Compositions and their use in methods for treating intestinal inflammation - Patents.com

Abstract

Described herein are methods for preventing or treating inflammatory bowel disease (IBD) by administering binding immunoglobulin protein (BiP) or a functional analog thereof.

Description

[Technical field]

[0001] FIELD OF THEINVENTION Embodiments of the present invention relate to compositions and methods for the treatment of inflammatory bowel disease (IBD) using binding immunoglobulin protein (BiP) or functional analogs thereof. [Background technology]

[0002] 2. Background of the Invention Inflammatory bowel disease (IBD), primarily represented by ulcerative colitis and Crohn's disease but also including non-infectious inflammation of the intestine, has been troubling gastroenterologists and immunologists since it was first described in modern medicine approximately 75–100 years ago. IBD is characterized by chronic inflammation of the gastrointestinal (GI) tract, resulting in damage to the GI tract. In 2017, there were 6.8 million cases of IBD worldwide. The burden of IBD is increasing worldwide, with the age-adjusted prevalence increasing from 79.5 per 100,000 population in 1990 to 84.3 per 100,000 population in 2017. 1 Most people with IBD are diagnosed in their 20s and 30s. Adults with IBD have higher health care utilization, including doctor visits, medication prescriptions, emergency department visits, hospitalizations, and surgeries, than adults without IBD. 2

[0003] IBD treatment usually involves either drug therapy or surgery. Anti-inflammatory drugs are often the first step in the treatment of inflammatory bowel disease (e.g., corticosteroids and aminosalicylates, such as mesalamine, balsalazide, and olsalazine). However, anti-inflammatory drugs are often insufficient to provide adequate and sustainable relief of IBD. Alternative therapies include immunosuppressants (e.g., azathioprine, mercaptopurine, and methotrexate), biologics that target neutralizing proteins that cause inflammation (e.g., infliximab (Remicade), adalimumab (Humira), golimumab (Simponi), certolizumab (Cimzia), vedolizumab (Entyvio), and ustekinumab (Stelara)), and, as a last resort, surgery. Over the past two decades, advances in biologics and small molecule therapeutics have led to a rapid increase in the IBD treatment armamentarium. Despite these advances, Crohn's disease and ulcerative colitis remain chronic and progressive diseases, and one of the main reasons for the persistent inflammation and intestinal damage is the failure of current medical therapies.

[0004] Currently, there is no cure or effective treatment for patients diagnosed with functional gastrointestinal disease such as IBD.The main goal of current therapy for IBD is to induce clinical remission by targeting symptoms, and then maintain it for a long time to achieve the best possible quality of life.Since current therapy has limited effectiveness, new therapies are needed to treat inflammatory and chronic pain in the intestine of subjects with IBD.Therefore, there is a need for improved therapies for the treatment of IBD. Summary of the Invention

[0005] The present invention provides a method for regulating inflammatory responses associated with intestinal inflammation. In some embodiments, the present invention provides a method for preventing or treating inflammatory bowel disease (IBD), comprising administering a therapeutically effective amount of binding immunoglobulin protein (BiP) or a functional analog thereof to a subject at risk of or suffering from IBD. In some embodiments, BiP or a functional analog thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, or a conservative amino acid substitute thereof. In an alternative embodiment, BiP or a functional analog thereof has an amino acid sequence consisting of SEQ ID NO:1. In some embodiments, the IBD is ulcerative colitis, Crohn's disease, or non-infectious inflammation of the intestine.

[0006] In some embodiments, the therapeutically effective amount of BiP-related polypeptide is administered once a month. In some embodiments, the BiP-related polypeptide is administered every 1, 2, 3, 4, 6, or 8 weeks. In some embodiments, the BiP-related polypeptide is administered intermittently, starting at the onset of IBD symptoms and stopping at the remission of IBD symptoms.

[0007] In some embodiments, the therapeutically effective amount of BiP-related polypeptide is 1-30 mg, 5-25 mg, 10-20 mg, 12-18 mg, or 15 mg by intravenous injection. In alternative embodiments, the therapeutically effective amount of BiP-related polypeptide is 25-75 mg, 30-70 mg, 40-60 mg, 45-55 mg, or 50 mg by intravenous injection. In yet other alternative embodiments, the therapeutically effective amount of BiP-related polypeptide is 50-200 mg, 75-150 mg, 85-125 mg, 90-110 mg, 95-105 mg, or 100 mg by intravenous injection.

[0008] Other implementations are also described and listed herein. [Brief description of the drawings]

[0009] For illustrative purposes, specific embodiments of the invention are shown in the drawings described below, but it should be understood that the invention is not limited to the precise arrangements, dimensions, and equipment shown.

[0010] [Figure 1] The amino acid sequence of the BiP analog IRL201805 (SEQ ID NO:1) is provided. [Diagram 2] The amino acid sequence of the His-tagged BiP analog expressed by plasmid pQE2 (SEQ ID NO:2) is provided. [Figure 3A] 3A-D provide the amino acid sequence of the BiP analog (SEQ ID NO:3). [Figure 3B] FIG. 3B is a continuation of FIG. 3A. [Figure 3C] This is a figure showing a continuation of Figure 3B. [Figure 3D] This is a figure showing a continuation of Figure 3C. [Figure 4A] 4A-D provide the amino acid sequence of the BiP analog (SEQ ID NO:4). [Figure 4B] This is a figure showing a continuation of Figure 4A. [Figure 4C] This is a figure showing a continuation of Figure 4B. [Figure 4D] This is a figure showing a continuation of Figure 4C. [Diagram 5] The amino acid sequence of native BiP (SEQ ID NO:5) is provided. [Figure 6] Figure 6A-E provides an overview of the procedures used to evaluate the effects of ex vivo human primary immune cells in inflamed and non-inflamed colon tissue treated with IRL201805. [Figure 7] FIG. 1 shows the effect of 12 hour treatment with 20 μg / mL IRL201805 on CD69+ / CD39+ Tregs in inflamed and non-inflamed colon tissue from IBD patients (Crohn's disease or ulcerative colitis) as determined by flow cytometry. [Figure 8]Shown is the mean fluorescence intensity of CD69 and CD39 surface expression on Tregs derived from peripheral blood mononuclear cells (PBMCs) cultured with 20 µg / mL of IRL201805 for 20–72 h at 37 °C. [Figure 9A] Figures 9A-B show the effect of 20 μg / mL IRL201805 on the JAK-STAT signaling pathway at the transcript and protein levels. Figure 9A provides an RNAseq analysis of gene transcript levels of key signaling genes involved in CD69 activation. Figure 9B provides an immunoblot of p-STAT5 levels in cell lysates from untreated and IRL1805-treated PBMCs. [Figure 9B] Please see the legend to FIG. 9A. [Figure 10] Figure 10A provides a schematic diagram of CD69-associated ligands. Figure 10B provides a bar graph showing the effect of IRL201805 on CD69 ligands, galectin-1 (Gal-1), and S100A8 / A9, as detected by TMT quantitative proteomics. Figure 10B provides a graphical representation of the increase in the post-translational modification trimethylation on threonine 113 on S100A9 following IRL201805 treatment. [Figure 11] Figures 11A-C provide bar graphs showing the effect of IRL201805 on the expression of surface CD154 and CD69 activation markers on the following T lymphocytes from PBMC: Tregs (Figure 11A), effector T cells (Teffs; Figure 11B), and CD8 T cells (Figure 11C). Results are from PBMCs isolated from four healthy donors. Mean ± SD is shown. P values ​​represent paired sample t-test. NS = not significant. [Figure 12] Schematic diagram of the effect of IRL201805 on CD69 and CD69-associated ligands. Abbreviations: APC: antigen-presenting cell, TCR: T cell receptor, Treg: regulatory T cell, P: phosphorylation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Detailed Description of the Invention The subject innovation will now be described with reference to the drawings, wherein like reference numerals are used to refer to like elements throughout. In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the invention. It may be apparent, however, that the invention may be practiced without these specific details. In other instances, well-known structures and devices are shown in block diagram form to facilitate description of the invention. It will be appreciated that certain aspects, modes, embodiments, variations and features of the invention are described below at varying levels of detail in order to provide a substantial understanding of the invention.

