Patent Grant
12174200
A Sequence Listing is provided herewith as a text file, “MKonaAbSeq_ST25.txt,” created on Jun. 20, 2017 and having a size of 5 KB. The contents of the text file are incorporated by reference herein in their entirety.
Inflammatory bowel disease (IBD) is the 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 inflammation goes beyond the lining and into affected tissue. Crohn's disease can affect the small intestine, large intestine, or both.
UC and CD affect an estimated 1.6 million people in the US alone with associated annual health care costs of over $6.3 billion. While UC and CD are both types of IBDs, differences between patients having UC or CD have major implications. Currently, clinicians use inexact combined classifications for patients having IBD, which include clinical, endoscopy, radiological, and histopathology in an effort to diagnose CD and UC. Nonetheless, differentiating patients having UC or CD among patients suffering from IBD remains challenging, so much so that cases of patients having IBD that are difficult to classify as UC or CD are classified as having indeterminate colitis (“IC”). A significant subgroup of IBD patients are misdiagnosed or have a correct diagnosis delayed despite use of a state-of-the-art classification system applying clinical, endoscopic, radiologic, and histologic tools. Indeed, it is estimated that 30% of patients suffering from IBD cannot currently be accurately diagnosed as CD or UC.
In addition, 15% of colonic IDB cases that undergo ileal pouch anal anastomosis surgery, as they are diagnosed with UC, will subsequently have their original diagnosis changed to CD based on their postoperative follow-up visits, clinical and histopathology changes, and development of de novo CD in the ileal pouch. Ileal pouch anal anastomosis, a treatment normally suitable for UC but not CD, restores gastrointestinal continuity after surgical removal of the colon and rectum, and involves the creation of a pouch of small intestine to recreate the removed rectum.
Implications of distinguishing cases of UC and CD include choice of medical treatment, timing of surgery, prognosis, whether to offer the patient an ileal pouch anal anastomosis, and lifestyle expectations. For these reasons, there is a need for improving the diagnosis, and subsequent treatment, of subjects having IBD.
It has been discovered that the DEFA5 protein (e.g., HD5), and the expression of the DEFA5 gene, may serve as a biomarker for determining whether a patient suffering from IBD has UC or CD. In particular, an anti-DEFA5 antibody has been identified and discovered that has high specificity for binding with DEFA5 while not binding with other defensin proteins that is highly advantageous for identifying DEFA5 as a biomarker in subjects.
In a first aspect, a method of measuring DEFA5 protein in a patient suffering from or at risk of inflammatory bowel disease (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.
In a second aspect, a method of treating a patient suffering from 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 and performing an intervention on the patient to treat Crohn's disease. The method may further comprise comparing the expression of DEFA5 or the concentration of DEFA5 in the sample to a benchmark value that is typical of a subject not suffering from Crohn's disease; and diagnosing Crohn's disease if the expression of DEFA5 or the concentration of DEFA5 in the sample significantly exceeds the benchmark value.
In a third aspect, a method diagnosing a subject suffering from or at risk of CD is provided, comprising measuring the level of DEFA5 or DEFA5 expression in a sample from the subject using an anti-DEFA5 antibody; comparing the expression of DEFA5 or the concentration of DEFA5 in the sample to a benchmark value that is typical of a subject not suffering from Crohn's disease; and diagnosing Crohn's disease if the expression of DEFA5 or the concentration of DEFA5 in the sample significantly exceeds the benchmark value.
In a fourth aspect, a method for treating a patient suffering from or at risk of UC is disclosed. The method comprises performing the method of measuring DEFA5 in the patient according to the first aspect; and performing an intervention on the patient to treat ulcerative colitis. The method may further comprise comparing the expression of DEFA5 or the concentration of DEFA5 in the sample to a benchmark value that is typical of a subject not suffering from Crohn's disease; and diagnosing ulcerative colitis if the expression of DEFA5 or the concentration of DEFA5 in the sample does not significantly exceed the benchmark value.
In a fifth aspect, a kit for measuring DEFA5 in a sample is provided. The kit comprises an assay comprising an anti-DEFA5 antibody; and a sample container configured to contain a sample selected from: a stool sample, a blood sample, a bowel tissue sample, and a serum sample. The kit may be for the diagnosis, and subsequent treatment of, inflammatory bowel disease. The kit comprises any of the anti-DEFA5 antibodies disclosed herein as part of immunoassay. The antibodies may be tagged, conjugated, truncated, or otherwise modified to function in the assay as is known in the art. The kit may further comprise one or more of a sample container and a sampling tool. The container and sampling tool may be configured to collect and store various types of samples including a stool sample, a blood sample, a serum sample, a rectal lavage sample, and a biopsy sample. The sampling tool may be any of a biopsy instrument, a rectal lavage kit, a swab, a blood sampler, and a vacutainer.
In a sixth aspect, a method of diagnosing and treating Crohn's disease in a subject suffering from 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 either of human DEFA1 or human DEFA6; comparing the concentration of DEFA5 in the sample to a benchmark value that is typical of a subject not suffering from Crohn's disease; diagnosing Crohn's disease if the concentration of DEFA5 in the sample significantly exceeds the benchmark value; and treating the subject for Crohn's disease by way of a non-surgical intervention.
The above presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key or critical elements or to delineate 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 is presented later.
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art of this disclosure. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and should not be interpreted in an idealized or overly formal sense, unless expressly so defined herein. Well-known functions or constructions may not be described in detail for brevity or clarity.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms “a”, “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The term “consisting essentially of” means that, in addition to the recited elements, what is claimed may also contain other elements (steps, structures, ingredients, components, etc.) that do not adversely affect the operability of what is claimed for its intended purpose as stated in this disclosure. This term excludes such other elements that adversely affect the operability of what is claimed for its intended purpose as stated in this disclosure, even if such other elements might enhance the operability of what is claimed for some other purpose.
The terms “about” and “approximately” shall generally mean an acceptable degree of error or variation for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error or variation are within 20%, preferably within 10%, and more preferably within 5% of a given value or range of values. For biological systems, the term “about” refers to an acceptable standard deviation of error, preferably not more than 2-fold of a given value. Numerical quantities in this detailed description are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.
