Compositions and their use in methods for treating intestinal inflammations

Abstract

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

Description

Technical Field

[0001] Embodiments of the present invention relate to compositions and methods for treating inflammatory bowel disease (IBD) with binding immunoglobulins (BiPs) or functional analogs thereof. Background Art

[0002] Inflammatory bowel disease (IBD), represented primarily by ulcerative colitis and Crohn's disease but also encompassing non-infectious inflammation of the intestine, has posed a mystery to gastroenterologists and immunologists since its first modern description approximately 75–100 years ago. IBD is characterized by chronic inflammation of the gastrointestinal (GI) tract, causing GI damage. In 2017, there were 6–8 million cases of IBD worldwide. The burden of IBD is rising globally; the age-standardized prevalence increased 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 use than adults without IBD, including physician visits, medication prescriptions, emergency department visits, hospitalizations, and surgeries. 2

[0003] IBD treatment usually involves drug therapy or surgery. Anti-inflammatory drugs are often the first step in the treatment of inflammatory bowel disease (for example, corticosteroids and aminosalicylate, such as mesalamine, balsalazide and olsalazine). However, anti-inflammatory drugs are often insufficient to provide sufficient and sustainable relief of IBD. Alternative therapies include immunosuppressive drugs (for example, azathioprine, mercaptopurine and methotrexate), biological agents for neutralizing proteins that cause inflammation (for example, infliximab (Remicade), adalimumab (Humira), golimumab (Simponi), certolizumab (Cimzia), vedolizumab (Entyvio) and ustekinumab (Stelara)), and surgery as a last resort. In the past two decades, the progress of biological agents and small molecule therapeutic agents has caused a rapid increase in IBD treatment equipment. Despite these advances, Crohn's disease and ulcerative colitis remain chronic and progressive diseases. One of the main reasons for persistent inflammation and intestinal damage is the failure of current drug therapies.

[0004] Currently, there is no cure or effective treatment for patients diagnosed with functional gastrointestinal diseases such as IBD. The main goal of current IBD therapies is to induce clinical remission by focusing on symptoms and then maintain clinical remission for a long time in order to achieve the best quality of life that can be achieved. Due to the limited efficacy of current therapies, new therapies for treating inflammatory and chronic pain in the intestines of subjects with IBD are needed. Therefore, there is a need for improved therapies for the treatment of IBD. Summary of the invention

[0005] Embodiments of the present invention provide a method for regulating an inflammatory response 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 a binding immunoglobulin (BiP) or a functional analog thereof to a subject at risk of or suffering from IBD. In some embodiments, the BiP or its functional analog 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 their conservative amino acid substitutions. In an alternative embodiment, the BiP or its functional analog 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 enteritis.

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

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

[0008] Other implementations are also described and listed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] For the purpose of illustration, certain embodiments of the invention are shown in the accompanying drawings described below. It should be understood, however, that the invention is not limited to the precise arrangements, dimensions and instruments shown. In the drawings:

[0010] Figure 1 The amino acid sequence of the BiP analog IRL201805 (SEQ ID NO: 1) is provided. 3

[0011] Figure 2 The amino acid sequence of the His-tagged BiP analog (SEQ ID NO: 2) expressed by plasmid pQE2 is provided. 4

[0012] Figure 3A-3D The amino acid sequence of a BiP analog (SEQ ID NO: 3) is provided. 5

[0013] Figure 4A-4D The amino acid sequence of a BiP analog (SEQ ID NO: 4) is provided. 6

[0014] Figure 5 The amino acid sequence of native BiP (SEQ ID NO: 5) is provided. 7

[0015] Figure 6 A- Figure 6 E provides an overview of the procedures used to evaluate the effects of ex vivo human primary immune cells within inflamed and non-inflamed colon tissues treated with IRL201805.

[0016] Figure 7 Shown is the effect of 12 h 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.

[0017] Figure 8 Depicted are the mean fluorescence intensity of CD69 and CD39 surface expression on Tregs of peripheral blood mononuclear cells (PBMCs) cultured with 20 μg / mL IRL201805 for 20-72 hours at 37°C.