[0012] definition For convenience, the meanings of some terms and phrases used in the specification, examples, and the appended claims are provided below. Unless otherwise stated or implied from the context, the following terms and phrases include the meanings provided below. Since the scope of the present invention is limited only by the claims, the definitions are provided to help explain certain embodiments and are not intended to limit the invention of the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. If there is an apparent discrepancy between the use of a term in the art and its definition provided herein, the definition provided herein shall prevail.

[0013] As used in this specification and the appended claims, the singular forms include plural references unless the content clearly dictates otherwise. For example, a reference to "a cell" includes a combination of two or more cells, and the like.

[0014] As used herein, the term "approximately" or "about" with respect to a value or parameter is generally interpreted to include numbers within 5%, 10%, 15%, or 20% in either direction (greater or less) of that number, unless otherwise stated or clear from the context (except where such number is less than 0% or more than 100% of a possible value). As used herein, reference to "approximately" or "about" a value or parameter includes embodiments that are directed to (and describe) that value or parameter. For example, a description that refers to "about X" includes a description of "X."

[0015] As used herein, the term "or" means "and / or." The term "and / or" as used in phrases such as "A and / or B" herein is intended to include both A and B, A or B, A (single), and B (single). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A (single), B (single), and C (single).

[0016] As used herein, the term "comprising" means that other elements may be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation.

[0017] The term "consisting of" refers to compositions, methods, and each component described herein that does not include any element not recited in that description of the embodiment.

[0018] As used herein, the term "consisting essentially of" refers to that component required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the invention.

[0019] The terms "statistically significant" or "significant" refer to statistical significance, generally meaning a difference of 2 standard deviations (2 SD) or greater.

[0020] As used herein, the term "subject" refers to a mammal, including, but not limited to, a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate. A subject may be, but is not limited to, a pet (e.g., dog, cat), an agricultural livestock animal (e.g., cow, horse, pig, chicken, etc.), or a laboratory animal (e.g., mouse, rat, rabbit, etc.). A subject includes a human subject. A human subject may be a pediatric, adult, or geriatric subject. A human subject may be of either gender.

[0021] As used herein, the terms "effective amount" and "therapeutically effective amount" include an amount sufficient to prevent or ameliorate the symptoms of a disease or medical condition, such as rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, rejection of organ, skin, tissue, blood, serum, plasma transplants, inflammatory bowel disease, or immune disorders, including Crohn's disease. Of course, there are many methods known in the art for determining the effective amount for a given application. For example, pharmacological methods for dose determination may be used in therapeutic situations. In the context of therapeutic or prophylactic applications, the amount of the composition administered to a subject will depend on the type and severity of the disease, as well as individual characteristics such as overall health, age, sex, weight, and tolerance to drugs. It will also depend on the extent, severity, and type of disease. Those skilled in the art will be able to determine the appropriate dosage depending on these and other factors. The composition may also be administered in combination with one or more additional therapeutic compounds.

[0022] As used herein, the terms "treatment" or "treat" or "palliative" or "palliating" refer to both (1) therapeutic measures that cure, slow, reduce symptoms, and / or stop the progression of a diagnosed disease or infection, and (2) prophylactic or preventative measures that prevent or delay the onset of a disease or infection. Symptoms of IBD may include, but are not limited to, persistent diarrhea, abdominal pain, rectal bleeding / bloody stool, fecal urgency and incontinence, fatigue, fever, anemia, and / or weight loss. Malnutrition and growth retardation may also occur in subjects who develop IBD during childhood.

[0023] As used herein, the terms "treatment", "treating", or "treatment" or "palliative" when used in relation to a disease, disorder, or medical condition refer to therapeutic treatment for a condition, the purpose being to reverse, alleviate, ameliorate, inhibit, slow down, or stop the progression or severity of a symptom or condition. The term "treating" includes reducing or alleviating at least one adverse effect of a symptom or condition. A treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of a condition is reduced or stopped. That is, "treatment" includes not only the improvement of a symptom or marker, but also the halting or at least slowing of the progression or worsening of a symptom that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, the alleviation of one or more symptoms, a reduction in the extent of the disorder, a stabilized (i.e., non-worsening) state of immune impairment, a delay or slowing of immune impairment, and an increase in lifespan compared to that expected in the absence of treatment.

[0024] As used herein, the term "intermittent" administration means that a therapeutic agent or drug is administered in the presence of IBD symptoms and administration is discontinued upon remission of the IBD symptoms.

[0025] As used herein, the term "long-term" administration means that therapeutic agent or drug is administered for a period of at least 12 weeks.This includes that therapeutic agent or drug is administered for a period of at least 12 weeks or to be effective for a period of at least 12 weeks, and this does not necessarily mean that administration itself is carried out for 12 weeks, for example, when a sustained release composition or a long-acting therapeutic agent or drug is used.Therefore, subject is treated for a period of at least 12 weeks.In many cases, long-term administration is at least 4, 5, 6, 7, 8, 9 months or more, or at least 1, 2, 3, 5, 7, or 10 years or more.

[0026] The administration of the compositions contemplated herein can be carried out in any convenient manner, including aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. In a preferred embodiment, the compositions are administered parenterally. As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrasternal, and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

[0027] The terms "reduce", "reduced", "reduction", or "inhibit" are all used herein to mean a statistically significant amount of reduction. In some embodiments, "reduce", "reduction" or "reduce" or "inhibit" generally means a reduction of at least 10% compared to a reference level (e.g., without administration of a given treatment or agent), and may include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, "reduce" or "inhibit" does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. Preferably, the decrease is to a level that is accepted as within the normal range for individuals without the given disorder.

[0028] The terms "increased," "increase," "enhance," or "activate" are all used herein to mean a statistically significant amount of increase. In some embodiments, the terms "increase," "increase," "enhance," or "activate" can mean an increase of at least 10% compared to a reference level, e.g., an increase of at least about 20% compared to a reference level, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase up to and including 100%, or any increase between 10 and 100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or more compared to a reference level. In the context of a marker or symptom, an "increase" is a statistically significant increase in such level.

[0029] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to designate a series of amino acid residues bound together by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein" and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylation, glycation, glycosylation, etc.) and amino acid analogs, regardless of their size or function. While "protein" and "polypeptide" are often used in reference to relatively large polypeptides, and the term "peptide" is often used in reference to small polypeptides, the use of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to gene products and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs of the foregoing.

[0030] In various embodiments described herein, it is further intended to encompass any variant (natural or otherwise), allele, homolog, conservatively modified variant, and / or conservatively substituted variant of the specific polypeptide described. With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that change a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants", which alterations result in the replacement of an amino acid with a chemically similar amino acid and retain the desired activity of the polypeptide. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles consistent with the present disclosure.

[0031] In some embodiments, the polypeptides described herein (or nucleic acids encoding such polypeptides) may be functional fragments of one of the amino acid sequences described herein. As used herein, a "functional fragment" is a peptide fragment or segment that retains at least 50% of the activity of a wild-type reference polypeptide according to the assays described below. Functional fragments may include conservative substitutions of the sequences disclosed herein.

