing the subject with ulcerative colitis if the level or expression of DEFA5 indicates ulcerative colitis. The sample can be obtained from any suitable source for measuring DEFA5 concentration and DEFA5 expression level, such as a tissue sample from the intestine, for example, from the large intestine or rectum. In this disclosure, the term “DEFA5 expression” should be understood to mean the expression of the DEFA5 gene; “DEFA5 level” should be understood to mean the concentration of the DEFA5 protein.

[0048] Samples may be taken from subjects who have IBD or are at risk of it. Subjects may exhibit one or more characteristic symptoms of IBD, such as severe diarrhea, abdominal pain, fatigue, and weight loss. In some embodiments of the method, subjects exhibit more than one of the described symptoms. In other embodiments, subjects exhibit two, three, or four of the described symptoms.

[0049] DEFA5 has been found to be differentially expressed in subjects with UC and CD. Used in this manner, DEFA5-targeting antibodies can be used to utilize and measure DEFA5 concentration and expression as biomarkers to differentiate UC and CD in IBD patients. Since ileal pouch-anal anastomosis is clinically much more successful in patients with UC than in those with CD, it can be used to treat patients identified as having DEFA5 levels indicative of UC or without CD. Indeed, since DEFA5 is produced solely by Paneth cells, it is not expected that DEFA5-secreting Paneth cells will be found in the colon. Paneth cells (secreting DEFA5) have been found in large numbers in subjects with CC. On the other hand, patients identified as having DEFA5 levels indicative of CD and DEFA5 expression can be treated with any suitable treatment for CD. In one implementation, diagnostic steps, such as diagnosing a subject with UC or CD, are optional.

[0050] Anti-DEFA5 antibodies can have similar properties to... Figure 2 The complementary determination region (CDR) is complementary to each or all of the DEFA5 sequence at the P, B, and M binding sites shown. As used herein, “complementary to” means that the CDR is capable of forming a stable complex with the target sequence (e.g., the P, B, or M binding site) under the intended binding conditions (e.g., physiological conditions).

[0051] In another embodiment, the antibody may be an antibody that has a certain degree of identity with the polypeptide sequence complementary to the P, B, and M binding sites of DEFA5. For example, the antibody may have at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 100% identity with the polypeptide sequence complementary to the polypeptide sequence of the P, B, and M binding sites of DEFA5.

[0052] Compared to other alpha defensins, some of the antibody implementations disclosed in this paper target DEFA5 more specifically. Figure 1 This demonstrates the specificity of commercially available DEFA5 antibodies for purified HD1-HD6 proteins relative to the Ponceau S control, as shown in the dot blot. As used herein, the term "specific" or similar term used in the context of antibodies, in relation to their target, refers to an antibody that specifically binds to the target antigen (as opposed to other antigens such as HD1, HD2, HD3, HD4, and HD6). This higher DEFA5 specificity of the antibodies of the present invention will allow, for example, easier and more accurate testing of DEFA5 levels or expression in samples from subjects.

[0053] It is believed that the DEFA5 activation pathway may be dysfunctional in patients with moderate to severe CD, thus an excess of the inactive form of DEFA5 is a potential mechanism of inflammation in patients with CD. This excess of the inactive form of DEFA5 can lead to increased damage to the epithelial lining and may even cause dysregulation of the level and composition of the gut microbiota.

[0054] The method may include a step of comparing DEFA5 levels to a baseline. The baseline may be a measure of central tendency based on levels observed in one or more populations of subjects identified as not having UC or CD. For example, the baseline may be the average level of gene expression or protein concentration observed in samples from a population of subjects not having UC, not having CD, or both. A population may be defined based on one or more of the patient's geographic location, age, ethnicity, sex, and medical history. The baseline may consider a combination of measures of variability and measures of central tendency. For example, the baseline may be the average level of gene expression or protein concentration observed in a given tumor population, plus or minus an error margin. The baseline may be based on a raw measurement (such as a fragment of mRNA or cDNA per kb of gene length per million reads) or a normalized measurement (such as a % of normal expression, or expression compared to constitutively expressed or widely expressed genes (with generally consistent expression), such as β-actin). An example of a suitable baseline is approximately 1 ng / mL DEFA5 or exactly 1 ng / mL DEFA5.

[0055] A baseline can also be established by analyzing control samples measured together with samples from the subjects. Examples of suitable control samples include: samples from subjects without UC, samples from subjects without CD, samples from subjects with UC (although not CD), samples from subjects with CD (although not UC), samples from subjects with diverticulitis (although not UC or CD), and samples from subjects without IBD. In some implementations, the baseline level may be a normal level, as described below.

[0056] In one implementation, a method for differentially diagnosing UC and CD in patients with IBD includes measuring the level of DEFA5 or DEFA5 expression present in a sample obtained from the patient. The concentration or expression level of DEFA5 or MMP-7 in tissue can be measured by any suitable peptide analysis. For example, the measurement steps may include one or more of enzyme-linked immunosorbent assay (ELISA), cation exchange, NMR analysis, whole-genome transcriptomics analysis, and mass spectrometry. The method may include comparing the concentration or expression of DEFA5 in the sample to a benchmark, and making a diagnosis if the concentration or expression of DEFA5 in the sample is significantly less than or significantly greater than the benchmark. For example, the method may include comparing the concentration or expression of DEFA5 in the sample to a benchmark, and making a CD diagnosis if the concentration or expression of DEFA5 in the sample is significantly greater than the benchmark. As another example, the method may include comparing the concentration or expression of DEFA5 in the sample to a benchmark, and making an UC diagnosis if the concentration or expression of DEFA5 in the sample is not significantly greater than the benchmark. The measurement of DEFA5 expression or concentration in the sample can be performed in vitro or ex vivo.

[0057] Based on any of a variety of known statistical tests for significance, a difference in expression or concentration can be considered significant. These are typically based on a set of measurements taken from a sample population and are affected by both population size and sample size. Such statistical tests are well known in the art and will not be described further in detail in this disclosure; external references may be relied upon to enable those skilled in the art to determine statistical significance, such as Rosener's Fundamentals of Biostatistics, 8 th ed. (2015), Cengage Learning, Boston, MA.