[0018] Figure 9A-9B The effects of 20 μg / mL IRL201805 on the JAK-STAT signaling pathway at the transcript and protein levels are illustrated. Fig.9A RNAseq analysis of gene transcript levels of key signaling genes involved in CD69 activation is provided. Fig. 9BProvided are immunoblots of p-STAT5 levels in cell lysates from untreated and IRL1805-treated PBMCs.

[0019] Fig.10 A provides a schematic diagram of CD69-related ligands. Fig.10 B 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. Fig.10 B provides a graphic representation of the increased trimethylation post-translational modification on threonine 113 on S100A9 after IRL201805 treatment.

[0020] Fig.11 A- Fig.11 C provides a bar graph showing the effect of IRL201805 on the expression of surface CD154 and CD69 activation markers on T lymphocytes from PBMCs: Treg ( Fig.11 A); effector T cells (Teff; Fig.11 B); and CD8 T cells ( Fig.11 C). Results are from PBMCs isolated from four healthy donors. Mean ± SD is shown. P = values ​​represent paired sample t-test. NS = not significant.

[0021] Fig.12 A schematic diagram of the effects of IRL201805 on CD69 and CD69-associated ligands is provided. Abbreviations: APC, antigen presenting cell; TCR, T cell receptor; Treg, regulatory T cell; P, phosphorylation. DETAILED DESCRIPTION

[0022] The present invention is now described with reference to the accompanying drawings, wherein the same reference numerals are used to refer to the same elements throughout. In the following description, for the purpose of explanation, many specific details are set forth in order to provide a thorough understanding of the present invention. However, it is apparent that the present invention can be practiced without these specific details. In other cases, for ease of describing the present invention, well-known structures and devices are shown in block diagram form. It should be understood that, in order to provide a basic understanding of the present invention, certain aspects, modes, embodiments, variations and features of the present invention are described below with different levels of detail.

[0023] definition

[0024] For convenience, the meanings of some terms and phrases used in the specification, examples and appended claims are provided below. Unless otherwise specified, or implied from the context, the following terms and phrases include the meanings provided below. These definitions are provided to help describe specific embodiments and are not intended to limit the claimed invention, as the scope of the present invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which the present invention belongs. If there is a clear difference between the use of the term in the art and its definition provided herein, the definition provided in the specification shall prevail.

[0025] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, etc.

[0026] As used herein, unless otherwise specified or otherwise apparent from the context (unless such a number would be a possible value of less than 0% or more than 100%), the term "about" or "approximately" with respect to a value or parameter is generally employed to include numbers falling within 5%, 10%, 15%, or 20% ranges in either direction (greater than or less than) of that number. As used herein, reference to "about" or "approximately" a value or parameter includes (and describes) embodiments for that value or parameter. For example, a description referring to "about X" includes a description of "X".

[0027] As used herein, the term "or" means "and / or". The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover 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).

[0028] As used herein, the term "comprising" means that there may be additional elements in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation.

[0029] The term "consisting of refers to the compositions, methods, and respective components thereof as described herein, excluding any elements not recited in this description of the embodiment.

[0030] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel features or functional characteristics of that embodiment of the invention.

[0031] The term "statistically significant" or "significant" refers to statistical significance and generally means a difference of two standard deviations (2SD) or greater.

[0032] As used herein, the term "subject" refers to a mammal, including but not limited to dogs, cats, horses, cattle, pigs, sheep, goats, chickens, rodents, or primates. The subject can be a domestic pet (e.g., dog, cat), agricultural livestock (e.g., cattle, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), but is not limited thereto. The subject includes a human subject. The human subject can be a child, an adult, or an elderly subject. The human subject can be of either gender.

[0033] As used herein, the terms "effective amount" and "therapeutically effective amount" include an amount sufficient to prevent or ameliorate the manifestation of a disease or medical condition, such as an immune disorder, including rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, organ, skin, tissue, blood, serum, plasma transplant rejection, inflammatory bowel disease or Crohn's disease. It should be understood that there are many known methods in the art to determine the effective amount for a given application. For example, pharmacological methods for dosage determination can be used in a therapeutic setting. In the case of therapeutic or preventive applications, the amount of the composition administered to the subject will depend on the type and severity of the disease and the characteristics of the individual, such as overall health, age, sex, weight and tolerance to drugs. This also depends on the extent, severity and type of the disease. A skilled technician will be able to determine the appropriate dosage based on these and other factors. These compositions can also be administered in combination with one or more additional therapeutic compounds.