[0032] In some embodiments, the polypeptides described herein may be variants of the sequences described herein. In some embodiments, the variants are conservatively modified variants. Conservative substitution variants can be obtained, for example, by mutation of the native nucleotide sequence. As referred to herein, a "variant" is a polypeptide that is substantially homologous to a native or reference polypeptide, but has an amino acid sequence that differs from the amino acid sequence of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. A DNA sequence encoding a variant polypeptide includes a sequence that encodes a variant protein or fragment thereof that contains one or more additions, deletions, or substitutions of nucleotides when compared to the native or reference DNA sequence, but retains activity. A wide variety of PCR-based site-directed mutagenesis approaches are known in the art and can be applied by one of ordinary skill in the art.

[0033] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. A nucleic acid may be either single-stranded or double-stranded. A single-stranded nucleic acid may be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, it may be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one embodiment, the nucleic acid may be DNA. In another embodiment, the nucleic acid may be RNA. Suitable DNA may include, for example, genomic DNA or cDNA. Suitable RNA may include, for example, mRNA.

[0034] In some embodiments of any aspect, the polypeptide, nucleic acid, or cell described herein may be engineered. As used herein, "engineered" refers to an aspect that has been manipulated by the hand of man. For example, a polypeptide is considered to be "engineered" if at least one aspect of the polypeptide, such as its sequence, has been manipulated by the hand of man to be different from the aspect in which it exists in nature. As is common practice and understood by those skilled in the art, the descendants of engineered cells typically continue to be referred to as "engineered", even though actual manipulation has been performed on the previous entity.

[0035] In some embodiments, the nucleic acid encoding the polypeptide (e.g., antibody or antibody reagent) described herein is contained in a vector. In some aspects described herein, the nucleic acid sequence encoding a given polypeptide described herein, or any module thereof, is operably linked to a vector. Vectors can include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, chromosomes, viruses, virions, and the like.

[0036] As used herein, the term "expression vector" refers to a vector that directs the expression of an RNA or polypeptide from a sequence linked to a transcriptional regulatory sequence on the vector. The sequence to be expressed is often, but not necessarily, heterologous to the cell. An expression vector may contain additional elements, for example, an expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example, human cells for expression, and in a prokaryotic host for cloning and amplification. The term "expression" refers to the cellular processes involved in the production of RNA and proteins, and optionally secretion of proteins, including, for example, but not limited to, transcription, transcription processing, translation, and protein folding, modification, and processing, as applicable. An "expression product" includes RNA transcribed from a gene, and a polypeptide obtained by translation of mRNA transcribed from a gene. The term "gene" refers to a nucleic acid sequence (DNA) that is transcribed into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions preceding and following the coding region, such as 5' untranslated (5'UTR) or "leader" and 3'UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0037] As used herein, the term "isolated" or "partially purified" refers to a nucleic acid or polypeptide that has been separated from at least one other component (e.g., nucleic acid or polypeptide) that, in the case of a nucleic acid or polypeptide, is present with the nucleic acid or polypeptide as found in its natural source and / or that may be present with the nucleic acid or polypeptide when expressed by a cell or, in the case of a secreted polypeptide, when secreted. A chemically synthesized nucleic acid or polypeptide or a nucleic acid or polypeptide synthesized using in vitro transcription / translation is considered "isolated". The term "purified" or "substantially purified" refers to an isolated nucleic acid or polypeptide that is at least 95% by weight, e.g., at least 96%, at least 97%, at least 98%, at least 99% by weight, or more, of the subject nucleic acid or polypeptide. In some embodiments, the antibodies, antigen-binding portions thereof, or chimeric antigen receptors (CARs) described herein are isolated. In some embodiments, the antibodies, antibody reagents, antigen-binding portions thereof, or CARs described herein are purified.

[0038] As used herein, "engineered" refers to an aspect that has been manipulated by the hand of man. For example, an antibody, an antibody reagent, its antigen-binding portion, a CAR, or a bispecific antibody is considered to be "engineered" if the sequence of the antibody, the antibody reagent, its antigen-binding portion, a CAR, or a bispecific antibody is manipulated by the hand of man so that it is different from the sequence of the antibody when it exists in nature. As is common practice and understood by those skilled in the art, the descendants and copies of engineered polynucleotides and / or polypeptides typically continue to be referred to as "engineered", even though actual manipulation has been performed on the previous entity.

[0039] Pharmaceutical Compositions The compositions and methods of the present invention can be utilized to treat individuals in need thereof. In certain embodiments, the individual is a mammal, such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or compound is preferably administered as a pharmaceutical composition, for example, comprising the compound of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions, such as water or physiologically buffered saline, or other solvents or vehicles, such as glycols, glycerol, oils, such as olive oil, or injectable organic esters. In a preferred embodiment, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes such as injection or implantation that avoid transport or diffusion through epithelial barriers), the aqueous solution is pyrogen-free or substantially pyrogen-free. The excipient can be selected, for example, to provide delayed release of the drug or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition may be in unit dosage form such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophilized powders for reconstitution, powders, solutions, syrups, suppositories, injections, etc. The composition may also be present in a transdermal delivery system, such as a skin patch. The composition may also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0040] A pharma- ceutically acceptable carrier may contain a physiologically acceptable agent that acts to stabilize, increase the solubility, or increase the absorption of a compound, such as the compound of the present invention. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers or excipients. The choice of a pharma- ceutical acceptable carrier that includes a physiologically acceptable agent depends, for example, on the route of administration of the composition. The formulation or pharmaceutical composition may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (formulation) may also be a liposome or other polymer matrix, into which, for example, the compound of the present invention may be incorporated. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively simple to prepare and administer.

[0041] The phrase "pharmacologically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, and other problems and complications, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0042] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not damaging to the patient. Some examples of materials that can function as pharma-ceutically acceptable carriers include: (1) sugars (such as lactose, glucose, and sucrose), (2) starches (such as corn starch and potato starch), (3) cellulose and its derivatives (such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate), (4) powdered tragacanth, (5) malt, (6) gelatin, (7) talc, (8) excipients (such as cocoa butter and suppository wax), (9) oils (peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, etc.), (10) cellulose, cellulose acetate, cellulose esters, cellulose esters, cellulose acetate ... , corn oil, and soybean oil), (10) glycols, (such as propylene glycol), (11) polyols (such as glycerin, sorbitol, mannitol, and polyethylene glycol), (12) esters (such as ethyl oleate and ethyl laurate), (13) agar, (14) buffers (such as magnesium hydroxide and aluminum hydroxide), (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) phosphate buffers, and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0043] The pharmaceutical compositions (formulations) may be administered to a subject by any of a number of routes of administration, including, for example, orally (e.g., as a drench, tablet, capsule (including sprinkle capsule and gelatin capsule), bolus, powder, granule, paste for application to the tongue, such as in an aqueous or non-aqueous solution or suspension); absorption through the oral mucosa (e.g., sublingual); subcutaneously; transdermally (e.g., as a patch applied to the skin); and topically (e.g., as a cream, ointment or spray applied to the skin). The compounds may also be formulated for inhalation. In certain embodiments, the compounds may simply be dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable therefor can be found, for example, in U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970, and 4,172,896, and the patents cited therein.

[0044] The formulations may be conveniently provided in unit dosage form and may be prepared by any method known in the art of pharmacy. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form varies depending on the host treated, the particular mode of administration. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form is generally the amount of compound that produces a therapeutic effect. Generally, out of 100 percent, this amount will range from about 1 percent to about 99 percent of active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent.