The terms “individual,” “subject,” or “patient” as used herein refer to any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, primates, and humans. The terms may specify male or female or both, or exclude male or female.
The terms “treatment”, “treat”, and “treating”, as used herein, refer to a course of action (such as administering a compound or pharmaceutical composition) initiated after the onset of a clinical manifestation of a disease state or condition so as to eliminate or reduce such clinical manifestation of the disease state or condition.
Such treating need not be absolute to be useful.
The terms “first”, “second”, and the like are used herein to describe various features or elements, but these features or elements should not be limited by these terms. These terms are only used to distinguish one feature or element from another feature or element. Thus, a first feature or element discussed below could be termed a second feature or element, and similarly, a second feature or element discussed below could be termed a first feature or element without departing from the teachings of the present disclosure.
DEFA5 is a small, microbicidal innate immune system protein belonging to the alpha defensin family of mammalian defensin peptides. DEFA5 is expressed in various tissues and particularly on mucosal surfaces. DEFA5 is encoded by the gene DEFA5. DEFA5 is involved in host defense mechanisms and is highly expressed in secretory granules of Paneth cells of the small intestine (ileum). Like most secreted proteins, DEFA5 is synthesized as prepro-DEFA5 (1-94) that undergoes proteolytic processing first, to the inactive pro-DEFA5s (20-94), DEFA5 (23-94), and DEFA5 (29-94). DEFA5 (23-94) and DEFA5 (29-94) are found within tissues, while DEFA5 (20-94) is the predominant intracellular form. The pro-DEFA5s are then processed to two active or mature forms. DEFA5 (56-94) and DEFA5 (63-94) with DEFA5 (63-94) being the most abundant form. These mature forms of DEFA5 are cysteine-rich host defense peptides which exert broad-spectrum antimicrobial activity and contribute to innate immunity in the human gut. As used herein, DEFA5 may refer to exclusively mature forms of DEFA5.
Methods of using an anti-DEFA5 antibody are described herein, whereby the anti-DEFA5 antibody is used in the detection, measurement, and/or treatment of patients having IBD. The anti-DEFA5 antibody forms a complex with DEFA5 that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1×10−6 M or less (e.g., a smaller KD denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. An anti-DEFA5 antibody may, however, exhibit cross-reactivity to other antigens such as DEFA5 molecules from other species. Moreover, multi-specific antibodies (e.g., bispecifics) that bind to DEFA5 and one or more additional antigens are nonetheless considered anti-DEFA5 antibodies, as used herein. As used herein, an “anti-DEFA5 antibody” is an antibody that forms a stable complex with DEFA5 under expected binding conditions (e.g., physiological conditions).
The anti-DEFA5 antibody may bind to DEFA5 at various levels of affinity. One embodiment of the anti-DEFA5 antibody is a high affinity anti-DEFA5 antibody. The term “high affinity” antibody refers to an antibody having a binding affinity to DEFA5 of at least 10−10 M; preferably 10−11 M; even more preferably 10−12 M, as measured by surface plasmon resonance, e.g., BIACORE™ or solution-affinity ELISA.
The anti-DEFA5 antibody may bind to DEFA5 with high affinity (“high-affinity anti-DEFA5 antibody”). As used herein, a “high affinity anti-DEFA5 antibody” is an antibody that has a high binding affinity. “High binding affinity” refers to a high strength with which the epitope binds to an individual paratope (antigen-binding cite). Antibodies that have a high binding affinity bind more quickly to the antigen, permit greater sensitivity in assays, and better maintain a bond with the paratope when compared to an antibody having a lower affinity. The anti-DEFA5 antibody described herein may have a binding affinity to DEFA5 of at least as low as 10−7, 10−8, 10−9, 10−10, 10−11, or 10−12 KDs (M), or any range or subvalue thereof, The term “KD” refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, or the equilibrium dissociation constant of an antibody, antibody binding fragment, or molecular interaction. The equilibrium dissociation may be calculated by obtaining the dissociation rate constant (koff value) of a particular antibody-antigen interaction, with the association rate constant of a particular antibody-antigen interaction. A lower KD value indicates a higher binding affinity.
The anti-DEFA5 antibody described herein may also have a high specificity to DEFA5. A “specificity” refers to the ability to bind to a particular antigen, but not other antigens. Some embodiments of the anti-DEFA5 antibody display an affinity for DEFA5 that exceeds a displayed affinity to 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. The canonical human neutrophil defensin 1 protein (DEFA1) is described at UniProt Accession No. P59665, the sequence of which is provided herein as SEQ ID NO: 2. The canonical human neutrophil defensin 2 protein (DEFA2) is described at UniProt Accession No. P59665, the sequence of which is provided herein as SEQ ID NO: 3. The canonical human neutrophil defensin 3 protein (DEFA3) is described at UniProt Accession No. P59666, the sequence of which is provided herein as SEQ ID NO: 4. The canonical human neutrophil defensin 4 protein (DEFA4) is described at UniProt Accession No. P12838, the sequence of which is provided herein as SEQ ID NO: 5. The canonical human neutrophil defensin 6 protein (DEFA6) is described at UniProt Accession No. P12838, the sequence of which is provided herein as SEQ ID NO: 6. Further embodiments of the high specificity anti-DEFA5 antibody display a higher affinity to DEFA5 than to DEFA1, DEFA6, or both. In an embodiment, the anti-DEFA5 antibody has a high specificity to DEFA5 and does not bind, or substantially does not bind (i.e., has a low or no binding affinity), to HD1 and HD6. The anti-DEFA5 antibody may have a binding affinity to HD1 and/or HD6 of greater than about 10−10, 10−9, 10−8, 10−7, 10−6, 10−5, 10−4, 10−3, 10−2, 10−1 KDs (M), or any range or subvalue thereof. The anti-DEFA5 antibody may have a KDs (M) with one or both of DEFA1 and DEFA6 that is greater than one of the following values: 10−10, 10−9, 10−8, 10−7, 10−6, 10−5, 10−4, 10−3, 10−2, and 10−1. The anti-DEFA5 antibody may recognize an epitope binding region having at least 80%, 85%, 90%, 92%, 94%, 96%, or 98% sequence identity to positions 51-94 of SEQ ID NO: 1. The anti-DEFA5 antibody may recognize an epitope binding region having 100% sequence identity to positions 51-94 of SEQ ID NO: 1. Some embodiments of the anti-DEFA5 antibody do not recognize an epitope binding region having at least 80%, 85%, 90%, 92%, 94%, 96%, or 98% sequence identity to positions 1-49 of SEQ ID NO: 1. A specific embodiment of the anti-DEFA5 antibody does not recognize an epitope binding region having 100% sequence identity to positions 1-49 of SEQ ID NO: 1.