[0034] As used herein, the terms "treat" or "alleviate" refer to (1) therapeutic measures that cure, slow, alleviate, and / or interrupt the progression of a diagnosed disease or infection, and (2) prophylactic or preventive measures that prevent or slow the progression 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 during childhood.

[0035] As used herein, the term "treatment" or "improvement" refers to the therapeutic treatment of a condition when used in reference to a disease, disorder or medical condition, wherein the purpose is to reverse, alleviate, improve, inhibit, slow down or terminate the progression or severity of a symptom or condition. The term "treatment" includes reducing or alleviating at least one harmful effect or symptom of a condition. If one or more symptoms or clinical markers are reduced, the treatment is usually "effective". Alternatively, if the progression of the condition is reduced or stopped, the treatment is "effective". That is, "treatment" includes not only the improvement of symptoms or markers, but also includes stopping or at least slowing down the progression or deterioration of symptoms expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, relief of one or more symptoms, reduction in the degree of defect, stabilization of the state of an immune disorder (i.e., no deterioration), delay or slowing of an immune disorder, and an increase in life expectancy compared to that expected in the absence of treatment.

[0036] As used herein, the term "intermittent" administration means that the therapeutic agent or drug is administered when IBD symptoms are present and administration is stopped when the IBD symptoms are alleviated.

[0037] As used herein, the term "long-term" administration means that the therapeutic agent or drug is administered for at least 12 weeks. This includes administering a therapeutic agent or drug so that it is effective for at least 12 weeks or continues to be effective for at least 12 weeks, but does not necessarily mean that the administration itself lasts for 12 weeks, for example, if a sustained release composition or a long-acting therapeutic agent or drug is used. Thus, the subject receives treatment for at least 12 weeks. In many cases, long-term administration lasts for at least 4, 5, 6, 7, 8, 9 months or longer, or for at least 1, 2, 3, 5, 7 or 10 years or longer.

[0038] The administration of the compositions contemplated herein can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, infusion, 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 intestinal and topical administration, usually administered by injection, and include but are not limited to intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal 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.

[0039] The terms "reduce", "reduced", "reduce" or "inhibit" are all used herein to mean a statistically significant reduction. In some embodiments, "reduce" or "reduce" or "inhibit" generally means a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent), and can 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. For individuals without a given disease, reduction can preferably be reduced to an acceptable level within the normal range.

[0040] The terms "increased", "increase", "enhanced" or "activity" are all used herein to mean an increase in a statistically significant amount. In some embodiments, the terms "increased", "increase", "enhancement" or "activity" may mean an increase of at least 10% compared to a reference level, such as an increase of at least about 20%, 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 up to and including an increase of 100% compared to a reference level or any increase between 10% and 100%, or 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 compared to a reference level, or any increase between 2 times and 10 times or greater. In the case of a marker or symptom, an "increase" is a statistically significant increase in such a level.

[0041] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to refer to a series of amino acid residues that are interconnected by peptide bonds between the α-amino and carboxyl groups of adjacent residues. The terms "protein" and "polypeptide" refer to polymers of amino acids, including modified amino acids (e.g., phosphorylation, saccharification, glycosylation, etc.) and amino acid analogs, regardless of their size or function. "Protein" and "polypeptide" are often used to refer to relatively large polypeptides, while the term "peptide" is often used to refer to small polypeptides, but the use of these terms in the art overlaps. When referring to gene products and fragments thereof, the terms "protein" and "polypeptide" are used interchangeably herein. Therefore, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologues, orthologues, paralogues, fragments and other equivalents, variants, fragments and analogs of the aforementioned substances.