[0045] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the present invention, with the carriers and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0046] Formulations of the present invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), lyophilized, powder, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, as an elixir or syrup, or as a pastille (using an inert base, such as gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. The composition or compound may also be administered as a bolus, electuary, or paste.

[0047] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharma- ceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders (such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid), (2) binders (such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia), (3) humectants (such as glycerol), (4) disintegrating agents (such as glycerol), (5) glycerol-containing agents (such as glycerol), (6) glycerol-containing agents (such as glycerol), (7) glycerol-containing agents (such as glycerol), (8) glycerol-containing agents (such as glycerol), (9) glycerol-containing agents (such as glycerol), (10) glycerol-containing agents (such as glycerol), (11) glycerol-containing agents (such as glycerol), (12) glycerol-containing agents (such as glycerol), (13) glycerol-containing agents (such as glycerol), (14) glycerol-containing agents (such as glycerol), (15) glycerol-containing agents (such as glycerol), (16) glycerol-containing agents (such as glycerol), (17) glycerol-containing agents (such as glycerol), (18) glycerol-containing agents (such as glycerol), (19) glycerol-containing agents (such as glycerol), (20) glycerol-containing agents (such as glycerol), (21) glycerol-containing agents ( agents (such as agar-agar, calcium carbonate and potato or tapioca starch, alginic acid, certain silicates, sodium carbonate), (5) solution retarders (such as paraffin), (6) absorption enhancers (such as quaternary ammonium compounds), (7) wetting agents (such as cetyl alcohol and glycerol monostearate), (8) absorbents (such as kaolin and bentonite clay), (9) lubricants (such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof), (10) complexing agents (such as modified and unmodified cyclodextrins), and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets, and pills, the pharmaceutical compositions may also include buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules, using such excipients as lactose or milk sugar, and high molecular weight polyethylene glycols.

[0048] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surface active agents or dispersing agents. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0049] Tablets and other solid dosage forms of pharmaceutical compositions, such as, for example, dragees, capsules (including sprinkle capsules and gelatin capsules), pills, and granules, can be optionally scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They can also be formulated to provide sustained or controlled release of the active ingredient therein, for example, using various proportions of hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres to provide the desired release profile. They can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or any other sterile injectable medium immediately before use. These compositions can also optionally contain emulsifying agents, and can optionally be compositions that release the active ingredient in a delayed manner, only, or preferentially, in a certain part of the gastrointestinal tract. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form, where appropriate, with one or more of the above-mentioned excipients.

[0050] Liquid dosage forms useful for oral administration include pharma- ceutically acceptable emulsion, lyophilized agent for reconstitution, microemulsion, solution, suspension, syrup, and elixir.In addition to active ingredient, liquid dosage forms may contain inert diluents commonly used in the art (such as water or other solvents), cyclodextrin and its derivatives, solubilizers and emulsifiers (such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oil (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof).

[0051] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0052] In addition to the active compounds, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0053] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants.The active compound may be mixed under sterile conditions with a pharma- ceutically acceptable carrier, and any preservatives, buffers, or propellants that may be required.

[0054] The ointments, pastes, creams, and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0055] Powders and sprays can contain, in addition to the active compounds, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons, and volatile unsubstituted hydrocarbons, such as butane and propane.

[0056] Transdermal patch has the additional advantage of providing controlled delivery of the compound of the present invention into the body.These dosage forms can be prepared by dissolving or dispersing active compound in suitable medium.Absorption enhancers can also be used to increase the flux of compound across the skin.The rate of this flux can be controlled by either providing a rate-controlling membrane or dispersing compound in a polymer matrix or gel.

[0057] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intraocular (e.g., intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrasternal, and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration include combinations of one or more active compounds with one or more pharma- ceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into a sterile injectable solution or dispersion immediately prior to use, which may include antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0058] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical composition of the present invention include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical composition of the present invention include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants. Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants.

[0059] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial action can be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the composition. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0060] In some cases, it is desirable to delay the absorption of drugs from subcutaneous or intramuscular injections in order to prolong the effect of drugs.This can be achieved by using liquid suspensions of crystalline or non-crystalline materials with low water solubility.The absorption rate of the drug then depends on its dissolution rate, which can depend on crystal size and crystalline form.Alternatively, the delayed absorption of drug forms in parenteral administration can be achieved by dissolving or suspending the drug in an oil vehicle.

[0061] Injectable depot forms are prepared by forming microencapsulated matrices of the subject compound in biodegradable polymers such as polylactide-polyglycolide.Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides).Injectable depot formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0062] For use in the methods of the invention, the active compound may be provided per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably 0.5 to 90%) of the active ingredient in combination with a pharma- ceutically acceptable carrier.

[0063] The introduction method may also be provided by a rechargeable or biodegradable device.For the controlled delivery of drugs, including proteinaceous biopharmaceuticals, various sustained release polymeric devices have been developed and tested in vivo in recent years.Various biocompatible polymers, including both biodegradable and non-degradable polymers (including hydrogels), can be used to form implants for sustained release of compounds at specific target sites.

[0064] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0065] The selected dosage level will depend on a variety of factors including the activity of the particular compound or combination of compounds, or esters, salts thereof or amides thereof, used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular compound used, the age, sex, weight, condition, overall health and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0066] A physician or veterinarian of ordinary skill in the art can easily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can start the dosage of the pharmaceutical composition or compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. A therapeutically effective amount refers to the concentration of the compound sufficient to induce the desired therapeutic effect. It is generally understood that the effective amount of the compound varies according to the subject's weight, sex, age, and medical history. Other factors that affect the effective amount include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent administered together with the compound of the present invention. A larger total dose can be delivered by multiple administrations of the agent. Methods for determining efficacy and dosage are known to those skilled in the art. See, for example, Isselbacher et al. (1996). 8

[0067] In general, a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0068] If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more subdoses administered separately at appropriate intervals throughout the day, optionally in unit dosage form. In certain embodiments of the present invention, the active compound may be administered two or three times a day. In other embodiments, the active compound is administered once a day.

[0069] The patient undergoing this treatment may be any animal in need, including primates, particularly humans, as well as other mammals such as cows, pigs, sheep, cats and dogs, poultry, and pets in general.

[0070] In certain embodiments, the compounds of the invention may be used alone or may be administered in combination with another type of therapeutic agent.

[0071] The present disclosure includes the use of pharma- ceutically acceptable salts of the compounds of the present invention in the compositions and methods of the present invention. In certain embodiments, the contemplated salts of the present invention include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkyl ammonium salts. In certain embodiments, the contemplated salts of the present invention include, but are not limited to, L-arginine, benentamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, the contemplated salts of the present invention include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts.In certain embodiments, salts contemplated by the present invention include, but are not limited to, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gensulfonic acid, methyl ... Thidic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippo, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanate, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenate.

[0072] Pharmaceutically acceptable acid addition salts may also exist as various solvates with water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates may also be prepared. The source of such solvates may be from the solvent of crystallization inherent to the solvent of preparation or crystallization, or may be exogenous to such solvent.

[0073] Wetting agents, emulsifying agents, and lubricating agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the composition.

[0074] Examples of pharma- ceutically acceptable antioxidants include: (1) water-soluble antioxidants (such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.); (2) oil-soluble antioxidants (such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like); and (3) metal chelators (such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.).