[0058] In one embodiment, the assay for differential diagnosis of UC and CD in patients with IBD includes measuring the level of DEFA5 or DEFA5 expression present in samples obtained from the patient. The level of DEFA5 or DEFA5 expression in tissues can be measured by an enzyme-linked immunosorbent assay (ELISA) using an antibody targeting DEFA5 disclosed herein. The method includes diagnosing the patient with UC if DEFA5 or DEFA5 expression is at any level indicating that the patient does not have CD, such as less than 5x the normal level for DEFA5, or DEFA5 or DEFA5 expression is less than about 5x-30x the normal level. In one embodiment, if the level of DEFA5 expression is less than 3x10^6 ng / 10 ng RNA... 6If a patient has 100 DEFA5 mRNA transcripts, they are diagnosed with UC. If the DEFA5 expression level is at any level indicative of CD, such as approximately 3 x 10^10 mRNA transcripts per 10 ng of RNA, the patient is diagnosed with UC. 6 Up to 1.2×10 8 If a patient has at least one DEFA5 mRNA transcript, the diagnosis can be made to indicate that the patient has CD. The term "normal level" of DEFA5 or DEFA5 expression as used herein refers to the level of DEFA5 or DEFA5 expression in the digestive tract tissue of subjects without CD or UC, or subjects with IBD (and specifically UC). Normal DEFA5 expression can be defined as 1 x 10^10 mRNA per 10 ng RNA. 5 -9x10 5 One DEFA5 mRNA transcript, or approximately 6 x 10^10 mRNA per 10 ng RNA. 5 One DEFA5 mRNA transcript.

[0059] In one embodiment, the assay includes determining the subject's status regarding the activity and / or expression of DEFA5 or the activity and / or expression of a DEFA5-regulated peptide. In one embodiment, the method includes determining the level of expression or activity of DEFA5 or a DEFA5-regulated peptide in a sample from the subject using a DEFA5-targeting antibody disclosed herein. The method may further include collecting a sample from the subject. As used herein, the biological sample being tested is a sample derived from the subject and includes (but is not limited to) any biological material, such as body fluids. Examples of body fluids include (but are not limited to) whole blood, serum, saliva, tissue infiltrates, pleural effusion, bronchoalveolar lavage fluid, etc. The biological fluid may be a cell culture medium or supernatant for culturing cells. For example, the sample may be intestinal tissue, feces, blood, or serum. In one embodiment, the biological sample is collected from the subject's colon.

[0060] Some embodiments of this method involve measuring the concentration of DEFA5 by selectively staining or dyeing a sample from a subject and measuring the signal from the staining agent. The staining agent or dye may contain any anti-DEFA5 antibody disclosed herein. The staining agent or dye may also contain a reporter molecule, such as a chromogenic group, radionuclide, stable isotope, fluorophore, chromophore, enzyme, magnetic particles, and quantum dots. The concentration of DEFA5 can then be measured by observing the signal from the reporter molecule, for example, by microscopy, colorimetry, radiometry, fluorescence examination, magnetotaxis, or any combination thereof. In one specific embodiment of this method, the concentration of DEFA5 is measured by immunostaining a sample with an immunostaining agent that recognizes DEFA5 and counting the number of stained cells by microscopy. This method has the advantage of relative simplicity and requires only the types of equipment already available in typical clinical laboratories. Diagnosis can be made based on a threshold number of staining-positive cells (e.g., at least 10%, 20%, and 30%). If the number of DEFA5-stained cells is significantly higher than the threshold, a diagnosis of CD can be made; however, if the number of DEFA5-stained cells is significantly lower than the threshold, a diagnosis of UC can be made.

[0061] Subjects whose DEFA5 activity and / or expression differed from the control or baseline (increased or decreased) or whose activity of the DEFA5-regulated peptide differed from the control or baseline were identified as having or being at risk of a disease state or condition associated with or characterized by increased or decreased DEFA5 activity.

[0062] Assay techniques for determining the levels of expression or activity in a sample are known. Such assays include (but are not limited to) radioimmunoassays, reverse transcriptase PCR (RT-PCR) assays, immunohistochemistry assays, in situ hybridization assays, competitive binding assays, Western blotting, ELISA assays and proteomics methods, two-dimensional gel electrophoresis (2D electrophoresis), and non-gel-based methods such as mass spectrometry or protein-protein interaction analysis. Assays also include (but are not limited to) competitive and non-competitive assay systems using techniques such as radioimmunoassays, enzyme immunoassays (EIA), enzyme-linked immunosorbent assays (ELISA), sandwich immunoassays, precipitin reactions, gel diffusion reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradioassays, fluorescence immunoassays, protein A immunoassays, and immunoelectrophoresis. For examples of immunoassay methods, see U.S. Patent Nos. 4,845,026 and 5,006,459. Any anti-DEFA5 antibody disclosed herein may be used in the assay.

[0063] Anti-DEFA5 antibodies can be incorporated into ELISA assays for diagnostic purposes. Additionally, reporter antibodies are typically prepared. The reporter antibody is linked to a detectable reagent, such as a radioactive, fluorescent, or enzymatic reagent, like horseradish peroxidase or alkaline phosphatase. In one embodiment of the ELISA, to perform the ELISA, the anti-DEFA5 antibody is incubated on a solid support containing the antibody. Any free protein binding sites on the dish are then covered by incubation with nonspecific proteins. Next, the sample to be analyzed is incubated with the solid support, during which the anti-DEFA5 antibody binds to DEFA5. Unbound samples are washed away with buffer. A reporter antibody specifically targeting the antigen and linked to a detectable reagent is introduced, causing the reporter antibody to bind to any antibody that binds to the antigen. Unlinked reporter antibodies are then washed away. A reagent for detecting the presence of the reporter antibody is then added. The detectable reagent is then measured to determine the amount of antigen present. In an alternative embodiment, the antigen is incubated with a solid support, followed by incubation with one or more antibodies, at least one of which contains a detectable reagent. Quantitative results can be obtained using a reference standard curve.

[0064] Methods for treating IBD in patients may include: (a) measuring the level of DEFA5 or DEFA5 expression present in a sample obtained from the patient using an anti-DEFA5 antibody to obtain the level of DEFA5 or DEFA5 expression; (b) if the level of DEFA5 or DEFA5 expression is below the level indicating that the patient does not have CD, then treating the patient's IBD with medical treatment appropriate for UC; if the level of DEFA5 or DEFA5 expression is below the level indicating that the patient has CD, then treating the patient's IBD with medical treatment appropriate for CD.