Examples of commercially available anti-DEFA5 antibodies include: Anti-alpha 5 Defensin antibody [EPR14309(B)] from ABCAM, Cambridge, United Kingdom; Anti-alpha 5 Defensin antibody (ab167591) from ABCAM, Cambridge, United Kingdom; Anti-alpha 5 Defensin antibody [8C8] (Catalogue #ab90802) from ABCAM, Cambridge, United Kingdom; Defensin 5 Monoclonal Antibody (8C8) (Catalogue #MA1-46026) from THERMO FISHER SCIENTIFIC INC., Waltham, MA; Anti-Alpha Defensin-5 (DEFA5) Antibody, clone 8C8 (Catalogue #MABF31) from MILLIPORESIGMA, Burlington, MA; Defensin 5 Antibody LS-C50934 (Catalogue #LS-C50934-100) from LSBIO, Seattle, WA; Defensin alpha 5 Antibody (8C8) (Catalogue #NB110-60002/NB110-60002SS) from NOVUS BIOLOGICALS, Littleton, CO; Defensin alpha 5 Antibody (8C8) (Catalogue #NBP1-84282) from NOVUS BIOLOGICALS, Littleton, CO; Defensin alpha 5 antibody (Catalogue #orb156565) from BIORBYT, Cambridge, United Kingdom; Defensin alpha 5 Antibody (Catalogue #bs-4313R) from BIOSS INC., Woburn, MA; Defensin alpha 5 antibody [N1C3] (Catalogue #GTX116079) from GENETEX, INC., Irvine, CA; Anti-DEFA5 Antibody (HPA015775) from ATLAS ANTIBODIES, Bromma, Sweden; DEFA5 antibody (catalogue number 972207.111 or CSL 1450400) from R&D SYSTEMS, Minneapolis, MN; and α-defensin 5 antibody (catalogue #53997) from SANTA CRUZ BIOTECHNOLOGY, INC., Dallas, TX, among many others.
In one embodiment, the anti-DEFA5 antibody is α-defensin 5 antibody (catalogue #53997) from SANTA CRUZ BIOTECHNOLOGY, INC., Dallas, TX Surprisingly, it has been discovered that the α-defensin 5 antibody (catalogue #53997) is particularly advantageous for measuring the levels of DEFA5 and DEFA5 expression in a sample. In particular, the α-defensin 5 antibody (catalogue #53997) displays a high affinity and high specificity for DEFA5, including the high affinity and high specificity for HD5 and low to no affinity and specificity for other defensins, such as HD1-HD4 or HD6. The anti-DEFA5 antibody may be a kappa light chain polypeptide subunit. In some embodiments, the anti-DEFA5 antibody is a mammalian antibody, such as a human antibody or a canine antibody.
A method of 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 a anti-DEFA5 antibody, and diagnosing the subject as suffering from Crohn's disease if the level or expression of DEFA5 is indicative of a subject having Crohn's disease; or diagnosing the subject as suffering from ulcerative colitis if the level or expression of DEFA5 is indicative of a subject having ulcerative colitis. The sample can be taken from any suitable source for measuring DEFA5 concentration, DEFA5 expression levels, such as tissue samples from the intestine, such as from the large intestine or rectum. In this disclosure the term “expression of DEFA5” should be interpreted to mean the expression of the DEFA5 gene; “levels of DEFA5” should be interpreted to mean the concentration of DEFA5 protein.
The sample may be taken from a subject who is suffering from or at risk of IBD. The subject may display one or more symptoms characteristic of IBD, such as severe diarrhea, abdominal pain, fatigue, and weight loss. In some embodiments of the method, the subject displays more than one of said symptoms. In further embodiments the subject displays two, three, or four of said symptoms.
It has been discovered that DEFA5 is differentially expressed in subjects having UC and CD. Used in this way, DEFA5 concentration and DEFA5 expression can be utilized and measured, using the targeted DEFA5 antibody, as a biomarker for distinguishing UC and CD in patients having IBD. As ileal pouch anal anastomosis is clinically much more successful in patients having UC than in patients suffering from CD, patients identified as having levels of DEFA5 indicative of UC, or not having CD, may be treated with ileal pouch anal anastomosis. Indeed, as DEFA5 is produced by Paneth cells only, one would not expect to find Paneth cells that secret DEFA5 in the colon. It has been discovered that Paneth cells (secreting DEFA5) are abundantly found in subjects having CC. On the other hand, patients identified as having levels of DEFA5 and DEFA5 expression indicative of CD may be treated with any suitable treatment for CD. In an embodiment, a diagnosing step, such as diagnosing a subject with UC or CD, is optional.
The anti-DEFA5 antibody may have a complementarity determining region (CDR) that is complementary to each of, or all of, the DEFA5 sequence of the P, B, and M binding sites of DEFA5, as shown in
In another embodiment, the antibody may be an antibody having a certain degree of identity to a polypeptide sequence complementary to the P, B, and M binding sites of DEFA5. For example, the antibody may be at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 100% identical to a complementary polypeptide sequence to polypeptide sequence of the P, B, and M binding sites of DEFA5.
Some embodiments of the antibody disclosed herein more specifically target DEFA5 than they do other alpha defensins.