[0075] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art to which this disclosure belongs. It is to be understood that the present invention is not limited to the specific methodology, protocols, and reagents, etc. described herein, as such may vary. The terms used herein are for the purpose of describing particular embodiments only, and are not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in the Merck Manual of Diagnostic and Therapy, 9 The Encyclopedia of Molecular Cell Biology and Molecular Medicine 10 , Molecular Biology and Biotechnology: a Comprehensive Desk Reference, 11 Immunology, 12 Janeway's Immunobiology, 13 Lewin's Genes XI, 14 Molecular Cloning: A Laboratory Manual, 15 Basic Methods in Molecular Biology, 16 Laboration Methods in Enzymology, 17Current Protocols in Molecular Biology(CPMB) 18 ,Current Protocols in Protein Science(CPPS) 19 and Current Protocols in Immunology (CPI). 20

[0076] In some embodiments of any of the aspects, the disclosure described herein does not relate to processes for cloning humans, processes for modifying the genetic identity of a human germ line, use of human embryos for industrial or commercial purposes, or processes for modifying the genetic identity of animals that have no substantial medical benefit to the human or animal, and animals resulting from such processes.

[0077] Other terms are defined herein within the description of various aspects of the invention.

[0078] Inflammatory bowel disease (IBD) The enteric nervous system (ENS) controls or regulates important gastrointestinal functions, including motility, secretion, local immunity, and inflammation, and represents the largest collection of autonomic neurons outside the brain. Disorders involving the ENS (such as IBD) are common and a major contributor to the health burden worldwide.

[0079] IBD, associated with damage to the ENS, is characterized by chronic severe inflammation of the small intestine and / or colon leading to recurrent diarrhea and abdominal pain. Crohn's disease (CD) and ulcerative colitis (UC) are the two major clinicopathological subtypes of IBD. Although both are chronic and recurrent inflammatory diseases of the intestine, they can be differentiated by the location of inflammation within the gastrointestinal tract and by the nature of histological changes in the intestinal wall. Anatomically, CD commonly affects the terminal ileum and colon, although it can affect the entire gastrointestinal tract from the mouth to the anus. UC is limited to the rectum, colon, and cecum. Microscopically, CD is transmural and often discontinuous, whereas UC affects only the intestinal mucosa in a continuous pattern.

[0080] IBD is a highly disabling disease due to fatigue associated with inflammatory symptoms and due to the chronic pain suffered by patients. The pathogenesis of IBD is only partially understood and involves a variety of environmental and host factors (e.g., genetic, epithelial, immune, and non-immune factors). Complex interactions between the immune system, intestinal commensal bacteria / pathogens, and host genotype are thought to underlie the development of IBD. These relapsing chronic inflammatory disorders are thought to be caused by environmental factors and / or overly aggressive T cell responses directed against a subset of commensal bacteria / pathogens that inhabit the distal ileum and colon of genetically susceptible hosts.

[0081] Currently, there is no cure or effective treatment for patients diagnosed with functional gastrointestinal disease such as IBD.The main goal of current therapy for IBD is to induce clinical remission by targeting symptoms, and then maintain it for a long time to achieve the best possible quality of life.Since current therapy has limited effectiveness, new therapies for treating inflammatory and chronic pain in the intestine of subjects with IBD are clinically important.

[0082] Binding immunoglobulin proteins (BiP) and related polypeptides Intracellular BiP is known to be an antiapoptotic protein with binding selectivity for hydrophobic residues exposed by both denatured or unfolded nascent polypeptides. BiP correctly folds nascent polypeptides but also protects cells by binding to denatured proteins and, as an endoplasmic reticulum (ER) resident protein, is tethered to the ER by a KDEL amino acid sequence at the 3' end of the protein that binds to ERD2.

[0083] Native endogenous BiP, also known as glucose-regulated protein 78 (GRP78), is a stress protein (SP) and a member of the heat shock protein (HSP) 70 family. BiP, present in all nucleated cells, is released from sequestering and upregulated upon activation of the unfolded protein response (UPR). This is initiated by the intracellular accumulation of unfolded proteins and nascent polypeptides, specifically triggered by cellular stresses such as reduced oxygen and / or glucose, as well as increased reactive oxygen species. These conditions are characteristic of chronic inflammatory pathologies, such as the inflamed synovium of patients with rheumatoid arthritis (RA).

[0084] In common with other SPs or HSPs with important intracellular functions, these molecular chaperones, including BiP, are expressed on the cell surface and secreted upon upregulation, allowing for unexpected and distinct extracellular functions, which has led to the increasing recognition that these highly conserved proteins may provide a link between the innate and adaptive immune systems.

[0085] IRL201805 (or 1805) is a modified analog of GRP78 that is being developed for the treatment of autoimmune diseases. During extensive investigation, rhuBiP (IRL201805, lacking the C-terminal KDEL sequence) has been shown to be able to treat and inhibit disease progression in animal models of arthritis. Adoptive cell transfer studies using spleen and lymph node cells from rhuBiP / IRL201805-treated mice in collagen-induced arthritis (CIA) models show that rhuBiP / IRL201805 has therapeutic properties that are long-lived in the absence of rhuBiP / IRL201805 and are associated with changes in immune cell populations.

[0086] Thus, rhuBiP / IRL201805 shifts the cellular immune response towards an anti-inflammatory TH2 profile and immune homeostasis. Further post-biomarker studies in patients confirmed the stabilization and increased efficacy of regulatory T cells, as well as a tendency to shift dendritic cell differentiation towards an indoleamine 3,2 oxygenase (IDO)+, more anti-inflammatory phenotype.

[0087] These data indicate that IRL201805 has significant activity in reducing inflammatory responses associated with rheumatoid arthritis, while its mechanism of action suggests potential efficacy in a variety of immune-based disorders.

[0088] CD69 is a transmembrane glycoprotein that contains a C-type lectin domain (CTLD). 21,22,23 , has recently been identified as a key regulator of intestinal inflammation and a new target molecule for IBD treatment. 24 CD69 is not expressed at detectable levels on naive leukocytes, but its surface expression is rapidly induced upon activation. 25、26 In human disease, CD69 expression is increased on leukocytes at sites of inflammation. 27,28,29 Furthermore, early in vitro studies demonstrated that CD69 is associated with intracellular Ca 2+ Influx induced proinflammatory molecules, lymphocyte proliferation, and production of proinflammatory mediators, such as IL-2, tumor necrosis factor-(TNF-)α, and nitric oxide (NO), have been described. 30,31,32,33 CD69 is also required for cell contact-dependent stimulation of macrophages by T cells. 34 However, recent in vivo studies using transgenic mice have demonstrated that CD69 can limit immune responses and proposed a regulatory function for CD69. CD69 has been shown to play a role in leukocyte migration, among the functions of regulatory T cells and resident tissue memory T cells. 35 Thus, stable induction of CD69 expression results in reduced lymphocyte migration into intestinal LP and CD69 + It has been suggested that this should lead to the generation of Treg cells. 36

[0089] As described below, the present disclosure provides evidence that BiP-related polypeptides induce CD69 expression and CD69-associated ligands, thereby providing methods of regulating inflammatory responses associated with intestinal inflammation and methods of preventing or treating inflammatory bowel disease (IBD).

[0090] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure, as one of ordinary skill in the relevant art would recognize. For example, while steps or functions of a method are presented in a given order, alternative embodiments may perform the functions in a different order or perform the functions substantially simultaneously. The teachings of the present disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified as appropriate to adopt compositions, functions and concepts of the above references and applications to provide still further embodiments of the present disclosure. Furthermore, in consideration of biological functional equivalence, some changes can be made to protein structures without affecting biological or chemical action in type or amount. These and other changes can be made to the present disclosure in consideration of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0091] Specific elements of any of the foregoing embodiments may be combined with or substituted for elements of other embodiments. Additionally, although advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily need to exhibit such advantages in order to fall within the scope of the present disclosure.