[0065] Medical treatments suitable for UC include ileal pouch-anal anastomosis or the administration of medications or their salicylates. Suitable medications may include one or more of the following: iron supplements; oral 5-aminosalicylates, such as mesalazine, balasalamine, and olsalazine; anti-inflammatory drugs; corticosteroids; immunosuppressants, such as azathioprine, mercaptopurine, methotrexate, and cyclosporine; anti-TNF-α antibodies, such as infliximab, adalimumab, and golimumab; anti-α4-integrin antibodies, such as vedolizumab; and antimicrobial antibiotics, such as ciprofloxacin and metronidazole. Surgical procedures sometimes used to treat UC include total rectocele and ileal pouch-anal anastomosis. Note that, in contrast to UC, ileal pouch-anal anastomosis is considered relatively ineffective in treating CD. It should also be noted that while cyclosporine and golimumab are currently approved for the treatment of UC in the United States, they are not currently approved for the treatment of CD. Some implementations of this method involve interventions that are effective in treating UC but ineffective in treating CD or have not yet been approved by regulatory agencies for the treatment of CD.

[0066] Medical treatment for CD includes the administration of medications or their salts. Suitable medications include: oral 5-aminosalicylates, such as mesalazine; vitamin supplements, such as vitamin B-12 and vitamin D supplements; mineral supplements, such as calcium supplements; anti-inflammatory drugs; corticosteroids, such as prednisone and budesonide; immunosuppressants, such as azathioprine, tacrolimus, methotrexate, and mercaptopurine; anti-TNF-α antibodies, such as infliximab, adalimumab, and pegylated cetrusuzumab; anti-α4-integrin antibodies, such as natezumab and vedolizumab; anti-interleukin antibodies, such as uterotumab; and antimicrobial antibiotics, such as metronidazole and ciprofloxacin. Although pegylated cetrusuzumab, methotrexate, and natezumab are approved in the United States for the treatment of CD, they are not currently approved for the treatment of ulcerative colitis (UC). Surgical methods are sometimes used to treat severe cases of CD. This type of surgery includes stoma placement, colostomy, ileostomy, bowel resection, colectomy, rectocolectomy, and stricture repair. In some implementations of this method, subjects are treated with a diet that is beneficial for CD management but not necessarily for UC management. One such diet is a low-fat diet. Some implementations of this method involve interventions that are effective in treating CD but ineffective in treating UC or have not yet been approved by regulatory agencies for the treatment of UC. Interventions may include the administration of medication but do not include surgery. The administration of medication may be selected from the following: vitamin supplements, anti-inflammatory drugs, corticosteroids, prednisolone, methylprednisolone, oral budesonide, 5-aminosalicylic acid, immunosuppressants, azathioprine, mercaptopurine, anti-TNF-α antibodies, infliximab, adalimumab, pegylated cerutuzumab, methotrexate, anti-α4-integrin antibody, natezumab, vedolizumab, anti-interleukin antibody, eutecticumab, antibacterial antibiotics, ciprofloxacin, metronidazole anticholinergics, propylthiophene, bicycloamine, hyoscyamine, bile acid sequestrants, cholestyramine, colestipol, and colesvelam. The administration of medication may also be selected from the following: vitamin B12, vitamin D, calcium, pegylated cerutuzumab, methotrexate, and natezumab. Interventions can be enteral nutrition therapy, including basal and non-basal diets, such as feeding via a nasogastric tube. In one implementation, in patients who may be diagnosed with UC but are diagnosed with IC upon measurement of DEFA5 or DEFA5 expression levels, DEFA5 or DEFA5 expression levels may be elevated to above normal. These patients can be treated with any medical treatment suitable for UC. Interventions may involve subjecting the subject to a low-fat or high-fiber diet.

[0067] Suitable interventions for UC may include the administration of medications selected from the following: iron supplements, anti-inflammatory drugs, corticosteroids, hydrocortisone, cortisone, prednisolone, 5-aminosalicylic acid, immunosuppressants, azathioprine, mercaptopurine, cyclosporine, anti-TNF-α antibodies, infliximab, adalimumab, golimumab, methotrexate, anti-α4-integrin antibody, vedolizumab, antibiotics, ciprofloxacin, metronidazole, mesalazine suppositories, mesalazine enemas, olsalazine, balsalazine, budesonide enemas, tacrolimus, and any combination of the foregoing. Suitable interventions for UC may also include the administration of medications selected from cyclosporine and golimumab.

[0068] A kit is provided for measuring DEFA5 in a subject. This kit can be used in several of the methods provided above, as well as other methods. The kit can be used, for example, for the diagnosis of inflammatory bowel disease. The kit includes an assay for measuring at least one of DEFA5 concentration and DEFA5 expression. The kit may include an assay containing an anti-DEFA5 antibody; and a sample container configured to hold a sample selected from: fecal samples, blood samples, intestinal tissue samples, and serum samples. The first assay may include a sample collector selected from: a fecal sample collector, a blood sample collector, a serum sample collector, and an intestinal tissue collector.

[0069] Figure 1 The diagram shows a dot blot of the specificity of a commercially available DEFA5 antibody for purified HD1-HD6 proteins relative to the Ponceau S control. It is believed that the DEFA5-targeting antibodies of the present invention have higher specificity than these commercially available antibodies, such that, for example, purification of DEFA5 in the sample is unnecessary or minimally purified. As used herein, the term "specificity" or similar term used in the context of an antibody, in relation to its target, refers to an antibody that specifically binds to a target antigen (as opposed to other antigens such as HD1, HD2, HD3, HD4, and HD6). This higher DEFA5 specificity of the antibodies of the present invention will allow, for example, easier and more accurate testing of DEFA5 levels or expression in samples from subjects.

[0070] Figure 2 A shows the alignment of the primary sequence of DEFA5 with the primary sequences of HD1 and HD6. Figure 2 B is a schematic diagram showing the DEFA5 antibody epitope to distinguish pre-DEFA5 from mature protein in the serum of IBD patients. Figure 2 C is a sandwich ELISA model used to detect pre-DEFA5 and mature DEFA5 in the serum of IBD patients. Figure 3 A and 3B illustrate the problems of diagnostic uncertainty and inaccuracy in the clinical setting of IBD. Figure 3A showed that 21 patients with IC were followed up for approximately ten years. At the end of the 10-year period, it was still impossible to make a precise diagnosis of UC or CC in 28.5% of the patients. Figure 3 B shows 67 patients who underwent UC RPC surgery and were subsequently re-evaluated after a mean follow-up of 9.4 years (range 8–13 years). Of these patients, 30% required a re-diagnosis of Crohn's disease.