It is believed that there may be a dysfunction in the activation pathway of DEFA5 in patients suffering from moderate and severe CD, and thus, an excess amount of inactive form DEFA5 is a potential mechanism for inflammation in patients suffering from CD. This excessive amount of inactive form DEFA5 may cause increased damage to the epithelial lining and potentially even a dysregulation in the levels and make-up of gut flora.
The methods may include a step of comparing the level of the DEFA5 to a benchmark value. The benchmark value may be a measure of central tendency based on levels observed in one or more populations of subjects that are established to be unafflicted by either UC or CD. For example, the benchmark value may be a mean level of the gene expression or protein concentration observed in samples from a population of subjects who are unafflicted by UC, unafflicted by CD, or both. The population may be defined by one or more of the patient's geography, age, ethnicity, sex, and medical history. The benchmark value may take into account a measure of variation combined with a measure of central tendency. For example, the benchmark value may be a mean level of the gene expression or protein concentration observed in a given tumor population, plus or minus a margin of error. The benchmark may be based on raw measurements (such as fragments of mRNA or cDNA per kb gene length per million reads) or normalized measurements (such as % of normal expression, or expression compared to a constitutively expressed or widely expressed gene with generally consistent expression, such as β-actin). An example of a suitable benchmark value is about 1 ng/mL DEFA5 or exactly 1 ng/mL DEFA5.
The benchmark may also be established by analysis of a control sample that is measured alongside the sample from the subject. Examples of suitable control samples are: a sample from a subject unafflicted with UC, a sample from a subject unafflicted with CD, a sample from a subject afflicted with UC (although unafflicted with CD), a sample from a subject afflicted with CD (although unafflicted with UC), a sample from a subject afflicted with diverticulitis (although unafflicted with either UC or CD), and a sample from a subject unafflicted from IBD. In some embodiments, the benchmark value level may be a normal level, such as described, infra.
In an embodiment, an assay method of differentially diagnosing UC and CD in a patient suffering from IBD includes measuring the level of DEFA5 or DEFA5 expression present in a sample obtained from the patient. The level of DEFA5 or MMP-7 concentration or expression in the tissue may be measured by any suitable peptide analysis. For example, the measuring step may include one or more of enzyme-linked immunosorbent assay (ELISA), cation-ion exchange, NMR analysis, genome-wide transcriptome analysis, and mass spectrometry. The method may include comparing the concentration or expression of DEFA5 in the sample to the 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 value. For example, the method may comprise comparing the concentration or expression of DEFA5 in the sample to the benchmark, and making a diagnosis of CD if the concentration or expression of DEFA5 in the sample is significantly greater than the benchmark value. As another example, the method may comprise comparing the concentration or expression of DEFA5 in the sample to the benchmark, and making a diagnosis of UC if the concentration or expression of DEFA5 in the sample is not significantly greater than the benchmark value. The measurement of the expression of DEFA5 or the concentration of DEFA5 in the sample may be measured in the same ex vivo or in vitro.
The difference in expression or concentration may be considered significant based on any of a variety of known statistical tests for significance. These are generally based on a collection of measurements made from a sampled population, and are affected by both the population size and the sampling size. Such statistical tests are well known in the art and are not further elaborated upon in this disclosure; outside references can be relied upon to enable those skilled in the art to determine statistical significance, such as Rosener's Fundamentals of Biostatistics, 8th ed. (2015), Cengage Learning, Boston, MA.
In an embodiment, an assay method of differentially diagnosing UC and CD in a patient suffering from IBD includes measuring the level of DEFA5 or DEFA5 expression present in a sample obtained from the patient. The level of DEFA5 or DEFA5 expression in the tissue may be measured by an enzyme-linked immunosorbent assay (ELISA) that uses the targeted DEFA5 antibody disclosed herein. The method includes diagnosing the patient as having UC if DEFA5 or DEFA5 expression is at any level that is indicative of a patient not having CD, such as less than 5× normal levels of DEFA5, or from less than about 5×-30× normal levels of DEFA5 or DEFA5 expression. In an embodiment, the patient is diagnosed as having UC if DEFA5 expression is at a level of less than 3×106 DEFA5 mRNA Transcript per 10 ng RNA. The diagnosing may diagnose the patient as having CD if the level of DEFA5 expression is at any level indicative of a patient having CD, such as from about 3×106 to 1.2×108 DEFA5 mRNA Transcript per 10 ng RNA. As used herein, a “normal level” of DEFA5 or DEFA5 expression means a level of DEFA5 or DEFA5 expression in the digestive tract tissue from a subject not having CD or UC, or a subject suffering from IBD and specifically UC. Normal DEFA5 expression may refer to from 1×105 to 9×105 DEFA5 mRNA Transcript per 10 ng RNA, or about 6×105 DEFA5 mRNA Transcript per 10 ng RNA.
In one embodiment, the assay methods involve determining the status of a subject with respect to the activity and/or expression of DEFA5 or the activity and/or expression of a polypeptide regulated by DEFA5. In one embodiment, such methods comprise determining the level of expression or activity of DEFA5 or a polypeptide regulated by DEFA5 in a sample from the subject with the targeted DEFA5 antibody disclosed herein. The method may further comprise collecting the sample from the subject. As used herein, a biological sample which is subjected to testing is a sample derived from a subject and includes, but is not limited to, any biological material, such as a bodily fluid. Examples of bodily fluids include, but are not limited to, whole blood, serum, saliva, tissue infiltrate, pleural effusions, lung lavage fluid, bronchoalveolar lavage fluid, and the like. The biological fluid may be a cell culture medium or supernatant of cultured cells. For example, the sample can be intestinal tissue, stool, blood, or serum. In embodiment, the biological sample is collected from the colon of a subject.
