Composition and its use in methods for treating intestinal inflammation

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 Invention Embodiments of the present invention relate to compositions and methods for the treatment of inflammatory bowel disease (IBD) using binding immunoglobulin proteins (BiPs) or their functional analogues. [Background technology]

[0002] Background of the Invention Inflammatory bowel disease (IBD), primarily characterized by ulcerative colitis and Crohn's disease but also including non-infectious inflammation of the intestines, has plagued gastroenterologists and immunologists since its first modern medical description approximately 75 to 100 years ago. IBD is characterized by chronic inflammation of the gastrointestinal tract (GI), leading to damage to the GI tract. In 2017, there were 6.8 million cases of IBD worldwide. The suffering caused by IBD is increasing globally, with the age-adjusted prevalence rising from 79.5 per 100,000 people in 1990 to 84.3 per 100,000 people in 2017. 1 Most IBD patients are diagnosed in their 20s or 30s. Adults with IBD utilize medical services more frequently than adults without IBD, including doctor visits, medication prescriptions, emergency room visits, hospitalization, and surgical procedures. 2

[0003] Treatment for IBD typically involves either medication or surgery. Anti-inflammatory drugs are often the first step in treating inflammatory bowel disease (e.g., corticosteroids and aminosalicylates, e.g., mesalamine, valsalazid, and orsalazine). 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), biological agents that target inflammatory neutralizing proteins (e.g., infliximab (Remicade), adalimumab (Humira), golimumab (Simponi), certolizumab (Cimzia), vedolizumab (Entyvio), and ustekinumab (Stelara)), and, as a last resort, surgery. Over the past 20 years, advances in biological agents and small molecule therapies have led to a rapid increase in IBD treatment facilities. Despite these advances, Crohn's disease and ulcerative colitis remain chronic and progressive diseases. One of the main reasons for persistent inflammation and bowel damage is the failure of current medical treatments.

[0004] Currently, there is no cure or effective treatment for patients diagnosed with functional gastrointestinal disorders such as IBD. The main goal of current therapies for IBD is to induce clinical remission by targeting symptoms and then maintain it over the long term to achieve the best possible quality of life. Because current therapies have limited efficacy, there is a need for new therapies to treat the inflammation and chronic pain in the intestines of individuals with IBD. Therefore, there is a need for improved therapies for the treatment of IBD. [Overview of the project]

[0005] Embodiments of the present invention provide methods for modulating inflammatory responses associated with intestinal inflammation. In some embodiments, embodiments of the present invention provide a method for preventing or treating inflammatory bowel disease (IBD), the method comprising administering a therapeutically effective amount of conjugated immunoglobulin protein (BiP) or a functional analogue thereof to a subject at risk of or suffering from IBD. In some embodiments, BiP or a functional analogue 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 conserved amino acid substituent thereof. In alternative embodiments, BiP or a functional analogue thereof has the amino acid sequence consisting of SEQ ID NO: 1. In some embodiments, IBD is ulcerative colitis, Crohn's disease, or non-infectious inflammation of the intestine.

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

[0007] In some embodiments, the therapeutically effective dose 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 dose 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 another alternative embodiment, the therapeutically effective dose 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 enumerated herein. [Invention 1001] A method for preventing or treating inflammatory bowel disease (IBD), comprising administering a therapeutically effective amount of conjugated immunoglobulin protein (BiP) or a functional analogue thereof to a subject at risk of or suffering from IBD. [Invention 1002] The method of the present invention 1001, wherein BiP or a functional analog thereof has an amino 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 its conserved amino acid substituents. [Invention 1003] The method of the present invention 1002, wherein BiP or a functional analog thereof has the amino acid sequence of SEQ ID NO: 1. [Invention 1004] Any method of the present invention 1001 to 1003, wherein IBD is selected from the group consisting of ulcerative colitis, Crohn's disease, and non-infectious inflammation of the intestine. [Invention 1005] A therapeutically effective amount of conjugated immunoglobulin protein (BiP) or a functional analogue thereof for use in the prevention or treatment of inflammatory bowel disease (IBD). [Invention 1006] A therapeutically effective amount of conjugated immunoglobulin protein (BiP) or a functional analogue thereof for use in the present invention 1005, wherein BiP or a functional analogue 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 its conserved amino acid substituents. [Invention 1007] A therapeutically effective amount of conjugated immunoglobulin protein (BiP) or a functional analog thereof for use in the present invention 1006, wherein BiP or a functional analog thereof has the amino acid sequence of SEQ ID NO: 1. [Invention 1008] A therapeutically effective amount of conjugated immunoglobulin protein (BiP) or a functional analogue thereof for use in any of the inventions 1005-1007, selected from the group consisting of IBD, ulcerative colitis, Crohn's disease, and non-infectious inflammation of the intestine.

Brief Description of the Drawings

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

[0010] [Figure 1] The amino acid sequence of IRL201805 (SEQ ID NO: 1), which is a BiP analog, is provided. 3 [Figure 2] The amino acid sequence of the His-tagged BiP analog (SEQ ID NO: 2) expressed by the plasmid pQE2 is provided. 4 [Figure 3A] Figures 3A - D provide the amino acid sequence of a BiP analog (SEQ ID NO: 3). 5 [Figure 3B] It is a figure showing the continuation of Figure 3A. [Figure 3C] It is a figure showing the continuation of Figure 3B. [Figure 3D] It is a figure showing the continuation of Figure 3C. [Figure 4A] Figures 4A - D provide the amino acid sequence of a BiP analog (SEQ ID NO: 4). 6 [Figure 4B] It is a figure showing the continuation of Figure 4A. [Figure 4C] It is a figure showing the continuation of Figure 4B. [Figure 4D] It is a figure showing the continuation of Figure 4C. [Figure 5] Provides the amino acid sequence of natural BiP (SEQ ID NO: 5). 7 [Figure 6] Figures 6A–E provide an overview of the procedure used to evaluate the effects of ex vivo human primary immune cells in inflammatory and non-inflammatory colon tissue treated with IRL201805. [Figure 7] This study demonstrates the effect of a 12-hour treatment with 20 μg / mL of IRL201805 on CD69+ / CD39+ Treg cells in inflammatory and non-inflammatory colon tissue from IBD patients (Crohn's disease or ulcerative colitis), as determined by flow cytometry. [Figure 8] This shows the average fluorescence intensity of CD69 and CD39 surface expression on Treg cells derived from peripheral blood mononuclear cells (PBMCs) cultured at 37°C for 20–72 hours in 20 μg / mL IRL201805. [Figure 9A] Figures 9A and 9B show the effects of 20 μg / mL of IRL201805 on the JAK-STAT signaling pathway at the transcript and protein levels. Figure 9A provides RNA-seq analysis at the gene transcript level of key signaling genes involved in CD69 activation. Figure 9B provides immunoblots of p-STAT5 levels in cell lysates from untreated and IRL201805-treated PBMCs. [Figure 9B] Please refer to the explanation in Figure 9A. [Figure 10] Figure 10A provides a schematic diagram of CD69-related ligands. Figure 10B provides a bar graph showing the effects of IRL201805 on CD69 ligand, galectin-1 (Gal-1), and S100A8 / A9 as detected by TMT quantitative proteomics. Figure 10B provides a schematic representation of the increased trimethylation of posttranslational modifications on threonine 113 on S100A9 after 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 derived from PBMCs: Treg (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-tests. NS = not significant. [Figure 12] This diagram provides a schematic representation of the effects of IRL201805 on CD69 and CD69-related ligands. Abbreviations: APC: antigen-presenting cell, TCR: T cell receptor, Treg: regulatory T cell, P: phosphorylation. [Modes for carrying out the invention]