[0071] Figure 4 A-4D shows that DEFA5 level can be used to determine a patient's eligibility for IPAA. Figure 4 Figure A shows representative results from a patient who underwent RPC surgery, whose diagnosis remained unchanged post-surgery and underwent molecular testing using the DEFA5 IHC. Figure B shows representative results from patients who underwent UC RPC and IPAA surgery, whose diagnoses were indeed changed from UC to recurrent Crohn's disease, and who underwent molecular testing using the DEFA5 IHC. Figure 4 C shows the NL-ileum, control. Figure 4 D shows the quantitative NEARASDEFA5 IHC staining spot counts of patients who underwent UC RPC and IPAA surgery without altering their original diagnosis, compared with those who changed from UC to recurrent Crohn's disease. (Ctrl 1 – staining control, UC – ulcerative colitis, CC – Crohn's colitis, DV – diverticulitis, DVL – diverticulosis).

[0072] Figure 5 A-5I shows histological staining of the typical morphological appearance of Paneth cells (PCs) on parallel sections, including the presence of dense, apical eosinophilic granules. Above: Figure 5 A, diverticulitis (DV, without PC); Figure 5 B, diverticulosis (DVL, without PC); Figure 5 C, Normal (NL-colon, control, no PC). Middle image: Figure 5 D, UC (precursor PC found in a patient, arrow). Figure 5 E, CC, shows a large number of PCs distributed throughout the basal recesses of the colon (arrow). Figure 5 F, normal (NL-ileum, control), with abundant PCs. Below: IHC detection of Paneth cell markers α-defensin 5 (DEFA5) and lysozyme (LYZ) in the colon. Figure 5 G, NL-colon; Figure 5 H, CC; and Figure 5 I, NL-ileum, control.

[0073] Figure 6 A-6I showed double staining of PC, lysosomes, and DEFA5. This indicates a recurrence of Crohn's disease. Figure 6 A and 6D) and normal ileum / control ( Figure 6 Double staining analysis of G). Vertically displayed images were deconvolved to evaluate lysozyme-specific permanent red (G). Figure 6 B, 6E and 6H) and DEFA5α-specific DAB ( Figure 6 C, 6F, and 6I).

[0074] Working Example 1

[0075] Working Example 1 demonstrates that human UC and CC can be molecularly distinguished by examining DEFA5 levels in human colectomy tissue, colonic biopsy, and / or serum using the DEFA5 antibody described herein. Furthermore, Working Example 1 depicts the potential mechanisms underlying the subtle differences between UC and CC. The ability to accurately differentiate CC from UC is significant and clinically important, particularly for gastroenterologists and colorectal surgeons, especially before determining the need for restorative rectocele surgery in IBD patients.

[0076] method

[0077] The inability to accurately distinguish Crohn's disease (CC) from ulcerative colitis (UC) leads to inaccurate diagnoses presented as IC, significantly impacting patient medical and surgical care. Preliminary assessments of DEFA5 expression were conducted in a trial cohort of IC patients and in UC patients undergoing recurrent prostatectomy (RPC). This showed elevated DEFA5 levels, and to a lesser extent, elevated DEFA6 levels, in samples from CC patients. Preliminary data suggest that detecting DEFA5 in tissues from IC patients or from UC patients undergoing RPC (which are actually CC) more accurately distinguishes CC from UC in patients otherwise misdiagnosed.

[0078] Clinical samples

[0079] To demonstrate that aberrant DEFA5 expression in IBD patients is a more reliable diagnostic method for differentiating CC from UC, we explored the potential of detecting DEFA5 as a biomarker for CC in IBD patient samples diagnosed with UC, CC, and IC tissues. Figure 11 A and Figure 11 B shows the distribution of fresh frozen tissue and blood samples collected from IBD and non-IBD patients by sex and race. Figure 11 A shows the classification of tissue samples by female, male, white, and black race, displaying tissue samples (732 samples), and Figure 11B shows the classification of serum samples (186 samples) by female, male, white, and black race. Samples were stored at -80°C. Immunohistochemistry (IHC) and semi-quantitative RT-PCR were performed on patient samples diagnosed with UC, CC, and IC tissue to evaluate the potential of detecting DEFA5 as a biomarker for CC in IBD patient samples diagnosed with UC, CC, and IC tissue. Figure 11 As shown in A and 11B, a total of 732 tissue samples and 186 serum samples were collected from IBD and healthy individuals distributed by race and sex. These tissues were from surgical colectomy tissues of consenting adults with a clear and well-defined diagnosis of UC and CC, as well as those diagnosed with IC, at Vanderbilt University Medical Center (VUMC). Collection of these patient samples was approved by Meharry Medical College (MMC) and the VUMC IRB Committees. The tissues were analyzed at MMC and VUMC by the pathology group according to the established criteria for IBD subtypes, considering the entire thickness of the tissues. For each sample, medical data related to patient demographics, pre- and post-operative variables, monitoring endoscopy and clinical findings, and medical and surgical treatment history were retrospectively reviewed. Experimental samples were taken from various portions of the colon.

[0080] Retrospective clinical studies of IBD patients have shown persistent diagnostic uncertainty.

[0081] Between 2000 and 2007, a retrospective study was conducted at the IBD center of VUMC on a cohort of 21 patients diagnosed with IC, with a mean follow-up of 8.7 ± 3.7 (range 4–14) years. In 2014, these patients were reassessed to determine whether the diagnosis was differentiated into UC or CC. Each patient was reassessed by three GI pathologists who were unaware of the initial clinical outcomes, and the new diagnosis was presented as consensus among the attending physicians. The pathological reassessment concluded that the diagnosis of IC remained in 6 patients (28.5%) because it could not yet be described as UC or CC. Meanwhile, 43% and 28.5% were differentiated into UC and CC, respectively. Figure 3 A). In another retrospective study, between 2001 and 2008, 120 patients with “deterministic” UC underwent RPC combined with IPAA. Of the 120 patients, after a mean follow-up time of 9.4 years (range 8–13) with functionally acceptable pouches, the diagnosis of 67 patients was reassessed. Figure 3 As shown in B, 30% of the initial UC diagnoses changed to recurrent Crohn's disease (recurrent CD). In summary, this highlights the ongoing diagnostic uncertainty in at least 30% of IBD cases, thus necessitating more reliable diagnostic procedures.