Some embodiments of the method comprise measuring the concentration of DEFA5 by selectively staining or dying the sample from the subject and measuring the signal from the stain. The stain or dye may comprise the any anti-DEFA5 antibody disclosed herein. The stain or dye may also comprise a reporter, such a colorimetric group, a radionuclide, a stable isotope, a fluorophore, a chromophore, an enzyme, a magnetic particle, and a quantum dot. The concentration of DEFA5 can then be measured by observing the signal from the reporter, such as by microscopy, colorimetry, radiometry, fluoroscopy, magnetotaxis, or any combination of the foregoing. In a specific embodiment of the method, the concentration of DEFA5 is measured by immunostaining the sample with an immunostain that recognizes DEFA5 and counting the number of stained cells by microscopy. This approach has the advantage of relative simplicity, and only requires the types of equipment that are already present in typical clinical laboratories. A diagnosis can be made based on a threshold number of cells that stain positive, such as at least 10%, 20%, and 30%. If the number of DEFA5 stained cells is significantly above the threshold value, than a diagnosis of CD can be made; whereas if the number of DEFA5 stained cells is significantly below the threshold value, than a diagnosis of UC can be made.
Those subjects in which DEFA5 activity and/or expression differs (increased or decreased) from a control or benchmark value or the activity of a polypeptide regulated by DEFA5 differs as compared to a control or benchmark value are determined to be suffering from or at risk for disease states and conditions associated with or characterized by increased or decreased DEFA5 activity.
Assay techniques that can be used to determine levels of expression or activity in a sample are known. Such assay methods include, but are not limited to, radioimmunoassays, reverse transcriptase PCR (RT-PCR) assays, immunohistochemistry assays, in situ hybridization assays, competitive-binding assays, Western Blot analyses, ELISA assays and proteomic approaches, two-dimensional gel electrophoresis (2D electrophoresis) and non-gel based approaches such as mass spectrometry or protein interaction profiling. 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 assay (ELISA), sandwich immunoassays, precipitin reactions, gel diffusion reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and immunoelectrophoresis assays. For examples of immunoassay methods, see U.S. Pat. Nos. 4,845,026 and 5,006,459. Any of the anti-DEFA5 antibodies disclosed herein may be in the assay.
The anti-DEFA5 antibody can be incorporated into an ELISA assay for the diagnosing methods. In addition, a reporter antibody generally is prepared. The reporter antibody is attached to a detectable reagent such as a radioactive, fluorescent, or enzymatic reagent, for example horseradish peroxidase enzyme or alkaline phosphatase. In one embodiment of the ELISA, to carry out the ELISA, the anti-DEFA5 antibody is incubated on a solid support that binds the antibody. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein. Next, the sample to be analyzed is incubated with the solid support, during which time the anti-DEFA5 antibody binds to DEFA5. Unbound sample is washed out with a buffer. A reporter antibody specifically directed to the antigen and linked to a detectable reagent is introduced resulting in binding of the reporter antibody to any antibody bound to the antigen. Unattached reporter antibody is then washed out. Reagents for detecting the presence of the reporter antibody are then added. The detectable reagent is then determined in order to determine the amount of antigen present. In an alternate embodiment, the antigen is incubated with the solid support, followed by incubation with one or more antibodies, wherein at least one of the antibodies comprises a detectable reagent. Quantitative results may be obtained by reference to a standard curve.
A method of treating IBD in a patient suffering from IBD may include: (a) measuring the level of DEFA5 or DEFA5 expression present in a sample obtained from the patient with the anti-DEFA5 antibody, whereby a level of DEFA5 or DEFA5 expression is obtained; (b) if the level of DEFA5 or DEFA5 expression is at a level indicative of a patient not having CD, treating the IBD in the patient with a suitable medical treatment for UC; if the level of DEFA5 or DEFA5 expression is at a level indicative of a patient having CD, treating the IBD in the patient with a suitable medical treatment for CD.
Suitable medical treatments for UC include ileal pouch anal anastomosis or the administration of pharmaceutical agents or salts thereof. Suitable pharmaceutical agents may be one or more of: an iron supplement; an oral 5-aminosalicylate, such as mesalamine, balsalazide and olsalazine; an anti-inflammatory; a corticosteroid; an immunosuppressant such as azathioprine, mercaptopurine, methotrexate, and cyclosporine; an anti-TNF-alpha antibody such as infliximab, adalimumab, and golimumab; an anti-α4-integrin antibody such as vedolizumab; and an antibacterial antibiotic, such as ciprofloxacin and metronidazole. Surgeries that are sometimes used to treat UC include a total proctocolectomy, and an ileal pouch anal anastomosis. Note that ileal pouch anal anastomosis are recognized as relatively ineffective when used to treat CD, in contrast to UC. It should also be noted that cyclosporine and golimumab, while currently approved for the treatment of UC in the United States, are not currently approved for the treatment of CD. Some embodiments of the method involve performing an intervention that is effective to treat UC, but either ineffective to treat CD or not yet approved by regulatory authorities for the treatment of CD.