[0011] Detailed description of the invention Here, the innovativeness of the subject matter is illustrated with reference to the drawings, and similar reference numbers are used throughout to refer to similar elements. In the following description, for illustrative purposes, numerous specific details are provided to provide a complete understanding of the invention. However, it may be apparent that the invention can be carried out without these specific details. In other examples, well-known structures and devices are shown in block diagram form to facilitate the description of the invention. Naturally, specific aspects, modes, embodiments, variations, and features of the invention are described below at various levels of detail to provide a substantial understanding of the invention.

[0012] definition For convenience, the meanings of some terms and phrases used herein, in the examples and in the appended claims are provided below. Unless otherwise stated or implicitly suggested by the context, the following terms and phrases have the meanings provided below. Since the scope of the present invention is limited only by the claims, the definitions are provided to aid in describing specific embodiments and are not intended to limit the present invention in the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the present invention pertains. In the event of any obvious inconsistency between the use of a term in the art and its definition provided herein, the definition provided herein shall prevail.

[0013] As used herein and in the appended claims, the singular form includes plural references unless otherwise explicitly indicated. For example, a reference to “cell” includes combinations of two or more cells, etc.

[0014] Where used herein, the terms “approximately” or “about” with respect to a value or parameter are generally interpreted, unless otherwise stated or made clear from the context, to include a number that is within the range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) that number (except when such a number is less than 0% or greater than 100% of a possible value). Where used herein, a reference to “approximately” or “about” a value or parameter includes embodiments that apply 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". As used herein in phrases such as "A and / or B", the term "and / or" is intended to include both A and B, A or B, A (alone), and B (alone). Similarly, as used in phrases such as "A, B, and / or C", the term "and / or" is intended to include 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 (alone), B (alone), and C (alone).

[0016] As used herein, the term "including" means that other components may exist in addition to the defined components presented. The use of "including" indicates inclusion, not limitation.

[0017] The term "consisting of" refers to the compositions, methods, and each component described herein, excluding any elements not enumerated in the description of the embodiments.

[0018] As used herein, the term "essentially consisting of" refers to the components necessary for a given embodiment. This term allows for the presence of additional elements that do not substantially affect the basic, novel, or functional features of that embodiment of the invention.

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

[0020] As used herein, the term “subject” refers to mammals, including but not limited to dogs, cats, horses, cattle, pigs, sheep, goats, chickens, rodents, or primates. Subjects may, but are not limited to, pets (e.g., dogs, cats), farm animals (e.g., cattle, horses, pigs, chickens, etc.), or laboratory animals (e.g., mice, rats, rabbits, etc.). Subjects include human subjects. Human subjects may be children, adults, or elderly subjects. Human subjects may be of any sex.

[0021] As used herein, the terms “effective dose” and “therapeutic effective dose” include an amount sufficient to prevent or improve 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, or plasma transplants, inflammatory bowel disease, or immune disorders, including Crohn's disease. Naturally, there are many methods known in the art for determining an effective dose for a given use. For example, pharmacological methods for dose determination may be used in therapeutic situations. In the context of therapeutic or prophylactic use, the amount of composition administered to a subject depends on the type and severity of the disease, as well as individual characteristics such as overall health, age, sex, weight, and tolerance to the drug. It also depends on the degree, severity, and type of the disease. A person skilled in the art will be able to determine an appropriate dose according to 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 “procedure” or “to treat” or “to alleviate” or “to alleviate” mean both (1) therapeutic measures that cure, slow, reduce the symptoms of, and / or halt the progression of a diagnosed disease or infection, and (2) preventive or protective 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 stools, urgency and fecal incontinence, fatigue, fever, anemia, and / or weight loss. Malnutrition and growth retardation may also occur in subjects who develop IBD in childhood.

[0023] Where used herein, in relation to a disease, disorder, or medical condition, the terms “treatment,” “procedure,” or “relief” refer to a therapeutic intervention for a condition whose purpose is to reverse, alleviate, improve, inhibit, slow, or halt the progression or severity of the symptoms or condition. The term “to treat” includes reducing or mitigating at least one adverse effect of a symptom or condition. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced; or if the progression of a condition is reduced or halted. That is, “treatment” includes not only improvement of symptoms or markers but also halt or at least slow the progression or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, relief of one or more symptoms, a reduction in the degree of disorder, a stabilization (i.e., no worsening) of an immune disorder, a delay or slowing of an immune disorder, and an extension of life 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 discontinued when the IBD symptoms are in remission.

[0025] As used herein, the term “long-term” administration means that a therapeutic agent or drug is administered for a period of at least 12 weeks. This includes the administration of the therapeutic agent or drug in such a manner that it is effective over a period of at least 12 weeks or for a period of at least 12 weeks, and this does not necessarily mean that the administration itself takes place over 12 weeks, for example, when a sustained-release composition or a long-acting therapeutic agent or drug is used. Thus, the subject is treated over a period of at least 12 weeks. Often, long-term administration is at least 4, 5, 6, 7, 8, or 9 months or longer, or at least 1, 2, 3, 5, 7, or 10 years or longer.

[0026] The compositions intended herein may be administered in any convenient manner, including aerosol inhalation, injection, ingestion, blood transfusion, implantation, or transplantation. In preferred embodiments, the compositions are administered parenterally. As used herein, the terms “parenteral administration” and “administered parenterally” refer to modes of administration other than intestinal and topical administration, usually by injection, and include, but are not limited to, intravascular, intravenous, intramuscular, intraarterial, subarachnoid, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intrasternal, and intrasternal injections and infusions. In one embodiment, the compositions intended herein are administered to a target by direct injection into a tumor, lymph node, or site of infection.