[0082] Differential expression of DEFA5 in CC and UC.

[0083] Two methods, training and independent test sets, were used to identify differentially expressed genes or their products in UC and CC. In the training test set, whole transcriptome microarrays using RNA were performed using the Affymetrix gene expression array, extracted and pooled from full-thickness colon samples from UC and CC patients (n=5), according to the manufacturer's instructions (Affymetrix, Santa Clara, CA). Tissue from diverticulitis was used as a control. This analysis showed that a total of 484 genes were antimicrobial peptides and mucins that were upregulated or downregulated between the two diseases. In the test set analysis using microarray technology (Affymetrix, Santa Clara, CA), DEFA5 levels were increased the most: 31-fold in CC compared to UC (p<7.23E-05), Table 2. In the independent test set, gene expression profiling analyses were independently validated using a PCR array specifically targeting inflammatory genes (NanoString Technologies Inc., Seattle, WA). In different colon samples from UC and CC patients with the same disease activity as in the test set, DEFA5 was found to be 118-fold increased in CC compared to UC (p<0.001). Table 3. The only gene present in both the microarray and PCR array was DEFA5. Among the upregulated genes were α-defensin 5, other antimicrobial peptides, and mucins (Table 2). HD5 showed the largest increase: 31-fold in CC compared to UC (in previous tests, HD5 increased 118-fold in CC compared to UC – Table 3). A complete list of microarray results is available in Table 2. Table 2 shows a list of targets from the AFFYMETRIX cDNA microarray. A total of 484 genes are highlighted in the microarray as potential markers to differentiate UC from CC. The gene showing the largest fold change between the two diseases was human defensin 5 (HD5).

[0084] To further validate these data, DEFA5 expression was assessed by semi-quantitative RT-PCR using RNA extracted from moderate CC and moderate UC tissues (n=3). This analysis confirmed that DEFA5 mRNA levels were significantly higher in CC compared to UC. Figure 7A, SEM, p < 0.03). Using dot blot, bacterial lysates prepared from DEFA1-6 transformed bacteria were used to screen for commercially available antibodies against recombinant DEFA5. This led to the discovery of a monoclonal antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)—α-defensin 5 antibody (catalog number 53997)—as a highly specific and high-affinity DEFA5 antibody for the assay. Next, DEFA5 expression was assessed by Western blotting (n ≥ 10 for each disease). Figure 7 B) found that tissues from patients with moderate and severe CC expressed higher levels of DEFA5 compared to tissues from all other disease states. Figure 7 C, p < 0.0001). However, due to the use of full-thickness samples for Western blotting, the overall abundance of DEFA5 in the samples was low. Finally, DEFA5 expression in moderate IBD and control tissues was examined by IHC using FFPE sections. This analysis also indicated that CC tissues ( Figure 7 DEFA5 levels in G were higher than in DV, UC, and normal (NL) control tissues. Figure 7 DEFA5 levels in samples D, 7E, and 7F. Quantification of DEFA5 by IHC staining showed that DEFA5 levels in CC samples increased 5.6 times compared to UC samples. Figure 7 H, p<0.0001). Interestingly, IHC analysis of DEFA5 revealed localized DEFA5 staining at the base of a single colonic crypt. Figure 7 A-7H shows that DEFA is abnormally expressed in IBD. Figure 7 A shows the quantification of DEFA5 transcription levels in moderate UC and CC samples by semi-quantitative RT-PCR, confirming that the DEFA5 level in moderate CC was higher than that in moderate UC (p<0.05). Figure 7 B is a representative DEFA5 protein blot, showing higher DEFA5 levels in moderate and severe CC compared to all other IBD disease states. β-actin was used as a loading control. Figure 7 C shows a graph of DEFA5 levels (density assay) across various IBD disease states. Each point represents the ratio of DEFA5 to β-actin. DEFA5 levels were significantly higher in moderate and severe CC than in all other disease states (p<0.0001). Figure 7 D-7H shows representative IHC staining of DEFA5 in colon tissue using formalin-fixed paraffin-embedded (FFPE) thin sections. Figure 7 D shows diverticulosis, with no primary antibody control; Figure 7 E shows moderate diverticulitis; Figure 7F indicates moderate UC; Figure 7 G indicates moderate CC; and Figure 7 H shows the quantitative level of DEFA5 IHC staining in moderate UC relative to moderate CC. DEFA5 levels were increased in CC compared to UC (p<0.0001). These figures are drawn at 40x magnification.

[0085] DEFA5 detection in tissues from IBD colectomy patients was consistent with follow-up clinical patient results, suggesting it as a potential candidate for CC. Sexual diagnostic tools.

[0086] To test whether the DEFA5 assay could be used to differentiate between CC and UC, and whether this was consistent with clinical follow-up outcomes, an IHC study was conducted on 21 patients with IC. Figure 3 DEFA5 was detected in tissues A). Staining intensity was assessed using Nikon Element Advanced Research Analysis Software (NEARAS). Based on DEFA5 staining intensity, among the 6 patients with unchanged IC diagnosis and as shown in Table 1 below, 3 patients showed high DEFA5 staining and met the final diagnosis of CC (red circle), while 3 patients showed low DEFA5 staining and met the final diagnosis of UC (green circle).