Suitable medical treatments for CD include the administration of pharmaceutical agents or salts thereof. Suitable pharmaceutical agents include: an oral 5-aminosalicylate, such as mesalamine; a vitamin supplement, such as a vitamin B-12 supplement and a vitamin D supplement; a mineral supplement, such as a calcium supplement; an anti-inflammatory; a corticosteroid such as prednisone and budesonide; an immunosuppressant such as azathioprine, tacrolimus, methotrexate, and mercaptopurine; an anti-TNF-α antibody, such as infliximab, adalimumab, and certolizumab pegol; an anti-α-4-integrin antibody, such as natalizumab and vedolizumab; an anti-interleukin antibody, such as ustekinumab; and an antibacterial antibiotic, such as metronidazole, and ciprofloxacin. Although certolizumab pegol, methotrexate, and natalizumab are approved in the US for the treatment of CD, they are not currently approved for the treatment of UC. Surgical approaches are sometimes used to treat severe cases of CD. Such surgeries include ostomy, colostomy, ileostomy, bowel resection, colectomy, proctocolectomy, and strictureplasty. In some embodiments of the method, the subject is treated using a diet that is advantageous for the management of CD, but not necessarily advantageous in the management of UC. One such diet is a low fat diet. Some embodiments of the method involve performing an intervention that is effective to treat CD, but either ineffective to treat UC or not yet approved by regulatory authorities for the treatment of UC. The intervention may be administration of a drug, to the exclusion of a surgery. The administration of a drug may be administration of a drug selected from the group consisting of: a vitamin supplement, an anti-inflammatory, a corticosteroid, prednisolone, methyl-prednisolone, oral budesonide, a 5-aminosalicylate, an immunosuppressant, azathioprine, mercaptopurine, an anti-TNF-alpha antibody, infliximab, adalimumab, certolizumab pegol, methotrexate, an anti-α4-integrin antibody, natalizumab, vedolizumab, an anti-interleukin antibody, ustekinumab, an antibacterial antibiotic, ciprofloxacin, metronidazole, an anticholinergic agent, propantheline, dicyclomine, hyoscyamine, a bile acid sequestrant, cholestyramine, colestipol, and colesevalm. The administration of a drug may be administration of a drug, vitamin, or mineral selected from the group consisting of: vitamin B12, vitamin D, calcium, certolizumab pegol, methotrexate, and natalizumab. The intervention may be enteral nutrition therapy, including elemental and non-elemental diets, such as by nasogastric tube feeding. In an embodiment, the level of DEFA5 or DEFA5 expression may be elevated above normal levels in patients who are likely to be diagnosed UC but, at the time the DEFA5 or DEFA5 expression level is measured, diagnosed as having IC. These patients may be treated with any suitable medical treatments for UC. The intervention may be placing the subject on a low fat diet or a high fiber diet.
The intervention suitable for UC may be administration of a drug selected from the group consisting of: an iron supplement, an anti-inflammatory, a corticosteroid, hydrocortisone, cortisone, prednisolone, a 5-aminosalicylate, an immunosuppressant, azathioprine, mercaptopurine, cyclosporine, an anti-TNF-alpha antibody, infliximab, adalimumab, golimumab, methotrexate, an anti-α4-integrin antibody, vedolizumab, an antibacterial antibiotic, ciprofloxacin, metronidazole, suppository mesalazine, enema mesalazine, olsalazine, balsalazide, enema budesonide, tacrolimus, and a combination of any of the foregoing. The intervention suitable for UC may be administration of a drug selected from the group consisting of: cyclosporine, and golimumab.
A kit is provided for measuring DEFA5 in a subject. The kit may find use in several of the methods provided above, as well as others. The kit may be, for example, used for the diagnosis of inflammatory bowel disease. The kit comprises an assay for measuring at least one of DEFA5 concentration and DEFA5 expression. The kit may include an assay comprising an anti-DEFA5 antibody; and a sample container configured to contain a sample selected from: a stool sample, a blood sample, a bowel tissue sample, and a serum sample. The first assay may include a sample collector selected from the group consisting of: a stool sample collector, a blood sample collector, a serum sample collector, and a bowel tissue collector.
Working Example 1 shows that human UC and CC can be distinguished molecularly by examining DEFA5 levels in colectomy tissues, colon biopsies, and/or sera in humans using the DEFA5 antibodies described herein. Also, Working Example 1 delineates the underlying mechanisms for the subtle differences between UC and CC. The ability to accurately distinguish CC from UC is significant and of clinical importance, and is especially meaningful for gastroenterologists and colorectal surgeons, particularly before deciding whether restorative proctocolectomy surgery is required in a patient having IBD.
The inability to accurately distinguish Crohn's disease (CC) from UC leads to an inexact diagnosis denoted as IC, which greatly affects the medical and surgical care of the patients. A preliminary assessment of DEFA5 expression was performed in a pilot cohort of IC patients as well as in UC patients who underwent RPC surgery. This showed that DEFA5 levels and to a lesser extent DEFA6 levels were higher in CC patient samples. The preliminary data reveal that detection of DEFA5 in the tissues of the IC patients or those from the RPC surgery UC patients, who in fact were CC, were more accurately differentiated CC from UC in then otherwise misdiagnosed patients.
Clinical Samples.
To show that aberrant DEFA5 expression in IBD patients is a more reliable diagnostic approach to differentiate CC from UC, the potential of detection of DEFA5 as a biomarker for CC in IBD patient samples diagnosed as UC, CC and IC tissues was explored.
Clinical Retrospective Studies on IBD Patients Reveal Persistent Diagnostic Uncertainty.
A retrospective investigation was conducted on a cohort of 21 patients diagnosed with IC between 2000 and 2007 at the IBD Center at VUMC, with a mean follow-up period of 8.7±3.7 (range, 4-14) years. In 2014, these patients were re-evaluated to determine whether the diagnosis resolved to UC or CC. Three GI pathologists blinded to the initial clinical outcome re-evaluated each patient and the new diagnosis was presented as a consensus among the attending physicians. The pathology reevaluations concluded that the diagnosis of 6 patients, (28.5%) remained as IC because these could still not be delineated into UC or CC. Meanwhile 43% and 28.5% resolved into UC and CC respectively (
Differential Expression of DEFA5 in CC and UC.
Two approaches were used, training and Independent test sets, to identify genes or their products that are differentially expressed in UC versus CC. In a training test set, a whole-transcriptome microarray was performed using RNA extracted and pooled from full-thickness colon samples from UC and CC patients (n=5) using the Affymetrix gene expression array according to the manufacturer's instructions (Affymetrix, Santa Clara, CA). Tissues from diverticulitis were used as control. This analysis showed a total of 484 genes that were up- or down-regulated antimicrobial peptides, and mucins between the two diseases. In a test set analysis using microarray technologies (Affymetrix, Santa Clara, CA). DEFA5 levels increased the most: 31-fold in CC vs. UC (p<7.23E-05), Table 2. In an independent test set, the gene expression profiling was independently verified using a PCR array (NanoString Technologies Inc., Seattle, WA) that specifically targeted inflammatory genes. It was found that DEFA5 was also increased 118-fold in CC vs. UC (p<0.001) in different colon samples from UC and CC patients with same disease activity as in test set. Table 3. The only gene to show up in both the microarray and the PCR array was DEFA5. Among the upregulated genes were α-defensin-5, other antimicrobial peptides, and mucins (Table 2). HD5 was increased the most: 31-fold in CC vs. UC (in a previous test HD5 increase by 118-fold in CC versus UC—Table 3). A full list of the microarray results can be found in Table 2. Table 2 shows a list of targets from an AFFYMETRIX cDNA microarray. A total of 484 genes were highlighted in the microarray as potential markers for distinguishing UC from CC. The gene showing the largest fold change between the two diseases was Human Defensin 5 (HD5).