[0027] The terms “reduce,” “reduced,” “decrease,” or “inhibit” are all used herein to mean a reduction of a statistically significant amount. In some embodiments, “reduce,” “decrease,” or “decrease” or “inhibit” typically mean a reduction of at least 10% compared to a baseline level (e.g., without administration of a given treatment or drug), and may include reductions 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, “decrease” or “inhibit” does not include complete inhibition or reduction compared to a baseline level. “Complete inhibition” is 100% inhibition compared to a baseline level. The reduction should preferably be to a level that is acceptable as being within the normal range for an individual without a given disorder.

[0028] The terms “increased,” “increased,” “enhanced,” or “activated” are all used herein to mean an increase of a statistically significant amount. In some embodiments, the terms “increased,” “increased,” “enhanced,” or “activated” may mean an increase of at least 10% compared to a baseline level, for example, an increase of at least about 20% compared to a baseline 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 an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase between 2 times and 10 times or more. In the context of markers or symptoms, “increase” is a statistically significant increase of such a level.

[0029] As used herein, the terms “protein” and “polypeptide” are used interchangeably to specify a set of amino acid residues linked to one another by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. The terms “protein” and “polypeptide” refer to polymers of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. While “protein” and “polypeptide” are often used in reference to relatively large polypeptides, the term “peptide” is often used in reference to small polypeptides, although the use of these terms in the art is redundant. The terms “protein” and “polypeptide” are used interchangeably herein when referring to gene products and their fragments. Thus, exemplary polypeptides or proteins include gene products, native proteins, homologs, orthologues, paralogs, fragments, and other equivalents, variants, fragments, and analogs of those described above.

[0030] In the various embodiments described herein, it is further intended that any variant (native or otherwise), allele, homologue, conserved modified variant, and / or conserved substitution variant of any particular polypeptide described herein be included. With respect to amino acid sequences, those skilled in the art will recognize that any individual substitution, deletion, or addition to a nucleic acid, peptide, polypeptide, or protein sequence that alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conserved modified variant,” and that such alteration results in an amino acid substitution by a chemically similar amino acid and preserves the desired activity of the polypeptide. Such conserved modified variants are, in addition to, and not excluded from polymorphic variants, interspecific homologues, and alleles consistent with this disclosure.

[0031] In some embodiments, the polypeptides described herein (or nucleic acids encoding such polypeptides) may be functional fragments of the amino acid sequences described herein. As used herein, “functional fragment” is a peptide fragment or segment that retains at least 50% of the activity of the wild-type reference polypeptide, according to the assays described below. Functional fragments may include conserved 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 conserved modified variants. Conservative substitution variants can be obtained, for example, by mutations in the native nucleotide sequence. Where used herein, “variant” is a polypeptide that is substantially homologous to the native or reference polypeptide but has a different amino acid sequence from the native or reference polypeptide due to one or more deletions, insertions, or substitutions. The DNA sequence encoding a variant polypeptide includes sequences that encode a variant protein or fragment thereof that, when compared to the native DNA sequence or reference DNA sequence, contains one or more additions, deletions, or substitutions of nucleotides but retains activity. A wide variety of PCR-based site-directed mutagenesis approaches are known in the art and can be applied by those skilled in the art.

[0033] As used herein, the terms “nucleic acid” or “nucleic acid sequence” refer to any molecule, preferably a macromolecule, that incorporates units of ribonucleic acid, deoxyribonucleic acid, or analogues thereof. Nucleic acids may be single-stranded or double-stranded. A single-stranded nucleic acid may be one nucleic acid strand of denatured double-stranded DNA. Alternatively, it may be a single-stranded nucleic acid 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 DNAs include, for example, genomic DNA or cDNA. Suitable RNAs include, for example, mRNA.

[0034] In some embodiments of any of the features described herein, the polypeptides, nucleic acids, or cells described herein may be manipulated. As used herein, “manipulated” means a form that has been manipulated by human hands. For example, a polypeptide is considered “manipulated” if at least one aspect of it, such as its sequence, has been manipulated by human hands to be different from its naturally occurring form. As is common practice and understood by those skilled in the art, the offspring of a manipulated cell are typically still referred to as “manipulated,” even though the actual manipulation was performed on the previous entity.

[0035] In some embodiments, the nucleic acid encoding the polypeptide described herein (e.g., an antibody or antibody reagent) is contained within the vector. In some embodiments described herein, a nucleic acid sequence encoding a given polypeptide described herein, or any module thereof, is operably ligated into the vector. The vector may include, but is 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 RNA or polypeptides from a sequence ligated to a transcriptional regulatory sequence on the vector. The sequence to be expressed is often heterogeneous to the cell, but not necessarily. An expression vector may include additional elements; for example, an expression vector may have two replication systems, thus enabling maintenance in two organisms, e.g., human cells for expression and a prokaryotic host for cloning and amplification. The term “expression” means cellular processes involved in the production of RNA and proteins, and, if applicable, the secretion of proteins, including, but not limited to, transcription, transcriptional processing, translation and protein folding, modification and processing, where applicable. “Expression product” includes RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene. The term “gene” means a nucleic acid sequence (DNA) that is transcribed to RNA in vitro or in vivo when operably ligated to an appropriate regulatory sequence. A gene may or may not include regions before and after the coding region, such as a 5' untranslated (5'UTR) sequence or "leader" sequence and a 3'UTR sequence or "trailer" sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0037] As used herein, the terms “isolated” or “partially purified” refer to a nucleic acid or polypeptide isolated from at least one other component (e.g., a nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide as it is found in its natural source, and / or 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 nucleic acid or polypeptide synthesized chemically, or synthesized using in vitro transcription / translation, is considered “isolated.” The terms “purified” or “substantially purified” refer to an isolated nucleic acid or polypeptide that is at least 95% by weight of the nucleic acid or polypeptide in question, e.g., at least 96% by weight, at least 97% by weight, at least 98% by weight, at least 99% by weight, or more. In some embodiments, the antibodies, their antigen-binding moieties, or chimeric antigen receptors (CARs) described herein are isolated. In some embodiments, the antibodies, antibody reagents, their antigen-binding moieties, or CARs described herein are purified.

[0038] As used herein, “manipulated” means an embodiment that has been manipulated by human hands. For example, an antibody, antibody reagent, its antigen-binding portion, CAR, or bispecific antibody is considered “manipulated” if the sequence of the antibody, antibody reagent, its antigen-binding portion, CAR, or bispecific antibody has been manipulated by human hands to differ from the sequence of the antibody as it naturally exists. As is common practice and understood by those skilled in the art, the offspring and copies of a manipulated polynucleotide and / or polypeptide are typically referred to as “manipulated” even though the actual manipulation was performed on a prior entity.