[0087] Table 1

[0088]

[0089] Also to Figure 3 DEFA5 staining was evaluated in patients with clinical changes to re-CD after RPC and IPAA surgery (n=20) and those with no changes in diagnosis (n=47) as described in B. Figure 4 This demonstrates how DEFA5, as disclosed herein, can be used to determine a patient’s eligibility for IPAA. Figure 4 A shows colectomy tissue from a patient whose diagnosis remained unchanged after RPC surgery. Molecular testing of the tissue was performed using DEFA5 IHC. Figure 4 B shows colectomy tissue from a patient who underwent UC RPC and IPAA surgery, and whose diagnosis was changed from UC to re-CD. Molecular testing of the tissue was performed using DEFA5 IHC. Figure 4 C shows the NL-ileum, control. Figure 4 D shows quantitative comparisons of NEARASDEFA5 IHC staining in tissues from patients who underwent UC RPC and IPAA surgery without change in their original diagnosis with NEARASDEFA5 IHC staining in tissues from patients whose diagnosis was changed from UC to re-CD. Figure 11B). (Ctrl 1 – Control, UC – Ulcerative colitis, CC – Crohn's colitis, DV – Diverticulitis, DVL – Diverticulosis). DEFA5 IHC revealed that the diagnosis remained unchanged, i.e., patients with UC showed only trace levels of DEFA5. Figure 4 A and 4D), while those patients whose clinical diagnosis changed from UC to re-CD showed significantly strong (p<0.0001) DEFA5 staining (A and 4D), while those patients whose diagnosis changed from UC to re-CD showed significantly strong (p<0.0001) DEFA5 staining (A and 4D). Figure 4 B and 4D). As expected, DEFA5 staining was high in normal ileum control tissue (B and 4D). Figure 4 C). Statistical analysis of the positive predictive value (PPV) of DEFA5 in patient tissues showed a value of 95.8% for CC and only 76.9% for UC. Chi-square analysis showed a significant correlation between high levels of DEFA5 and the diagnosis of CC (p<0.0001). These data suggest that DEFA5 is a candidate diagnostic biomarker for accurately distinguishing CC from UC and reliably reclassifying IC into CC and UC subtypes.

[0090] Establish the specificity and selectivity of the DEFA5 antibody.

[0091] Although DEFA5 was detected in IBD tissue by IHC ( Figure 4 A-4D) and RT-PCR data ( Figure 7 The A-7H values ​​are consistent, but commercially available antibodies can exhibit some cross-reactivity, particularly with PC-derived DEFA6. This prompted us to evaluate commercially available DEFA5 antibodies using dot blot analysis. A DEFA5 antibody (α-defensin 5 antibody catalog number sc-53997) from Santa Cruz Biotechnology was identified as a DEFA5-specific antibody. Evaluation of the antibody's cross-reactivity with DEFA6 indicated that it strongly detects DEFA5, while detecting DEFA6 to a lesser extent. Figure 10 A). Considering the possibility of cross-reactivity between antibodies against these proteins, the aim was to identify DEFA5-specific antibodies. A total of 11 monoclonal antibodies against DEFA5 were obtained from R&D Systems, and their ability to be used as specific DEFA5 detection antibodies was evaluated. Figure 10 C). A DEFA5 sandwich ELISA kit (OKEH01234) was obtained from AVIVA System Biology Inc. to determine whether DEFA5 could be detected in patient serum. Unexpectedly, DEFA5 was detected in serum from patients with data on mild CC and mild UC activity, and DEFA5 levels were found to be higher in serum from CC patients than in UC patients (p<0.05; R²=0.9938). Figure 10 B).

[0092] Predictive Example 2

[0093] Establish the specificity and selectivity of DEFA5 antibody for sandwich ELISA.

[0094] It is believed that the specificity of commercially available ELISA kits can be determined by using DEFA6 as the antigen. If the test proves nonspecific, immunoprecipitation (IP) using DEFA5 and DEFA6 expressed in bacteria, along with 11 monoclonal antibodies against DEFA5, is thought to identify antibodies that specifically form immune complexes with DEFA5 but not with DEFA6. It is believed that the DEFA5 antibody from R&D Systems, Minneapolis, MN (catalog number 972207.111 or CSL 1450400) can be biotinylated and used as the detection antibody for IP. It is believed that combinations of detection antibodies and optimal capture antibodies develop more specific sandwich ELISAs for detecting DEFA5 in serum. In general, purified DEFA5 expressed in bacteria is believed to be used to determine appropriate concentrations of DEFA5 antibodies for robust ELISAs and to compare them with commercially available ELISA kits.

[0095] Optimize DEFA5 sandwich ELISA to detect DEFA5 in serum from IBD patients and healthy subjects.

[0096] Since DEFA5 is not used in the clinical setting of IBD, the goal of this task is to establish a reference range of DEFA5 levels in normal blood and compare it with values ​​in the serum of IBD patients. Figure 10 As shown in Figure B, it is anticipated that DEFA5, a protein locally produced in the intestinal mucosal crypts, can be detected in circulating human serum by sandwich ELISA. Serum samples were collected from 117 IBD patients (40 UC, 52 CC, and 25 IC) spanning different races / ethnicities and both sexes, and from 69 non-IBD controls. Figure 11B). A sample size of 92 subjects (46 CCs and 46 UCs) will be used to detect a 19% clinically significant difference in positive predictive value between CCs and UCs using a one-tailed test of proportion between the two groups with 80% statistical power and a 5% significance level. This 19% difference represents a 96% probability that subjects with a positive screening test in the CC group actually have the disease, compared to a 77% probability in the UC group. To establish normal values ​​for DEFA5 in human serum, up to 120 serum samples from men of different ethnic backgrounds and a similar number of serum samples from women of different ethnic backgrounds from the MMC and VUMC outpatient clinics will be used. Serum samples from patients diagnosed with diseases that may affect the analysis will be disqualified. Preanalytical sampling and quantitative analysis, as well as the definition, establishment, and validation of DEFA5 reference intervals, will be performed according to previously established guidelines. Blood samples from healthy individuals at the VUMC Clinical Research Center (CRC), part of the Vanderbilt Institute for Clinical and Translational Research (VICTR), Clinical Chemistry Pathology Laboratories, will be analyzed. Additional blood samples will be collected with the consent of healthy adult volunteers and IBD patients as part of an ongoing process to increase serum collection. Figure 11 B).

[0097] DEFA5 expression in formalin-fixed paraffin-embedded (FFPE) IBD biopsies / tissues was detected by IHC.

[0098] During the previous R21 funding period, 205 FFPE blocks were collected from IBD patients. Of these samples, 83 were from UC patients, 75 from CC patients, and 47 from IC patients. Tissue from DV patients will be used as a non-IBD control. Thin FFPE sections from these samples will be stained with anti-DEFA5 antibody at the Translational Pathology Shared Resource (TPSR). Following IHC staining, the slides will be digitally scanned using an Ariol SL-50 digital high-resolution imaging system (Leica) and quantified using Tissue IA software from the Vanderbilt University Digital Histology Shared Resource (DHSR). This will allow for scoring of each slide based on staining intensity and the percentage of stained cells. This digital analysis of the IHC results will serve as an additional or alternative bioassay to DEFA5 detection in biopsies.