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 (
Detection of DEFA5 in IBD Colectomy Tissues Agrees with Follow-Up Clinical Patient Outcomes as a Potentially Selective Diagnostic Tool for CC.
To test if detection of DEFA5 could be used to discriminate CC from UC and if this agreed with the patient follow-up clinical outcomes, detection of DEFA5 was carried out in the tissues from the 21 IC patients (
DEFA5 staining was also evaluated for the RPC and IPAA-operated patients described in
Establish the Specificity, and Selectivity of DEFA5 Antibodies.
Although the detection of DEFA5 in IBD tissues by IHC (
Establish the Specificity, and Selectivity of DEFA5 Antibodies for Sandwich ELISAs.
It is believed that the specificity of the commercially available ELISA kit can be determined by using DEFA6 as the antigen. If test proves to be not specific, it is believed that immunoprecipitations (IPs) using DEFA5 and DEFA6 expressed in bacteria and the 11 monoclonal antibodies to DEFA5 can identify those that may specifically form immune complexes with DEFA5 but not DEFA6 can be conducted. It is believed that a R&D Systems DEFA5 antibody (catalogue number 972207.111 or CSL 1450400) from R&D SYSTEMS, Minneapolis, MN, can be biotinylated and used as the detection antibody for the IPs. It is believed that the combination of the detection and the best capture antibodies to develop a more specific sandwich ELISA to detect DEFA5 in sera. Overall, it is believed that purified DEFA5 expressed in bacteria can be used to determine the appropriate concentrations of the DEFA5 antibodies for a robust ELISA and compare this with the commercially available ELISA kits.
Optimize the DEFA5 Sandwich ELISA to Detect DEFA5 in Sera from IBD Patients and Normal Subjects.
Since DEFA5 has not been used in IBD clinical settings, the goal of this task will be to establish normal blood DEFA5 reference interval levels and compare these to the values in sera of IBD patients. As depicted in
Detect DEFA5 Expression in Formalin-Fixed, Paraffin-Embedded (FFPE) IBD Biopsies/Tissues by IHC.
205 FFPE blocks were collected from IBD patients during a prior R21 funding period. Of these samples, 83 are from UC, 75 are from CC, and 47 are from IC patients. Tissues from DV patients will be used as non-IBD controls. The 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 the Ariol SL-50 digital high resolution imaging system (Leica) and quantified using the Tissue IA software at the Digital Histology Shared Resource (DHSR) at Vanderbilt University. This will enable the scoring for each slide based on its staining intensity and percentage of stained cells. This digital analysis of IHC results will serve as either an additional or an alternative bioassay for DEFA5 detection in biopsies.
Determine Whether Differences in the Levels of DEFA5 in the Colonic Mucosa Tissues Correlate with Circulating Levels of DEFA5 in CC, UC or Normal Subjects.
To determine whether the level of circulating or secreted DEFA5 (sDEFA5) correlates with its level of expression in situ, biopsy samples collected from the three groups will be used to isolate mRNA to determine by real time PCR the levels of DEFA5 message present. If possible, simultaneous biopsies from areas of CC activity vs. normal, adjacent tissue will inform us regarding whether serum levels denote active disease. To that end, biopsy specimens from normal, adjacent mucosal, actively inflamed mucosa, and mucosa around the transitional zone will be examined for DEFA5 mRNA expression. A power analysis indicates that comparison of prevalence between case and control groups having 30 subjects per group would generally have 84% power to detect 40% differences (e.g. 40% vs. 80%, odds ratio 6.0) based on a two-sided test with 0.05 alpha level. In terms of precision for the prevalence estimates, when the sample size of each group is 30, a two-sided 95% confidence interval for a single proportion will be 18% from the observed rate expected to be 50%. Sample size requirements were calculated based on detecting differentially expressed proteins between two groups while controlling for the false discovery rate (FDR). The measure is the ratio of protein expression (or fold) in cases to controls for a particular protein. Effect sizes equal to 1.5 fold change with more than 80% power can be detected, based on algorithms from Jung S H, Bioinformatics 2005; 21:3097. This assumes a FDR of 0.001 and two-sided p-values, and is based on a sample of 30 cases and 30 controls. To determine the significance of sDEFA5 as a candidate biomarker of active CC and achieve a model with predictive accuracy, the models such as generalized linear model with regularization approaches and ensemble methods for feature identification including boosting, bagging, and random forest classifier will be used.
It is believed that DEFA5 specific assays to detect DEFA5 in sera and tissues of IBD patients will be shown. With this, quantitative standard numerical normal reference interval (RI) values can be determined and developed for DEFA5 in sera from healthy subjects and relate these to the levels in IBD patient sera. The RI approach will be based on the central 95% of laboratory test values observed for a reference population that is free of diseases. Based on the preliminary data, it is anticipated that DEFA5 expression will be higher in tissues and sera from CC patients than in those from UC patients and that all patients with IC can be reclassified as either CC or UC patients. Although the power calculation indicated 46 patients per disease subtype, up to 100 patient tissues and sera per disease will be used from varied ethnic backgrounds to validate the detection of DEFA5 as a diagnostic tool for CC in sera.
While it is possible that the sensitivity of the assays may be poor due to relatively low levels of DEFA5 in sera, the assays will be validated by using alkaline phosphatase-conjugated anti-DEFA5 monoclonal antibodies or modify the assay to direct ELISA or a radioimmunoassay. Peroxidase-conjugated streptavidin 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 the development of a sandwich assay and the antibodies will avoid cross reaction with DEFA6.