[0039] Pharmaceutical composition The compositions and methods of the present invention may be used to treat individuals in need. In certain embodiments, the individuals are mammals such as humans, or non-human mammals. When administered to animals such as humans, the compositions or compounds are preferably administered as a pharmaceutical composition comprising, for example, the compounds 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 oils such as glycols, glycerols, olive oil, or other solvents or vehicles such as injectable organic esters. In preferred embodiments, if such a pharmaceutical composition is for human administration, particularly for invasive routes of administration (i.e., routes such as injection or transplantation that avoid transport or diffusion through the epithelial barrier), the aqueous solution is pyrogenic or substantially pyrogenic. Excipients may be selected, for example, to delay the release of the drug or to selectively target one or more cells, tissues, or organs. Pharmaceutical compositions may be in unit dosage forms such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophilized reconstituters, powders, solutions, syrups, suppositories, and injections. Compositions may also be present in transdermal delivery systems, such as skin patches. Compositions may also be present in solutions suitable for topical administration, such as lotions, creams, or ointments.

[0040] A pharmaceutically acceptable carrier may contain a physiologically acceptable agent that acts to stabilize, increase the solubility of, or increase the absorption of a compound, such as the compound of the present invention. Examples of such physiologically acceptable agents include 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 selection of a pharmaceutically acceptable carrier containing 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, which may incorporate therein, for example, the compound of the present invention. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively easy to manufacture and administer.

[0041] The term "pharmaceutically acceptable" is used herein to mean a compound, material, composition, and / or dosage form that, within the bounds of sound medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, and other problems and complications, corresponding to a reasonable benefit-risk ratio.

[0042] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically 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 that it is compatible with the other components of the formulation and does not harm the patient. Some examples of materials that can function as pharmaceutically 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), and (9) oils (peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil). (10) Glycols (such as corn oil and soybean oil), (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) Water free of pyrogens, (17) Isotonic saline, (18) Ringer's solution, (19) Ethyl alcohol, (20) Phosphate buffer, and (21) Other non-toxic suitable substances used in pharmaceutical formulations.

[0043] Pharmaceutical compositions (formulations) can be administered to a target by any of a number of routes of administration, including, for example, oral (e.g., aqueous or non-aqueous solutions or suspensions, such as liquids, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption via the oral mucosa (e.g., sublingual); subcutaneous; transdermal (e.g., as a patch applied to the skin); and topical (e.g., as a cream, ointment or spray applied to the skin). Compounds may also be formulated for inhalation. In certain embodiments, compounds may simply be dissolved or suspended in sterile water. Details of suitable routes of administration and compositions therefor can be found, for example, in U.S. Patents 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 formulation may be conveniently provided in unit dosage forms and may be prepared by any method well known in the pharmaceutical art. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form varies depending on the host being treated and the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally the amount of compound that produces the therapeutic effect. Generally, out of 100 percent, this amount is in the range of about 1 percent to about 99 percent of the active ingredient, preferably about 5 percent to about 70 percent, and most preferably about 10 percent to about 30 percent.

[0045] Methods for preparing these formulations or compositions include the step of associating an active compound, such as the compound of the present invention, with a carrier and optionally one or more minor components. Generally, formulations are prepared by homogeneously and closely associating the compound of the present invention with a liquid carrier, or a finely divided solid carrier, 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 agents, powders, granules, or as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, as elixirs or syrups, or as pastels (using an inert base, e.g., gelatin and glycerin, or sucrose and acacia), and / or mouthwashes, each containing a predetermined amount of the compound of the present invention as the active ingredient. The composition or compound may also be administered as a bolus, lick, or paste.

[0047] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, sugar-coated tablets, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically 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, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia), (3) humectants (such as glycerol), (4) disintegrants. (1) Buffering agents (such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, etc.), (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 glycol, sodium lauryl sulfate, and mixtures thereof), (10) Complexing agents (such as modified cyclodextrin and unmodified cyclodextrin), and (11) Colorants. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets, and pills, the pharmaceutical composition may also contain buffering agents. Similar types of solid compositions may also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycol.

[0048] Tablets may be prepared by compression or molding with one or more optional minor components. Compressed tablets may be prepared using a binder (e.g., gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium glycolate starch or cross-linked carboxymethylcellulose sodium), a surfactant, or a dispersant. Molded tablets may be prepared by molding a mixture of powder compounds moistened with an inert liquid diluent using a suitable machine.

[0049] Tablets, and other solid dosage forms of pharmaceutical compositions such as sugar-coated tablets, capsules (including sprinkle capsules and gelatin capsules), pills, and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings known in pharmaceutical formulation technology. They may also be formulated using various proportions of hydroxypropyl methylcellulose, other polymer matrices, liposomes, and / or microspheres to provide a desired release profile, for example, to provide sustained-release or controlled release of the active ingredient therein. They may be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating a sterilizer in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injection medium immediately before use. These compositions may also optionally contain emulsifiers and may optionally release the active ingredient in a delayed manner, only in or preferentially in a specific portion of the gastrointestinal tract. Examples of embedding compositions that may be used include polymer substances and waxes. The active ingredient may also, where appropriate, be in the form of microcapsules together with one or more of the excipients described above.

[0050] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophilized reconstituted agents, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art (e.g., water or other solvents), cyclodextrins and their derivatives, solubilizers and emulsifiers (e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (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] In addition to inert diluents, oral compositions may also contain adjuvants such as humectants, emulsifiers and suspending agents, sweeteners, flavoring agents, colorants, flavoring agents and preservatives.

[0052] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, 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 pharmaceutically acceptable carrier and any preservatives, buffers, or propellants as needed.

[0054] In addition to the active compound, ointments, pastes, creams, and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0055] The powders and sprays may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powders, or mixtures thereof, in addition to the active compound. The sprays may also contain conventional propellants such as chlorofluorohydrocarbons, as well as volatile unsubstituted hydrocarbons such as butane and propane.

[0056] Transdermal patches offer the additional advantage of providing controlled delivery of the compounds of the present invention into the body. Such dosage forms can be prepared by dissolving or dispersing the active compound in a suitable medium. Absorption enhancers can also be used to increase the flow of the compound across the skin. The rate of such flow can be controlled either by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or gel.