[0092] The technology described herein is further illustrated by the following examples, which should not be construed as further limiting in any respect. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. EXAMPLES

[0093] The invention will now be generally described, which will be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to be limiting of the invention.

[0094] Example 1. Evaluation of the efficacy of binding immunoglobulin protein (BiP) analogs in a mouse model of intestinal inflammation Currently, there are over 50 animal models of human IBD. Although no model allows for complete phenotyping of human IBD, many models are useful for studying various aspects of the human disease, including disease onset and progression and wound healing responses.

[0095] In this study, we demonstrated that intestinal inflammation was induced by the transfer of syngeneic splenic CD4 + CD45RB 高 It is initiated by T cell adoptive transfer. 37 CD4 + CD45RB 高 The T cell population contains primarily naive T cells primed for activation, capable of inducing chronic small intestinal and colonic inflammation. This animal model allows for the modification of key experimental variables, including both innate and adaptive immune cell populations, to answer biologically relevant questions regarding disease pathogenesis. Furthermore, it provides a precise onset and a well-characterized experimental time course of disease development, which allows for kinetic studies of clinical features of disease progression in mice. The intestinal inflammation induced by this method shares many characteristics with human IBD, including chronic large and small intestinal transmural inflammation, a pathogenesis driven by cytokines such as TNF and IL-12, and systemic symptoms such as wasting. 38 It is therefore an ideal model system to evaluate the efficacy of BiP analogs such as IRL201805 (SEQ ID NO:1).

[0096] material and method material IRL201805 (SEQ ID NO:1) is manufactured according to GMP standards at CBC, Bristol, UK.

[0097] method Experimental Overview Male RAG2 - / - Mice are randomly assigned to experimental groups and allowed to acclimate for one week. On day 0, spleens are removed from donor C57Bl / 6N male mice and single cell suspensions generated according to CRL SOPs. On day 0, male RAG KO mice are inoculated with CD4 + CD45RB 高 T cells (CD45RB 低 T cells) are injected intraperitoneally.

[0098] From day 21 until the end of the experiment on day 49, animals are monitored daily for clinical signs of colitis, including weight loss, loose stools, and / or diarrhea. On day 49, animals are sacrificed, colons excised, and colon weights and lengths are measured. Colons from half of the animals in each group are transferred to tissue fixative and then processed for paraffin embedding and preserved for histopathological analysis. Lamina propria from half of the animals in each group are processed for flow cytometry.

[0099] Dosing schedule Treatments are administered according to the schedule in Table 1 below. All groups have n=10.

[0100] (Table 1) TIFF2025510966000002.tif43161Abbreviations: NA: Not applicable, IP: Intraperitoneal injection, IV: Intravenous injection, TBC: Confirmed target, CRL: Charles River regimen

[0101] Morbidity and mortality Morbidity and mortality are assessed daily. Animals are monitored for clinical signs including abnormal posture (e.g., hunched), abnormal hair condition (e.g., piloerection), change in color of hairless areas (including cyanosis or jaundice), presence of masses and / or swelling, abnormal breathing, abnormal movements, and decreased activity. Causes of death are recorded once identified. Survival rates are recorded.

[0102] body weight Animals are weighed at the start of the study (day 0) and periodically over the first 3 weeks. All animals are weighed daily from day 21 through the end of the study. Data are analyzed and graphed.

[0103] Clinical score Animals are monitored daily for clinical signs of colitis, including weight loss, loose stools, and / or diarrhea, from day 21 until the end of the experiment. The following scoring system is provided in Table 2.

[0104] (Table 2) TIFF2025510966000003.tif30128

[0105] Flow cytometry Single cell suspensions are prepared from colonic lamina propria cells isolated from half the animals in each group using the manufacturer's protocol (Miltenyi Biotec). Cells are harvested, restimulated with PMA / ionomycin, and stained with the following panel containing viability dyes and surface and intracellular markers: CD44, CD45, CD3, CD4, IFNγ, IL-17, CD25, and FOXP3. The number and percentage of each population is determined.

[0106] gross pathology At termination, the colon is excised and photographed so that length measurements can be performed on the digital images using ImageJ software, and the colon is weighed.

[0107] histopathology At termination, colons from half of the animals in each group are transferred to tissue fixative and then processed for paraffin embedding in a "Swiss roll" format. These samples are sectioned and stained with hematoxylin and eosin (H&E), and up to two sections per animal are scored by a qualified histopathologist according to a semiquantitative scoring system. Sections are scored for mucosal thickness, mucosal ulceration, lamina propria mononuclear cell infiltration, lamina propria granulocytic infiltration, and crypt abscess / distension / distortion, as described in Table 3. An overall severity score is calculated by adding the scores for each of the criteria.

[0108] (Table 3) TIFF2025510966000004.tif74132

[0109] result In this study, IRL201805 resulted in a significant reduction in clinical signs of colitis, including reduction in weight loss, loose stools, and / or diarrhea, and a significant reduction in severity scores based on sections scored for mucosal thickness, mucosal ulceration, lamina propria mononuclear cell infiltration, lamina propria granulocytic infiltration, and crypt abscesses / dilation / distortion.

[0110] These results suggest that administering binding immunoglobulin protein (BiP) or a functional analog to a subject at risk for or suffering from IBD is an effective method of preventing or treating IBD. BiP or functional analogs useful in the contemplated methods include, but are not limited to, polypeptides having an amino acid sequence selected from SEQ ID NOs: 1-5.

[0111] Example 2 Evaluation of the efficacy of a binding immunoglobulin protein (BiP) analog in a mouse model of dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) Perhaps the most widely used mouse model of colitis uses dextran sodium sulfate (DSS), a chemical colitogen with anticoagulant properties, to induce the disease. DSS is a water-soluble, negatively charged sulfated polysaccharide with a highly variable molecular weight ranging from 5 to 1400 kDa. The most severe mouse colitis, which most resembles human UC, is 39 This results from the administration of 40-50 kDa DSS in drinking water. The mechanism by which DSS induces intestinal inflammation is unclear, but is likely the result of damage to the epithelial monolayer lining the large intestine, which allows the penetration of pro-inflammatory intestinal contents (e.g., bacteria and their products) into the underlying tissue. The DSS colitis model is highly popular in IBD research because of its rapidity, simplicity, reproducibility, and controllability. Acute, chronic, and relapsing models of intestinal inflammation can be achieved by altering the concentration and frequency of administration of DSS.

[0112] In this study, intestinal inflammation is a DDS mouse model of UC used to evaluate the efficacy of BiP analogs such as IRL201805 (SEQ ID NO: 1).

[0113] material and method material IRL201805 (SEQ ID NO:1) is manufactured according to GMP standards at CBC, Bristol, UK.

[0114] method Experimental Overview Adult C57bl / 6 mice are randomly assigned to experimental groups and allowed to acclimate for one week. Starting on day -2, treatments are administered according to the following dosing schedule: On day 0, drinking water is replaced with 5% dextran sulfate sodium (DSS) in water (except for group 1, which continues to receive regular drinking water throughout). Animals are given free access to the 5% DSS solution. From day 0 until the end of the experiment on day 7, animals are monitored daily for clinical signs of colitis, including weight loss, loose stools and / or diarrhea, and the presence of occult or gross blood in the stool.

[0115] Blood samples are collected on days -2, 3 (in-life), and 7 (termination), processed to serum, and stored for analysis.

[0116] On day 7, animals are sacrificed, colons excised, and colon length measured. One sample of colon (distal) per animal is taken and fixed for histopathological analysis. A further sample of colon (proximal) is taken at termination and cultured in the presence or absence of LPS for 48 hours before analysis of cytokines. The remaining supernatant is saved for analysis.