Paneth cells are the differentiated progenies of the ileal epithelial stem cells (ISCs) that support the ISCs and provide antibacterial protection in mammals. Although IBD is inflammation prone, the notion that UC and CC are histologically different and require distinct surgical treatment options suggests that DEFA5 and/or specific pro-inflammatory cytokines play a major role in the pathogenesis of these diseases. It is believed that the high levels of DEFA5 in CC colectomy samples arise from aberrant metaplastic colonic crypt PCs; and that sera from patients with UC and CC contain 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 that pro-inflammatory cytokines such as TNF-α, IL-1β, and IFN-γ (Valdez I A et al., 2016) promote the differentiation of stem cells. However, whether bacterial enterotoxins or pro-inflammatory cytokines with or without and DEFA5 underlie the distinct pathologic features of CC relative to UC remain poorly understood. It is believed that DEFA5, bacterial enterotoxins and/or certain CC-associated pro-inflammatory cytokines promote the differentiation/expansion of colonic stem cells, and the distinct pathology associated with CC. To test this hypothesis, and in the absence of de facto animal models for CC, two different normal human colonic epithelial cell lines (NCM460 and NCM356), colonoids and/or enteroids from endoscopy biopsy tissues will be used to a) test the effects of purified DEFA5, DEFA6, and DEFA1 in the presence or absence of bacterial enterotoxins on the formation of metaplastic colonic PCs; b) assess the effects of CC- and UC-specific cytokines on DEFA5 secretion, the generation of ROS and cell viability. It is believed that DEFA5 and, to a lesser extent, DEFA6 will promote the secretion of CC-specific cytokines and the production of ROS, but attenuate both cell viability and tissue damage. It is also believed that the CC-specific cytokines will promote the synthesis/secretion of DEFA5 while the UC-specific cytokines will have the opposite effects.
Aberrantly expressed DEFA5 in CC patients is synthesized by metaplastic colonic crypt PCs. DEFA5 is predominantly synthesized by PCs. Therefore, it was determined whether PCs were present in the colon crypt of CC patients and to validate whether the pool of DEFA5 found in CC and in de novo CD colectomy samples originated from colonic epithelial crypts. All 20 UC samples from RPC-operated patients with de novo CD showed pools of colonic metaplastic crypt PCs, as demonstrated by H&E staining (
It was found that the PCs were the DEFA5 secreting cells by staining the colectomy tissue samples for DEFA5 and lysozyme (LYZ) to detect PCs. It was found that abundant crypt PCs were present in CC samples (
It is to be understood that any given elements of the disclosed embodiments of the invention may be embodied in a single structure, a single step, a single substance, or the like. Similarly, a given element of the disclosed embodiment may be embodied in multiple structures, steps, substances, or the like.
The foregoing description illustrates and describes the processes, machines, manufactures, compositions of matter, and other teachings of the present disclosure. Additionally, the disclosure shows and describes only certain embodiments of the processes, machines, manufactures, compositions of matter, and other teachings disclosed, but as mentioned above, it is to be understood that the teachings of the present disclosure are capable of use in various other combinations, modifications, and environments and are capable of changes or modifications within the scope of the teachings as expressed herein, commensurate with the skill and/or knowledge of a person having ordinary skill in the relevant art. The embodiments described hereinabove are further intended to explain certain best modes known of practicing the processes, machines, manufactures, compositions of matter, and other teachings of the present disclosure and to enable others skilled in the art to utilize the teachings of the present disclosure in such, or other, embodiments and with the various modifications required by the particular applications or uses. Accordingly, the processes, machines, manufactures, compositions of matter, and other teachings of the present disclosure are not intended to limit the exact embodiments and examples disclosed herein. Any section headings herein are provided only for consistency with the suggestions of 37 C.F.R. § 1.77, or otherwise to provide organizational queues. These headings shall not limit or characterize the invention(s) set forth herein.
This application cites and claims priority of U.S. Patent Application No. 62/522,652, filed Jun. 20, 2017.
This invention was made with government support under grant numbers R21DK095186; U54CA091408-09S1; U54CA091408-09S2; U54RR026140; U54MD007593; UL1RR024975; UL1TR000445; G12MD007586; U54CA163069; R24 DA036420; and S10RR0254970 awarded by the National Institute of Health. The government has certain rights in the invention. In this context “government” refers to the government of the United States of America.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/038582 | 6/20/2018 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/237064 | 12/27/2018 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 11427852 | M'Koma | Aug 2022 | B2 |
| 20090155788 | Abbas et al. | Jun 2009 | A1 |
| 20100004213 | Abbas | Jan 2010 | A1 |
| 20140179620 | Abbas | Jun 2014 | A1 |
| 20150253342 | Ataman-Onal et al. | Sep 2015 | A1 |
| Number | Date | Country |
|---|---|---|
| WO-2006063133 | Jun 2006 | WO |
| Entry |
|---|
| Arijs et al. Mucosal Gene Expression of Antimicrobial Peptides in Inflammatory Bowel Disease Before and After First Infliximab Treatment. PLoS ONE. 4(11): e7984: Published: Nov. 24, 2009 (Year: 2009). |
| Noble et al. Regional variation in gene expression in the healthy colon is dysregulated in ulcerative colitis. Gut. 57: 1398-1405; Published: Jun. 3, 2008 (Year: 2008). |
| Wehkamp et al. Reduced Paneth cell-defensins in ileal Crohn's disease. PNAS. 102(50): 18129-18134: Published: Dec. 13, 2005 (Year: 2005). |
| Emd Millipore, Anti-Alpha Defensin-5 (HD5) Antibody, clone 8C8, 2020, available at: http://www.emdmillipore.com/US/en/product/Anti-Alpha-Defensin-5-HD5-Antibody-clone-8C8,MM_NF-MABF31. |
| Williams et al. Human alpha defensin 5 is a candidate biomarker to delineate inflammatory bowel disease., PloS One, 2017, p. e0179710, vol. 12, No. 8. |
| Number | Date | Country | |
|---|---|---|---|
| 20210278417 A1 | Sep 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62522652 | Jun 2017 | US |