[0057] As used herein, the terms “parenteral administration” and “administered parenterally” refer to modes of administration other than intra-intestinal and topical administration, usually by injection, and include, but are not limited to, intravenous, intraocular (e.g., intravitreous), intramuscular, intra-arterial, subarachnoid, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intrasternal, and intrasternal injections and infusions. Pharmaceutical compositions suitable for parenteral administration include combinations of one or more active compounds and one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted into sterile injections or dispersions immediately before use, which may include antioxidants, buffers, bacteriostatic agents, solutes that make the intended recipient’s blood and preparation isotonic, or suspending agents or thickeners.

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

[0059] These compositions may also contain adjuvants such as preservatives, humectants, emulsifiers, and dispersants. Prevention of microbial action can be ensured by including various antimicrobial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. Furthermore, long-term absorption of injectable pharmaceutical forms can be achieved by including absorption-delaying agents such as aluminum monostearate and gelatin.

[0060] In some cases, it is desirable to delay the absorption of a drug from subcutaneous or intramuscular injection in order to prolong its effects. This can be achieved by using a liquid suspension of a crystalline or amorphous material with low water solubility. The absorption rate of the drug then depends on its dissolution rate, which may depend on the crystal size and crystalline form. Alternatively, delaying the absorption of a drug form in parenteral administration can be achieved by dissolving or suspending the drug in an oily vehicle.

[0061] Injectable depot formulations are prepared by forming a microencapsulation matrix of the target compound in a biodegradable polymer such as polylactide-polyglycolide. The drug release rate can be controlled depending on the drug-to-polymer ratio and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoester) and poly(anhydride). Injectable depot formulations can also be prepared by encapsulating the drug in liposomes or microemulsions compatible with body tissues.

[0062] For use in the method of the present invention, the active compound may be given by itself or as a pharmaceutical composition containing 0.1 to 99.5% (more preferably 0.5 to 90%) of the active ingredient in combination with, for example, a pharmaceutically acceptable carrier.

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

[0064] The actual dose level of the active ingredient in a pharmaceutical composition may be varied to obtain an amount of the active ingredient effective in achieving 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 various factors, including the activity of the specific compound or combination of compounds used, or its ester, salt, or amide; the route of administration; the time of administration; the rate of excretion of the specific compound used; the duration of treatment; other drugs; compounds and / or materials used in combination with the specific compound used; age; sex; weight; condition; the overall health and prior medical history of the patient being treated; and similar factors well known in the medical technology field.

[0066] A physician or veterinarian with general art in this field can easily determine and prescribe the therapeutically effective dose of the required pharmaceutical composition. For example, a physician or veterinarian can start with a dose of the pharmaceutical composition or compound at a level lower than the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. The therapeutically effective dose means a concentration of the compound sufficient to induce the desired therapeutic effect. It is generally understood that the effective dose of a compound varies depending on the subject's weight, sex, age, and medical history. Other factors that affect the effective dose 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 with the compound of the present invention. A larger total dose can be delivered by multiple administrations of the drug. Methods for determining efficacy and dosage are known to those skilled in the art. See, for example, Isselbacher et al. (1996). 8

[0067] Generally, the appropriate daily dose of the active compound used in the compositions and methods of the present invention is the amount of the compound that is the minimum effective dose to produce a therapeutic effect. Such an effective dose generally depends on 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 partial doses, separately at appropriate intervals throughout the day, 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] Patients receiving this treatment include primates, particularly humans, as well as any animals in need, including other mammals such as cattle, pigs, sheep, cats, and dogs, poultry, and pets in general.

[0070] In certain embodiments, the compounds of the present invention may be used alone or administered in combination with other types of therapeutic agents.

[0071] This disclosure includes the use of pharmaceutically acceptable salts of the compounds of the present invention in compositions and methods of the present invention. In certain embodiments, the salts intended by the present invention include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkylammonium salts. In certain embodiments, the salts intended by the present invention include, but are not limited to, L-arginine, venentamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydravamin, 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 salts intended by the present invention include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts.In certain embodiments, the salts intended for the present invention are not limited to, but include, 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, (+)-monosulfonic 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, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, and gen. This product contains tidic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphate, glycolic acid, hip phosphate, 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 can also exist as various solvates containing water, methanol, ethanol, dimethylformamide, and the like. Mixtures of these solvates can also be prepared. The source of these solvates may be derived from the crystallization solvent inherent to the solvent used for preparation or crystallization, or it may be exogenous to such solvent.

[0073] Wetting agents, emulsifiers, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the composition.

[0074] Examples of pharmaceutically acceptable antioxidants include the following: (1) water-soluble antioxidants (such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, 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 chelating agents (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 pertains. It should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents, etc. described herein and may therefore vary. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention, which is defined only by the claims. Definitions of common terms in immunology and molecular biology can be found in 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 found in Current Protocols in Immunology (CPI). 20

[0076] In some embodiments of any aspect, the disclosure described herein does not relate to the process of cloning a human, the process of altering the genetic identity of a human germline, the use of human embryos for industrial or commercial purposes, or the process of altering the genetic identity of an animal for which there is no substantial medical benefit to humans or animals, or to animals resulting from such processes.

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

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

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

[0080] IBD is a highly distressing disease due to fatigue associated with inflammatory symptoms and the chronic pain patients suffer from. The pathogenesis of IBD is only partially understood, involving a variety of environmental and host factors (e.g., genetic, epithelial, immune, and non-immune factors). A complex interaction between the immune system, gut symbiotic bacteria / pathogens, and host genotype is thought to underlie the development of IBD. These relapsing chronic inflammatory disorders are thought to be triggered by an overly aggressive T-cell response directed towards a subset of symbiotic bacteria / pathogens residing in the distal ileum and colon of the host that are environmentally and / or genetically susceptible.

[0081] Currently, there is no cure or effective treatment for patients diagnosed with functional gastrointestinal disorders such as IBD. The main goal of current therapies for IBD is to induce clinical remission by targeting symptoms and then maintain it over the long term to achieve the best possible quality of life. Because current therapies have limited efficacy, new therapies to treat inflammation and chronic pain in the intestines of patients with IBD are clinically important.

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

[0083] The innate endogenous BiP, also known as glucose regulatory protein 78 (GRP78), is a stress protein (SP) and a member of the heat shock protein (HSP) 70 family. Present in all nucleated cells, BiP is released from capture and upregulated upon activation of the endoplasmic reticulum stress response (UPR). This is initiated by the intracellular accumulation of denatured proteins and nascent polypeptides, particularly triggered by cellular stress such as a decrease in oxygen and / or glucose, as well as an increase in reactive oxygen species. These conditions are characteristic of chronic inflammatory pathologies, such as the inflamed synovium in patients with rheumatoid arthritis (RA).