[0099] Determine whether differences in DEFA5 levels in colonic mucosal tissue are related to DEFA5 circulation in CC, UC, or normal subjects. Horizontal correlation.

[0100] To determine whether the level of circulating or secreted DEFA5 (sDEFA5) is correlated with its in situ expression level, biopsy samples collected from these three groups were used to isolate mRNA for determining the presence of DEFA5 messenger levels via real-time PCR. Where possible, simultaneous biopsies from adjacent normal tissues in the active CC region will inform whether serum levels indicate active disease. For this purpose, DEFA5 mRNA expression in biopsy samples from normal adjacent mucosa, active inflamed mucosa, and mucosa surrounding the transition zone will be examined. Power analysis showed that, based on a two-sided test with 0.05α levels, the prevalence comparison between the case and control groups with 30 subjects per group generally had 84% power to detect a 40% difference (e.g., 40% vs. 80%, odds ratio 6.0). Regarding the precision of prevalence estimation, when the sample size is 30 per group, a two-sided 95% confidence interval for a single proportion would result in an expected 50% observation rate of 18%. Sample size requirements were calculated based on detecting differentially expressed proteins between the two groups, while controlling for false discovery rate (FDR). This metric is the ratio of protein expression (or fold change) of a specific protein in cases to that in controls. Based on the algorithm of Jung SH, Bioinformatics 2005; 21:3097, an effect size equal to a 1.5-fold change can be detected (with power exceeding 80%). This assumes an FDR of 0.001 and a two-sided p-value, and is based on samples from 30 cases and 30 controls. To determine the significance of sDEFA5 as a candidate biomarker for active CC and to build a model with predictive accuracy, a generalized linear model, such as one with regularization methods and ensemble methods for feature identification (including boosting, bagging, and random forest classifiers), will be used.

[0101] Expected outcomes, challenges, and alternative procedures.

[0102] This is believed to demonstrate a DEFA5-specific assay that detects DEFA5 in the serum and tissues of IBD patients. Using this assay, a quantitative standard numerical value for DEFA5 in the serum of healthy subjects can be determined and a normal reference range (RI) value can be developed and correlated with the level in the serum of IBD patients. The RI method will be based on the center 95% of laboratory test values ​​observed in a disease-free reference population. Based on preliminary data, DEFA5 expression in tissues and serum from CC patients is expected to be higher than that from UC patients, and all patients with IC can be reclassified as CC or UC patients. Although power calculations indicate 46 patients per disease subtype, up to 100 patient tissues and sera from each disease from diverse ethnic backgrounds will be used to validate the DEFA5 assay as a diagnostic tool for serum CC.

[0103] While the sensitivity of the assay may be poor due to the relatively low levels of DEFA5 in serum, it can be validated using an alkaline phosphatase-conjugated anti-DEFA5 monoclonal antibody, or modified to a direct ELISA or radioimmunoassay. Peroxidase-conjugated streptobibiotin can be used to develop a DEFA5 detection assay, using 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS) as a substrate. It is believed that developing a sandwich assay and antibody will avoid cross-reactivity with DEFA6.

[0104] Working Example 3

[0105] Paneth cells are differentiated progeny of ileal epithelial stem cells (ISCs), supporting ISCs and providing antimicrobial protection in mammals. Despite the inflammatory predisposition of IBD, the histological differences between UC and CC, and the need for distinct surgical treatment options, suggest that DEFA5 and / or specific pro-inflammatory cytokines play a major role in the pathogenesis of these diseases. High DEFA5 levels in CC colectomy samples are thought to be due to aberrant metaplastic colonic crypts (PCs); and serum from both UC and CC patients contains high levels of IBD subtype-specific pro-inflammatory cytokines. There is ample evidence supporting the possibility that bacterial enterotoxins such as staphylococcal enterotoxin C and cholera toxin (Xiao-Chen Wan et al., 2008; Androutsellis-Theotokis A et al., 2011) and pro-inflammatory cytokines such as TNF-α, IL-1β, and IFN-γ promote stem cell differentiation. However, little is known about whether the presence or absence of DEFA5-containing bacterial enterotoxins or pro-inflammatory cytokines underlies the unique pathological features of CC relative to UC. It is believed that DEFA5, bacterial enterotoxins, and / or certain CC-associated pro-inflammatory cytokines promote the differentiation / proliferation of colonic stem cells and the unique pathology associated with CC. To test this hypothesis and in the absence of actual animal models of CC, two different normal human colonic epithelial cell lines (NCM460 and NCM356), colonoids and / or enteroids from endoscopic biopsy tissues were used to a) test the effects of purified DEFA5, DEFA6, and DEFA1 on the formation of metaplastic colonic PC in the presence or absence of bacterial enterotoxins; and b) assess the effects of CC- and UC-specific cytokines on DEFA5 secretion, ROS production, and cell viability. It is believed that DEFA5, and to a lesser extent DEFA6, will promote the secretion of CC-specific cytokines and ROS production, but attenuate both cell viability and tissue damage. It is also believed that CC-specific cytokines will promote the synthesis / secretion of DEFA5, while UC-specific cytokines will have the opposite effect.

[0106] In patients with chronic colitis (CC), aberrantly expressed DEFA5 is synthesized via polypsyllosis (PC) in the colonic crypts. DEFA5 is primarily synthesized by PC. Therefore, this study aimed to determine the presence of PC in the colonic crypts of CC patients and to verify whether the pools of DEFA5 found in CC and re-CD colectomy samples originated from colonic epithelial crypts. As confirmed by H&E staining, all 20 UC samples from patients with re-CD who underwent RPC surgery showed pools of PC in the colonic metaplastic crypts (PC). Figure 8 A-8C). IHC staining of lysozyme in PCs confirmed that PC abundance was higher in the colonic crypts of the CC than in the UC. Figure 8 D-8F (arrow). Figure 8 A-8C show representative H&E staining of colectomed tissue. 8A, normal colon (NLC). 8B, UC, scattered PCs (arrows). 8C, CC, mature PCs within crypts (arrows). Figure 8 D-8F shows representative IHC assays of DEFA5 and lysozyme in the colon. 8D, NLC. 8E, UC (sporadic pre-PC in one patient). 8F, CC. Magnification: 40x.