[0117] Dosing schedule Treatments are administered according to the schedule in Table 1 below. All groups have n=10 unless otherwise stated.

[0118] (Table 1) TIFF2025510966000005.tif46161Abbreviations: TBC: not applicable, NA: not applicable, IV: intravenous, PO: oral.

[0119] Morbidity and mortality Morbidity and mortality are assessed daily. Animals are monitored for clinical signs including abnormal posture (e.g., hunched), abnormal hair condition (e.g., piloerection), change in color of hairless areas (including cyanosis or jaundice), presence of masses and / or swelling, abnormal breathing, abnormal movements, and decreased activity. Causes of death are recorded once identified. Survival rates are recorded.

[0120] body weight All animals are weighed daily from day 1 through the end of the study. Data are analyzed and graphed.

[0121] Clinical score From day 0 to the end of the experiment, animals are monitored daily for clinical signs of colitis, including weight loss, loose stools and / or diarrhea, and occult or gross blood in the stool. The following scoring system is provided in Table 2.

[0122] (Table 2) TIFF2025510966000006.tif29128

[0123] gross pathology At termination, the colon is excised and photographed so that length measurements can be performed on the digital images using ImageJ software, and the colon is weighed.

[0124] Measurement of colon explant cytokine levels Colon samples taken on day 7 are cultured for 48 hours in the presence or absence of LPS and then analyzed for cytokine content by Luminex. The following cytokines are analyzed by multiplex (8-plex) Luminex: sTNFRII, sIL-6R, IL-6, IL-10, IL-1b, TNF-a, IFN-γ, and MCP-1. Samples are analyzed in single specimens using multiplex xMAP bead technology, which utilizes microspheres as solid supports for sandwich immunoassays and the determination of multiple cytokines in the same sample (Luminex).

[0125] histopathology At termination, colons from half of the animals in each group are transferred to tissue fixative and then processed for paraffin embedding in a "Swiss roll" format. These samples are sectioned and stained with hematoxylin and eosin (H&E), and up to two sections per animal are scored by a qualified histopathologist according to a semiquantitative scoring system. Sections are scored for mucosal thickness, mucosal ulceration, lamina propria mononuclear cell infiltration, lamina propria granulocytic infiltration, and crypt abscess / distension / distortion, as described in Table 3. An overall severity score is calculated by adding the scores for each of the criteria.

[0126] (Table 3) TIFF2025510966000007.tif74132

[0127] result In this study, IRL201805 resulted in a significant reduction in clinical signs of colitis, including reduction in weight loss, loose stools, and / or diarrhea, and a significant reduction in severity scores based on sections scored for mucosal thickness, mucosal ulceration, lamina propria mononuclear cell infiltration, lamina propria granulocytic infiltration, and crypt abscesses / dilation / distortion.

[0128] These results suggest that administering binding immunoglobulin protein (BiP) or a functional analog to a subject at risk for or suffering from IBD is an effective method of preventing or treating IBD. BiP or functional analogs useful in the contemplated methods include, but are not limited to, polypeptides having an amino acid sequence selected from SEQ ID NOs: 1-5.

[0129] Example 3 Evaluation of the effect of binding immunoglobulin protein (BiP) analogs to CD69 in colon punch biopsies obtained from IBD patients In this study, punch biopsies (2 mm) of inflamed and non-inflamed colonic tissues were used as a control, as shown in Figure 6A-E. 2 ) were obtained from each consenting IBD patient following ethical approval from the hospital. Tissue biopsies were then incubated with or without IRL201805 (20 μg / mL) for 12–14 h and assessed for CD69+ / CD39+ regulatory T cells (Tregs).

[0130] As mentioned above, CD69 is known to have many biological functions. In regulatory T cells (Tregs), CD69 is thought to promote CD39 surface expression, FoxP3 transcription, and phosphorylation of STAT5, which leads to STAT3 inhibition. CD39 is a Ca 2+ and Mg 2+ CD39 is an integral membrane protein that phosphorylates ATP and, more inefficiently, ADP to AMP in a CD39-dependent manner. The adenosine generated is ultimately a potent activator of tolerogenic dendritic cells (DCs). CD39 becomes catalytically active upon its localization on the cell surface.

[0131] As shown in Figure 7, exposure of primary immune cells in inflamed colon tissue to IRL201805 induced a 20% increase in double positive CD69+ / CD39+ Tregs compared to the same treatment on non-inflamed tissue from the same patients. In this study, flow cytometry showed that IRL201805 also led to an increase in p-STAT5 in a subset of immune cells (data not shown). Previous unpublished data also demonstrated that BiP inhibits STAT3 phosphorylation in human lymphocytes. These data suggest that BiP-related peptides only enhance CD69 activity and the resulting cascade in inflamed tissues from IBD patients, but not in non-inflamed tissues.

[0132] Example 4 Evaluation of the effect of combined immunoglobulin protein (BiP) analogs on T cell activation in PBMCs CD69 and CD154 (CD40L) surface markers T cell activation can be monitored by detecting increased expression of several surface markers, typically CD69 and CD154 (CD40L). Expression of CD69 is usually transient on most T cells, particularly T effector cells, and declines within 24 hours.

[0133] In this study, PBMCs were isolated from two healthy donors. PBMCs were then incubated with IRL201805 (20 μg / mL) for 20–72 h at 37 °C. Flow cytometry was used to measure mean fluorescence intensity and evaluate the surface expression of CD69 and CD39 on Tregs from PBMCs.

[0134] As shown in Figure 8, IRL201805 treatment induced sustained expression of CD69 on CD154- cells, which was maintained for at least 72 hours. These results are indicative of T cell receptor (TCR)-activated Treg cells.

[0135] JAK-STAT signaling pathway The Janus kinase (JAK) / signal transducer and activator of transcription (STAT) (JAK / STAT) pathway orchestrates and controls many of the cytokine and differentiation pathways that control and regulate immune responses. Signaling through the JAK-STAT signaling pathway involves rapid transmission of the signal from the cell membrane to the nucleus, followed by a highly orchestrated response and then a controlled downregulation and attenuation of the initial signal. Suppressor of cytokine signaling (SOCS) proteins are the primary drivers of signal attenuation. They are induced by cytokine exposure (via STATs) and then act as negative feedback inhibitors to turn off the signaling cascade. SOCS3 is a potent regulator of cytokine signaling in monocytes / macrophages and T cells.

[0136] In this study, we assessed the effect of IRL201805 on the JAK-STAT signaling pathway at the transcript and protein levels.PBMCs were isolated from healthy subjects and treated with 20 μg / mL IRL201805 for 24 h.

[0137] RNAseq analysis was used to evaluate gene transcript levels of key signaling genes involved in CD69 activation. As shown in Figure 9A, IRL201805 treatment significantly increased gene transcript levels of JAK3 and SOCS3 by approximately 10-fold measured 24 hours after treatment. IRL201805 did not affect transcript levels of STAT5A or STAT5B in monocytes from three healthy individuals.

[0138] Immunoblotting of PBMC cell lysates with recombinant anti-STAT5 (phospho-Y694) antibody [E208] (ab32364) (Abcam, ab32364) was used to assess p-STAT5 levels from untreated and IRL1805-treated PBMCs. As shown in Figure 9B, PBMCs isolated from healthy subjects and treated with 20 μg / mL IRL201805 for 24 hours showed increased pf p-STAT5 levels compared to untreated PBMCs.