[0084] Like other SPs or HSPs with important intracellular functions, these molecular chaperones, including BiP, are expressed on the cell surface, secreted during upregulation, and enable unexpected and distinctly different extracellular functions. This has led to a growing 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 being developed for the treatment of autoimmune diseases. Extensive research has shown that rhuBiP (IRL201805, lacking the C-terminal KDEL sequence) may 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 a collagen-induced arthritis (CIA) model demonstrate that rhuBiP / IRL201805 possesses therapeutic properties associated with increased lifespan and changes in immune cell populations in the absence of rhuBiP / IRL201805.

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

[0087] These data demonstrate that IRL201805 exhibits significant activity in reducing inflammatory responses associated with rheumatoid arthritis, and its mechanism of action suggests potential efficacy in various immune-based disorders.

[0088] CD69 is a transmembrane glycoprotein that has a C-type lectin domain (CTLD). 21,22,23 It has recently been identified as an important regulator of intestinal inflammation and a novel target molecule for the treatment of IBD. 24 CD69 is not expressed at detectable levels on naive leukocytes, but its surface expression is rapidly induced upon activation. 25、26 In human diseases, CD69 expression increases on leukocytes at the site of inflammation. 27,28,29 Furthermore, early in vitro studies have shown that CD69's association with intracellular Ca 2+ The influx of pro-inflammatory molecules, lymphocyte proliferation, and the production of pro-inflammatory mediators, such as IL-2, tumor necrosis factor-(TNF-)α, and nitric oxide (NO), has been described. 30,31,32,33 CD69 is also required for T cell-mediated contact-dependent stimulation of macrophages. 34 However, recent in vivo studies using transgenic mice have shown that CD69 can limit the immune response and have proposed a regulatory function for CD69. CD69 has been shown to play a role in leukocyte migration within the functions of regulatory T cells and commensal tissue memory T cells. 35 Therefore, stable induction of CD69 expression reduces lymphocyte migration to the intestinal LP and CD69 + It has been suggested that this should lead to the generation of Treg cells. 36

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

[0090] The description of embodiments of this disclosure is not intended to be exhaustive or to limit the disclosure to the exact form disclosed. While specific embodiments and examples of this disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of this disclosure, as will be recognized by those skilled in the art. For example, while the steps or functions of a method are presented in a given order, alternative embodiments may perform the functions in a different order or substantially simultaneously. The teachings of this disclosure provided herein may be applied to other techniques or methods as needed. Further embodiments may be provided by combining the various embodiments described herein. Aspects of this disclosure may be modified, as needed, to adopt compositions, functions, and concepts of the above-mentioned references and applications to provide further embodiments of this disclosure. Furthermore, several modifications to the protein structure may be made without affecting the biological or chemical action in any way or quantity, taking into account biofunctional equivalence. These and other modifications may be made to this disclosure with regard to the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0091] Certain components of any of the embodiments described above may be combined with or substituted for components of other embodiments. Furthermore, while the advantages associated with specific embodiments of this disclosure have been described in the context of those embodiments, other embodiments may also demonstrate such advantages, and not all embodiments are required to demonstrate such advantages in order to fall within the scope of this disclosure.

[0092] The techniques described herein are further illustrated by the following examples, which should not be construed as further limitations in any respect. Similar or equivalent methods and materials may be used in the implementation or testing of the disclosure, but preferred methods and materials are described below. [Examples]

[0093] The present invention will be described in general terms here, but this will be more readily understood by referring to the following examples, which are included solely for illustrative purposes of specific aspects and embodiments of the invention and are not intended to limit the invention.

[0094] Example 1: Evaluation of the efficacy of a binding immunoglobulin protein (BiP) analog in a mouse model of enteritis. Currently, there are over 50 animal models of human IBD. While no model can fully analyze the phenotype of human IBD, many models are useful for studying various aspects of human disease, including disease onset and progression, as well as wound healing responses.

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

[0096] material and method material IRL201805 (Sequence ID 1) is manufactured in accordance with GMP standards at CBC in Bristol, UK.

[0097] method Experiment Overview Male RAG2 - / - Mice are randomly assigned to experimental groups and allowed to acclimate for one week. On day 0, the spleen is removed from donor C57Bl / 6N male mice and single-cell suspension prepared according to the CRL SOP. On day 0, male RAG KO mice are given CD4 cells resuspended in 500 μL of PBS. + CD45RB 高 T cells (CD45RB only for group 1) 低 T cells are injected intraperitoneally.

[0098] From day 21 until the end of the experiment on day 49, the animals were monitored daily for clinical signs of colitis, including weight loss, loose stools, and / or diarrhea. On day 49, the animals were sacrificed, the colon was dissected, and its weight and length were measured. Colons from half of the animals in each group were transferred to a tissue fixative, then processed for paraffin embedding and preserved for histopathological analysis. The lamina propria from half of the animals in each group were processed for flow cytometry.

[0099] Dosage schedule The treatment will be administered according to the schedule in Table 1 below. All groups have n=10.

[0100] (Table 1) TIFF0007898242000001.tif43161 Abbreviations: NA: Not applicable, IP: Intraperitoneal injection, IV: Intravenous injection, TBC: Confirmation required, CRL: Charles River regimen

[0101] Morphology and mortality rates Morphology and mortality rates are assessed daily. Animals are monitored for clinical signs including abnormal posture (e.g., crouching), abnormal hair condition (e.g., erect hair), changes in the color of hairless areas (including cyanosis or jaundice), the presence of lumps and / or swellings, abnormal respiration, abnormal movement, and decreased activity. Causes of death are recorded once identified. Survival rates are recorded.

[0102] body weight The animals will be weighed at the start of the study (day 0) and regularly throughout the first three weeks. From day 21 until the end of the study, all animals will be weighed daily. The data will be analyzed and graphed.

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

[0104] (Table 2) TIFF0007898242000002.tif30128

[0105] Flow cytometry Single-cell suspensions are prepared from lamina propria colon cells isolated from half of 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 as well as surface and intracellular markers: CD44, CD45, CD3, CD4, IFNγ, IL-17, CD25, and FOXP3. The number and percentage of each population are determined.

[0106] gross pathology At the end of the procedure, the colon is dissected and photographed using ImageJ software so that its length can be measured digitally. The colon is also weighed.

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

[0108] (Table 3) TIFF0007898242000003.tif74132

[0109] result This study shows that IRL201805 results in a significant reduction in clinical signs of colitis, including weight loss, loose stools, and / or diarrhea, as well as a significant reduction in severity scores based on sections scored for mucosal thickness, mucosal ulceration, lamina propria mononuclear cell infiltration, lamina propria granulocyte infiltration, and crypt abscess / dilation / deformity.