[0042] In the various embodiments described herein, variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservatively substituted variants of any particular polypeptide are further contemplated. With respect to amino acid sequences, one skilled in the art will recognize that a single substitution, deletion, or addition to a nucleic acid, peptide, polypeptide, or protein sequence that changes a single amino acid or a small portion of amino acids in the encoded sequence is a "conservatively modified variant," wherein the change results in the substitution of an amino acid with a chemically similar amino acid and retains 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.

[0043] 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 fragment or segment of a peptide that retains at least 50% of the activity of a wild-type reference polypeptide according to an assay described herein below. A functional fragment may comprise conservative substitutions of the sequences disclosed herein.

[0044] In some embodiments, the polypeptides described herein can be variants of sequences described herein. In some embodiments, the variants are conservatively modified variants. For example, conservative substitution variants can be obtained by mutations of natural nucleotide sequences. As referred to herein, "variants" are polypeptides that are substantially homologous to natural or reference polypeptides, but due to one or more deletions, insertions or substitutions, their amino acid sequences are different from the amino acid sequences of natural or reference polypeptides. The DNA sequence encoding the variant polypeptide encompasses sequences that include one or more nucleotide additions, deletions or substitutions compared to a natural or reference DNA sequence, but encodes variant proteins or fragments thereof that retain activity. A variety of PCR-based site-directed mutagenesis methods are known in the art and can be applied by ordinary technicians.

[0045] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, incorporating ribonucleic acid, deoxyribonucleic acid or its analog units. Nucleic acids can be single-stranded or double-stranded. A single-stranded nucleic acid can be a nucleic acid strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, for example, genomic DNA or cDNA. Suitable RNA can include, for example, mRNA.

[0046] In some embodiments of any aspect, the polypeptide, nucleic acid or cell described herein can be engineered. As used herein, "engineered" refers to aspects that have been artificially manipulated. For example, a polypeptide is considered "engineered" when at least one aspect of the polypeptide, such as its sequence, has been artificially manipulated to be different from the aspect when it exists in nature. As is commonly practiced and understood by those skilled in the art, the progeny of an engineered cell are still generally referred to as "engineered", even if the actual manipulation was performed on a previous entity.

[0047] In some embodiments, a nucleic acid encoding a polypeptide as described herein (e.g., an antibody or antibody reagent) is contained in a vector. In some aspects described herein, a nucleic acid sequence encoding a given polypeptide as described herein or any module thereof is operably linked to a vector. A vector may include, but is not limited to, a cloning vector, an expression vector, a plasmid, a phage, a transposon, a cosmid, a chromosome, a virus, a virion, etc.

[0048] As used herein, the term "expression vector" refers to a vector that guides RNA or polypeptides to be expressed from a sequence connected to a transcriptional regulatory sequence on a vector. The expressed sequence is often, but not necessarily, heterologous to the cell. The expression vector may contain additional elements, for example, the expression vector may have two replication systems, thereby allowing it to be maintained in two organisms, such as being maintained in human cells for expression and being maintained in a prokaryotic host for cloning and amplification. The term "expression" refers to a cell process involving the production of RNA and protein and secretion of protein when appropriate, including but not limited to, where applicable, such as transcription, transcription processing, translation and protein folding, modification and processing. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translating mRNA transcribed from a gene. The term "gene" means a nucleic acid sequence that is transcribed (DNA) into RNA in vitro or in vivo when operably linked to an appropriate regulatory sequence. The gene may or may not include regions before and after the coding region, such as a 5' untranslated region (5'UTR) or a "leader" sequence and a 3'UTR or a "trailing" sequence, as well as insertion sequences (introns) between each coding segment (exon).

[0049] In the case of nucleic acids or polypeptides, the term "isolated" or "partially purified" as used herein refers to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide), which is present together with the nucleic acid or polypeptide found in its natural source and / or when expressed by a cell, or when secreted in the case of a secretory polypeptide, it will be present together with the nucleic acid or polypeptide. Chemically synthesized nucleic acids or polypeptides or nucleic acids or polypeptides synthesized using in vitro transcription / translation are considered to be "isolated". The term "purified" or "substantially purified" refers to an isolated nucleic acid or polypeptide, which is at least 95% by weight of the subject nucleic acid or polypeptide, including, for example, at least 96%, at least 97%, at least 98%, at least 99% or more. In some embodiments, the antibodies described herein, their antigen binding portions, or chimeric antigen receptors (CARs) are isolated. In some embodiments, the antibodies, antibody reagents, their antigen binding portions, or CARs described herein are purified.