[0139] FOXP3 transcription and STAT5 phosphorylation In Tregs, CD69 is thought to promote CD39 surface expression, FoxP3 transcription, and STAT5 phosphorylation leading to inhibition of STAT3. This study evaluated the effect of IRL1805 on p-STAT5. IRL201805 led to an increase in p-STAT5 in a subset of immune cells. Treatment of PBMCs with IRL201805 led to a change in p-STAT5 status in CD4 / CD8 T cells and a three-fold increase in FoxP3 transcript levels (see Table 4).

[0140] (Table 4) TIFF2025510966000008.tif21157

[0141] CD69-related ligand Figure 10A provides a schematic of CD69-associated ligands. CD69 surface expression increases with APC-derived MHC class II-TCR activation. TCR engagement induces modest activation of the metabolic sensor mTOR, which, together with CD28 and IL-2 signaling, can promote T cell commitment to the generation of TH1, TH2, and TH17 effector cells. Membrane SiP1 / SiPR1 interaction can sustain mTOR activation and inhibit differentiation of FoxP3 cells. 40 DC-derived galectin-1 (Gal-1) 41 and S100A8 / A9 42 It can bind to CD69, which activates JAK3 / STAT5 phosphorylation. Phosphorylated STAT5 can translocate to the nucleus, promote the differentiation of FoxP3 Tregs, inhibit STAT3 activation, and prevent Th17 cell differentiation.

[0142] The potential ligands Gal-1 and S100A8 / A9 are known to bind to CD69. 43To investigate the effect on T cell expression and the ability of T cells to maintain the Treg phenotype, the present study investigated the effect of 1805 on the expression of Gal-1 and S100A8 / A9 in APCs (monocytes). Human monocytes isolated from healthy individuals (n=4) were exposed to 1805 for 1 h on both Gal-1 and S100A8 / A9 protein levels.

[0143] The abundance of Gal-1 and S100A8 / A9 proteins on the cell surface was detected by tandem mass tag (TMT) quantitative proteomics. As shown in Figure 10B, the expression of Gal-1 and S100A8 / A9 was not altered by 1805 on the cell surface.

[0144] However, 1805 treatment increased phosphorylation of S100A9 by 58% (Figure S10C). Mass spectrometry identified an increase in the trimethylation post-translational modification on threonine 113 in S100A9 following 1805 treatment, a modification known to promote active secretion of S100A9 from myeloid cells.

[0145] Surface CD154 and CD69 activation markers on T lymphocytes derived from PBMCs CD69 is commonly detected by flow cytometry as a marker of activated cells, most frequently on lymphocytes, and its appearance on the cell surface serves as a biomarker of MHC-TCR interactions between APCs and T cells. Both CD4 and CD8 T cells express CD69 under homeostatic conditions, indicating that a large number of these cells are constantly exposed to antigen challenge.

[0146] This study evaluated the effect of 1805 on the expression of surface CD154 and CD69 activation markers on T lymphocytes from PBMCs of Treg, Teff, and CD8 T cells.PBMCs isolated from four healthy donors were cultured with or without 20 μg / mL IRL201805 at 37°C for 20 h.

[0147] As shown in Figure 11, basal levels of cell surface CD69 expression were highest in CD8 T cells (42%, Figure 11C), followed by Tregs (24%, Figure 11A) and Teffs (15%, Figure 11B). These results indicate that various subsets of T cells exhibit CD69 on their cell surface. However, 1805 only produced a statistically significant 2-fold increase (p=0.008) on the cell surface of Tregs. These results suggest that Tregs exposed to 1805 are selectively activated.

[0148] IRL201805 - Possible mechanism of action Without being bound to a particular mechanism of action, Figure 12 provides a schematic of a possible mechanism of action for the effect of BiP-related peptides such as IRL201805 on CD69-associated ligands. The CD69 receptor is expressed on the membrane of activated regulatory T cells within 2-20 hours after presentation of the 1805 peptide to the TCR by APCs. The CD69 receptor binds to ligands (e.g., S100A8 / A9 or Gal-1) on the surface of APCs. The cytoplasmic tail of CD69 associates with Jak3 and Stat 5 proteins, causing phosphorylation of Stat5 and its translocation to the nucleus, where it activates the transcription factor FoxP3 and stimulates differentiation of regulatory T cells. 44 CD69 engagement can also induce the expression of IL-2, TGF-β, and CD39 on the cell surface. These cytokines and receptors can act in an autocrine manner to induce differentiation of regulatory T cells and tolerogenic APCs. CD69 can inhibit the Th17 differentiation pathway by at least two mechanisms: CD69-activated Stat5 directly inhibits Stat3 translocation to the nucleus and indirectly antagonizes Stat3-mediated RORγt activation via activation of FoxP3.

[0149] conclusion CD69 has recently been identified as a key regulator of intestinal inflammation and a novel target molecule for the treatment of IBD. 45Recent in vivo studies using transgenic mice have shown that CD69 can limit immune responses and have proposed a regulatory function for CD69. CD69 has been shown to play a role in leukocyte migration among the functions of regulatory T cells and resident tissue memory T cells. Thus, stable induction of CD69 expression reduces lymphocyte migration to the intestinal LP and inhibits CD69 expression. + It has been suggested that this should lead to the generation of Treg cells.

[0150] The present disclosure provides evidence that BiP-related polypeptides result in stable induction of CD69 expression only in inflamed colon tissue and induce activation of several CD69 activation markers and CD69-related ligands, thereby providing a method of regulating inflammatory responses associated with intestinal inflammation and methods of preventing or treating inflammatory bowel disease (IBD).

[0151] References TIFF2025510966000009.tif91161TIFF2025510966000010.tif215161TIFF2025510966000011.tif96160

[0152] All patents and other publications, including references, issued patents, published patent applications, and co-pending patent applications cited throughout this application, are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by prior invention or for any other reason. All statements as to dates or representations regarding the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0153] The foregoing written specification is deemed sufficient to enable one skilled in the art to practice the present aspects and embodiments. The present aspects and embodiments are intended as a single illustration of one aspect, and are not limited in scope by the examples provided, as other functionally equivalent embodiments are within the scope of the present disclosure. In addition to those shown and described herein, various modifications will be apparent to those skilled in the art from the foregoing description and are included within the scope of the appended claims. The advantages and objectives described herein are not necessarily encompassed by each embodiment. Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the scope of the appended claims.

Claims

1. 1. A method for preventing or treating inflammatory bowel disease (IBD), comprising administering to a subject at risk for or suffering from IBD a therapeutically effective amount of a binding immunoglobulin protein (BiP) or a functional analog thereof.

2. 2. The method of claim 1, wherein BiP or a functional analog thereof has an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, or conservative amino acid substitutes thereof.

3. 3. The method of claim 2, wherein BiP or a functional analog thereof has the amino acid sequence of SEQ ID NO:

1.

4. The method according to any one of claims 1 to 3, wherein the IBD is selected from the group consisting of ulcerative colitis, Crohn's disease, and non-infectious inflammation of the intestine.

5. A therapeutically effective amount of a binding immunoglobulin protein (BiP) or a functional analogue thereof for use in the prevention or treatment of inflammatory bowel disease (IBD).

6. 6. A therapeutically effective amount of a binding immunoglobulin protein (BiP) or a functional analog thereof for use according to claim 5, wherein BiP or a functional analog thereof has an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, or conservative amino acid substitutes thereof.

7. 7. A therapeutically effective amount of a binding immunoglobulin protein (BiP) or a functional analogue thereof for use according to claim 6, wherein BiP or a functional analogue thereof has the amino acid sequence of SEQ ID NO:

1.

8. A therapeutically effective amount of a binding immunoglobulin protein (BiP) or a functional analogue thereof for use according to any one of claims 5 to 7, wherein the IBD is selected from the group consisting of ulcerative colitis, Crohn's disease, and non-infectious inflammation of the intestine.

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