[0110] These results suggest that administering binding immunoglobulin proteins (BiPs) or functional analogues to subjects at risk of or suffering from IBD is an effective method for preventing or treating IBD. BiPs or functional analogues useful in this intended method include, but are not limited to, polypeptides having amino acid sequences selected from SEQ ID NOs: 1-5.

[0111] Example 2: Evaluation of the efficacy of a conjugated 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 sulfate sodium (DSS), a chemocoritogen 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, most similar to human UC, is: 39 This is caused by the administration of 40-50 kDa DSS in drinking water. The mechanism by which DSS induces colitis is unknown, but it is likely a result of damage to the epithelial monolayer lining the large intestine, which allows for the penetration of pro-inflammatory intestinal contents (e.g., bacteria and their products) into the basal tissue. The DSS colitis model is very popular in IBD research due to its speed, simplicity, reproducibility, and controllability. Acute, chronic, and recurrent models of colitis can be achieved by changing the concentration and frequency of DSS administration.

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

[0113] material and method material IRL201805 (Sequence ID 1) is manufactured in accordance with GMP standards at CBC in Bristol, UK.

[0114] method Experiment Overview Adult C57bl / 6 mice were randomly assigned to the experimental group and allowed to acclimate for one week. From day 2, treatment was administered according to the following dosing schedule. On day 0, drinking water was replaced with a 5% dextran sulfate sodium (DSS) aqueous solution (except for group 1, which continued to receive normal drinking water throughout). The animals were given free access to the 5% DSS solution. From day 0 until the end of the experiment on day 7, the animals were monitored daily for clinical signs of colitis, including weight loss, loose stools and / or diarrhea, and the presence of occult blood or gross bleeding in the feces.

[0115] Blood samples are collected on day 2, day 3 (while alive), and day 7 (end of treatment), processed into serum, and stored for analysis.

[0116] On day 7, the animals are slaughtered, the colon is dissected, and its length is measured. One sample (distal) of the colon is taken from each animal and fixed for histopathological analysis. A further sample (proximal) of the colon is taken at the end and cultured for 48 hours in or without LPS before cytokine analysis. The remaining supernatant is saved for analysis.

[0117] Dosage schedule The treatment will be carried out according to the schedule in Table 1 below. Unless otherwise noted, all groups have n=10.

[0118] (Table 1) TIFF0007898242000004.tif46161 Abbreviations: TBC: Not applicable, NA: Not applicable, IV: Intravenous, PO: Oral.

[0119] Morphology and mortality rates Morphology and mortality rates are assessed daily. Animals are monitored for clinical signs including abnormal posture (e.g., crouching), abnormal hair condition (e.g., erect hair), changes in the color of hairless areas (including cyanosis or jaundice), the presence of lumps and / or swellings, abnormal respiration, abnormal movement, and decreased activity. Causes of death are recorded once identified. Survival rates are recorded.

[0120] body weight All animals will be weighed daily from day 1 until the end of the experiment. The data will be analyzed and graphed.

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

[0122] (Table 2) TIFF0007898242000005.tif29128

[0123] gross pathology At the end of the procedure, the colon is dissected and photographed using ImageJ software so that its length can be measured digitally. The colon is also weighed.

[0124] Measurement of cytokine levels in extracolon transplants Colon samples collected on day 7 are cultured for 48 hours in or without LPS, and then cytokine content is analyzed using Luminex. The following cytokines are analyzed using multiplex (8-plex) Luminex: sTNFRII, sIL-6R, IL-6, IL-10, IL-1b, TNF-α, IFN-γ, and MCP-1. Samples are analyzed as single samples using sandwich immunoassays and multiplex xMAP bead technology, which utilizes microspheres as a solid support for determining multiple cytokines in the same sample (Luminex).

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

[0126] (Table 3) TIFF0007898242000006.tif74132

[0127] result This study shows that IRL201805 results in a significant reduction in clinical signs of colitis, including weight loss, loose stools, and / or diarrhea, as well as a significant reduction in severity scores based on sections scored for mucosal thickness, mucosal ulceration, lamina propria mononuclear cell infiltration, lamina propria granulocyte infiltration, and crypt abscess / dilation / deformity.

[0128] These results suggest that administering binding immunoglobulin proteins (BiPs) or functional analogues to subjects at risk of or suffering from IBD is an effective method for preventing or treating IBD. BiPs or functional analogues useful in this intended method include, but are not limited to, polypeptides having amino acid sequences selected from SEQ ID NOs: 1-5.

[0129] Example 3: Evaluation of the effect of a binding immunoglobulin protein (BiP) analog on CD69 in colon punch biopsies obtained from IBD patients. In this study, as shown in Figures 6A to 6E, punch biopsies (2 mm) were taken of inflamed and non-inflamed colon tissue. 2 Tissue biopsies were obtained from each informed IBD patient with hospital ethical approval. Subsequently, tissue biopsies were incubated for 12-14 hours with or without IRL201805 (20 μg / mL) and evaluated 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 STAT5 phosphorylation, which leads to STAT3 inhibition. CD39 is Ca 2+ and Mg 2+ CD39 is a membrane-bound protein that, in a dependent manner, phosphate hydrolyzes ATP and, more inefficiently, ADP to obtain AMP. Ultimately, the resulting adenosine is a potent activator of tolerogenic dendritic cells (DCs). CD39 becomes catalytically active upon its localization to the cell surface.

[0131] As shown in Figure 7, exposure of primary immune cells in inflamed colon tissue with IRL201805 induced a 20% increase in bipositive CD69+ / CD39+ Tregs compared to the same treatment of non-inflammatory tissue from the same patient. Flow cytometry in this study also showed that IRL201805 resulted in an increase in p-STAT5 in a subset of immune cells (data not shown). Previous unpublished data have also demonstrated that BiP inhibits STAT3 phosphorylation in human lymphocytes. These data suggest that BiP-related peptide enhances CD69 activity and the resulting cascade only in inflammatory tissue from IBD patients, but not in non-inflammatory tissue.

[0132] Example 4: Evaluation of the effect of binding 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, such as CD69 and CD154 (CD40L), which are commonly used. CD69 expression is usually transient on most T cells, especially T effector cells, and decreases within 24 hours.