[0107] PCs were detected by staining colectomy tissue samples for DEFA5 and lysozyme (LYZ), revealing that PCs are DEFA5-secreting cells. Numerous crypt PCs were also found in the CC samples. Figure 6 A and 6D). Normal ileum tissue was used as a control. Figure 6 G). Figure 6 A-6I shows co-expression of DEFA5 and lysozyme in crypt PCs within colectomycosis (CC). Double staining was performed on reconstituted Crohn's disease tissue (6A and 6D) and normal ileum (control) (6G) from two patients using lysozyme (6B, 6E, and 6F) and DEFA5 (6C, 6F, and 6I). Combined images are shown in 6A, 6D, and 6G. To determine if PCs are DEFA5-secreting cells, colectomy tissue samples were stained for DEFA5 and lysozyme (LYZ) to detect PCs. Numerous crypt PCs were found in the CC samples. Figure 6 A and 6D). Normal ileum tissue was used as a control. Figure 6 G).

[0108] Figure 9 A-9D shows the presence of DEFA5 in adjacent IBD tissue. Patients from CC (9A and 9B) and patients from UC (...) Figure 9IHC and H&E staining of DEFA5 in adjacent normal and diseased tissues from C and 9D. Note that DEFA5 staining was not prominent in diseased and normal tissues from UC patients. DEFA5 was detected in adjacent normal tissues from CC. Considering the lack of or sparse PC in normal healthy colonic tissue, an attempt was made to determine whether DEFA5 could be detected in normal tissues adjacent to diseased tissues in CC and UC patients. IHC for DEFA5 showed positive staining at the crypt base in both inflamed and normal adjacent tissues from CC patients. Figure 9 A). Figure 9 B describes H&E. Considering the co-location of DEFA5 and PC ( Figure 6 This suggests that PCs may be present in both diseased and normal adjacent tissues of patients with CC, but not in tissues from patients with UC. However, little is known about what causes PCs to appear in this subtype of IBD.

[0109] It should be understood that any given element of the disclosed embodiments of the present invention may be embodied in a single structure, a single step, a single substance, etc. Similarly, a given element of the disclosed embodiments may be embodied in multiple structures, steps, substances, etc.

[0110] The foregoing description illustrates and describes the processes, machines, manufacture, composition of matter, and other teachings of this disclosure. Furthermore, this disclosure shows and describes only certain embodiments of the disclosed processes, machines, manufacture, composition of matter, and other teachings; however, as mentioned above, it should be understood that the teachings of this disclosure are applicable to various other combinations, modifications, and environments, and can be varied or modified to the extent appropriate to the skill and / or knowledge of one of ordinary skill in the art within the scope of the teachings as set forth herein. The embodiments described above are further intended to explain certain best practices known in carrying out the processes, machines, manufacture, composition of matter, and other teachings of this disclosure, and to enable others skilled in the art to utilize the teachings of this disclosure and various modifications required for a particular application or use in such or other embodiments. Therefore, the processes, machines, manufacture, composition of matter, and other teachings of this disclosure are not intended to limit the precise embodiments and examples disclosed herein. Any section headings provided herein are merely for consistency with the recommendations of 37 C. FR § 1.77, or otherwise to provide organization. These headings should not limit or characterize the invention set forth herein.

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Claims

1. Use of an anti-DEFA5 antibody in the manufacture of a diagnostic reagent for measuring DEFA5 protein in a patient having or at risk of inflammatory bowel disease (IBD), comprising measuring at least one of expression of DEFA5 and concentration of DEFA5 in a sample from the patient using the anti-DEFA5 antibody; comparing the expression of DEFA5 or the concentration of DEFA5 in the sample to a benchmark value typical of subjects having ulcerative colitis; and (i) diagnosing Crohn's disease if the expression of DEFA5 or the concentration of DEFA5 in the sample significantly exceeds the benchmark value; or (ii) diagnosing ulcerative colitis if the expression of DEFA5 or the concentration of DEFA5 in the sample does not significantly exceed the benchmark value.

2. The use of claim 1, wherein the benchmark value is about 1 ng / mL DEFA5.

3. The use of any one of claims 1-2, further comprising diagnosing Crohn's disease if the expression of DEFA5 or the concentration of DEFA5 in the sample exceeds the benchmark value.

4. The use of any one of claims 1-3, wherein the sample is intestinal tissue and comprises measuring the concentration of DEFA5 by immunostaining the sample with an anti-DEFA5 immunostaining reagent comprising the anti-DEFA5 antibody; and measuring the percentage of cells that stain positive in the sample; wherein the concentration of DEFA5 in the sample significantly exceeds the benchmark value if the percentage of cells that stain positive in the sample is at least 20%.

5. The use of any one of claims 1-2, further comprising diagnosing ulcerative colitis if the expression of DEFA5 or the concentration of DEFA5 in the sample is below the benchmark value.

6. The use of any one of claims 1-2 or 5, wherein the sample is intestinal tissue and comprises measuring the concentration of DEFA5 by immunostaining the sample with an anti-DEFA5 immunostaining reagent comprising the anti-DEFA5 antibody; and measuring the percentage of cells that stain positive in the sample; wherein the concentration of DEFA5 in the sample does not significantly exceed the benchmark value if the percentage of cells that stain positive in the sample is less than 10%.

7. The use of any one of claims 1-6, wherein the anti-DEFA5 antibody recognizes an epitope binding region having 100% sequence identity to positions 51-94 of SEQ ID NO:

1.

8. The use of any one of claims 1-7, wherein the anti-DEFA5 antibody does not recognize an epitope binding region having at least 90% sequence identity to positions 1-49 of SEQ ID NO:

1.

9. The use of any one of claims 1-8, wherein the anti-DEFA5 antibody is used as part of an assay selected from the group consisting of a radioimmunoassay, an immunohistochemical assay, a competitive binding assay, a Western blot analysis, an ELISA assay, a two-dimensional gel electrophoresis, an enzyme immunoassay, a sandwich immunoassay, a precipitin reaction, a gel diffusion reaction, an immunodiffusion assay, an agglutination assay, a complement fixation assay, an immunoradioassay, a fluorescent immunoassay, a protein A immunoassay, and an immunoelectrophoresis assay.

10. The use of any one of claims 1-9, wherein the anti-DEFA5 antibody is used as part of an enzyme-linked immunosorbent assay (ELISA).

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