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

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

[0135] JAK-STAT signaling pathway The Janus kinase (JAK) / signaling and transcriptional activator (STAT) (JAK / STAT) pathway organizes and regulates differentiation pathways that control and modulate many cytokines and immune responses. Signaling via the JAK-STAT signaling pathway involves rapid transmission of signals from the cell membrane to the nucleus, followed by a highly organized response, and then controlled downregulation and attenuation of the initial signal. Cytokine signaling suppressor (SOCS) proteins are major drivers of signal attenuation. They are induced by cytokine exposure (via STAT) 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] This study evaluated the effects of IRL201805 on the JAK-STAT signaling pathway at both the transcript and protein levels. PBMCs were isolated from healthy subjects and treated with 20 μg / mL of IRL201805 for 24 hours.

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

[0138] p-STAT5 levels were evaluated from untreated and IRL1805-treated PBMCs using immunoblotting of PBMC cell lysates with recombinant anti-STAT5 (phospho Y694) antibody [E208] (ab32364) (Abcam, ab32364). 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 Treg cells, CD69 is thought to promote CD39 surface expression, FoxP3 transcription, and STAT5 phosphorylation, which leads to STAT3 inhibition. This study evaluated the effect of IRL1805 on p-STAT5. IRL201805 resulted in increased p-STAT5 in a subset of immune cells. Treatment of PBMCs with IRL201805 resulted in altered p-STAT5 status and a threefold increase in FoxP3 transcription levels in CD4 / CD8 T cells (see Table 4).

[0140] (Table 4) TIFF0007898242000007.tif21157

[0141] CD69-related ligands Figure 10A provides a schematic diagram of CD69-related ligands. CD69 surface expression increases with APC-derived MHC class II-TCR activation. TCR engagement induces moderate activation of the metabolic sensor mTOR, which is enhanced along with CD28 and IL-2 signaling, and this can promote T cell commitment to the generation of TH1, TH2, and TH17 effector cells. Membrane SiP1 / SiPR1 interactions can maintain mTOR activation and inhibit FoxP3 cell differentiation. 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] Potential ligands Gal-1 and S100A8 / A9 are known to bind to CD69. 43To investigate the effects of 1805 on T cell expression and the ability of T cells to maintain the Treg phenotype, this study examined the effects of 1805 on Gal-1 and S100A8 / A9 expression in APCs (monocytes). Human monocytes isolated from healthy individuals (n=4) were exposed to 1805 for 1 hour at levels of both Gal-1 and S100A8 / A9 proteins.

[0143] The abundance of Gal-1 and S100A8 / A9 proteins on the cell surface was detected by tandem mass-tagged (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, the 1805 treatment increased S100A9 phosphorylation by 58% (Figure 10C). Mass spectrometry identified increased post-translational modification of threonine 113 in S100A9 after the 1805 treatment, a modification known to promote the active secretion of S100A9 from bone marrow cells.

[0145] Surface CD154 and CD69 activation markers on PBMC-derived T lymphocytes 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 number of these cells are constantly exposed to antigen challenge.

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

[0147] As shown in Figure 11, the basal level of cell surface CD69 expression was 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 produced only a statistically significant twofold 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 While not bound by a specific mechanism of action, Figure 12 provides a schematic diagram of a possible mechanism of action for the effects of BiP-related peptides such as IRL201805 on CD69-related 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 the APC. The CD69 receptor binds to ligands on the surface of the APC (e.g., S100A8 / A9 or Gal-1). The cytoplasmic tail of CD69 associates with the Jak3 and Stat 5 proteins, causing phosphorylation of Stat 5 and its translocation to the nucleus, where it activates the transcription factor FoxP3 and stimulates the 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 may act autocrinely to induce differentiation of regulatory T cells and tolerant APCs. CD69 can inhibit the Th17 differentiation pathway by at least two mechanisms: CD69-activated Stat5 directly inhibits the nuclear translocation of Stat3 and indirectly antagonizes Stat3-mediated RORγt activation via FoxP3 activation.

[0149] conclusion CD69 has recently been identified as an important 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 the immune response and have proposed a regulatory function of CD69. CD69 has been shown to play a role in leukocyte migration within the function of regulatory T cells and commensal tissue memory T cells. Therefore, stable induction of CD69 expression may reduce lymphocyte migration to the intestinal LP and CD69 + It has been suggested that this should lead to the generation of Treg cells.

[0150] This disclosure provides evidence that BiP-related polypeptides produce stable induction of CD69 expression only in inflamed colon tissue, and provides methods for inducing activation of several CD69 activation markers and CD69-related ligands, thereby modulating inflammatory responses associated with intestinal inflammation and preventing or treating inflammatory bowel disease (IBD).

[0151] References TIFF0007898242000008.tif91161TIFF0007898242000009.tif215161TIFF0007898242000010.tif96160

[0152] All patents and other publications cited throughout this application, including references, issued patents, published patent applications, and concurrently pending patent applications, are expressly incorporated herein by reference for the purpose of describing and disclosing methodologies described in such publications that may be used, for example, in connection with the technology described herein. These publications were made available prior to the filing date of this application solely for the purpose of their disclosure. In this regard, it should not be construed that the inventors acknowledge that they do not have prior art or any other right to such disclosures. All statements regarding dates or representations relating to the contents of these documents are based on information available to the applicant and do not constitute any endorsement of the accuracy of the dates or contents of these documents.

[0153] The aforementioned written specification is deemed sufficient to enable a person skilled in the art to carry out the present aspects and embodiments. The present aspects and embodiments are intended as single examples of one aspect, and the scope is not limited by the examples provided, as other functionally equivalent embodiments are within the scope of this disclosure. In addition to those shown and described herein, various modifications will be apparent to a person skilled in the art from the foregoing description and are included in the appended claims. The advantages and objectives described herein are not necessarily encompassed by each embodiment. A person skilled in the art will be able to recognize or confirm many equivalents to the particular embodiments described herein by means of routine experimentation. Such equivalents are intended to be covered by the appended claims.

Claims

1. A pharmaceutical composition for preventing or treating inflammatory bowel disease (IBD) in subjects at risk of IBD or suffering from IBD, comprising a therapeutically effective amount of a conjugated immunoglobulin protein (BiP) consisting of the amino acid sequence of SEQ ID NO:

1.

2. The pharmaceutical composition according to claim 1, wherein IBD is ulcerative colitis.

3. The pharmaceutical composition according to claim 1, wherein IBD is Crohn's disease.

4. A pharmaceutical composition according to any one of claims 1 to 3 for intravenous administration.

5. Use of a conjugated immunoglobulin protein (BiP) comprising the amino acid sequence of SEQ ID NO: 1 in the preparation of a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease (IBD).

6. The use according to claim 5, wherein IBD is ulcerative colitis.

7. The use according to claim 5, wherein IBD is Crohn's disease.

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