[0050] As used herein, "engineered" refers to aspects that have been manipulated artificially. For example, when the sequence of an antibody, an antibody reagent, an antigen-binding portion thereof, a CAR, or a bispecific antibody is artificially manipulated to be different from the sequence of the antibody when it exists in nature, the antibody, antibody reagent, antigen-binding portion thereof, CAR, or bispecific antibody is considered to be "engineered". As is commonly practiced and understood by those skilled in the art, the progeny of engineered polynucleotides and / or polypeptides are still generally referred to as "engineered", even if the actual manipulation is performed on a previous entity.

[0051] Pharmaceutical composition

[0052] The compositions and methods of the present invention can be used to treat individuals in need. In certain embodiments, the individual is a mammal, such as a human or non-human mammal. When applied to an animal such as a human, the composition or compound is preferably applied as a pharmaceutical composition, which includes, for example, a compound of the present application 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 ethylene glycol, glycerol, oil (such as olive oil) or injectable organic esters. In a preferred embodiment, when such pharmaceutical compositions are used for human administration, particularly for invasive administration routes (i.e., avoiding routes of transport or diffusion through epithelial barriers, such as injection or implantation), the aqueous solution is pyrogen-free, or substantially pyrogen-free. Excipients can be selected, for example, to achieve delayed release of a medicament or selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form, such as tablets, capsules (including dispersed capsules and gelatin capsules), granules, lyophiles for reconstruction, 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.

[0053] A pharmaceutically acceptable carrier may contain a physiologically acceptable agent that, for example, acts to stabilize a compound (such as a compound of the present invention), increase its solubility, or increase its absorption. 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 selection of a pharmaceutically acceptable carrier including 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 (preparation) may also be a liposome or other polymer matrix, into which, for example, a compound of the present invention may be incorporated. For example, liposomes comprising phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively easy to prepare and administer.

[0054] The phrase "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, and with a reasonable benefit / risk ratio commensurate.

[0055] As used herein, the phrase "pharmaceutically acceptable carrier" refers to 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 of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can be used 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, ethyl cellulose and cellulose acetate; (4) astragalus powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, 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 solution); (19) ethanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0056] The pharmaceutical composition (preparation) can be administered to a subject by any of a variety of routes of administration, including, for example, oral administration (e.g., drenches in the form of aqueous or non-aqueous solutions or suspensions, tablets, capsules (including dispersible capsules and gelatin capsules), pills, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); 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 compound can also be formulated for inhalation. In certain embodiments, the compound can simply be dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable therefor can be found in, for example, U.S. Patent 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.

[0057] The formulation can be conveniently present in unit dosage form and can be prepared by any method well known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated, 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 the compound that produces the therapeutic effect. Typically, one hundred percent, the range of this amount will be about 1% to about 99% of the active ingredient, preferably about 5% to about 70%, and most preferably about 10% to about 30%.

[0058] The methods for preparing these formulations or compositions include the step of bringing the active compound (such as a compound of the present invention) into association with a carrier 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 a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0059] Formulations of the present invention suitable for oral administration may be in the form of capsules (including dispersible capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), lyophiles, powders, granules, or as a solution or suspension in an aqueous liquid or a non-aqueous liquid, or as an oil-in-water or water-in-oil emulsion, or 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, etc., each containing a predetermined amount of the compound of the present invention as the active ingredient. The composition or compound may also be administered in the form of a bolus, electuary or paste.

[0060] To prepare solid dosage forms for oral administration (capsules (including dispersible capsules and gelatin capsules), tablets, pills, dragees, 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, for example, carboxymethyl cellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and / or gum arabic; (3) wetting agents such as Such as glycerol; (4) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retardants, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (8) absorbents, such as kaolin and bentonite; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, 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 dispersible capsules and gelatin capsules), tablets and pills, the pharmaceutical composition may also contain a buffer. Similar types of solid compositions can also be used as fillers in soft-filled and hard-filled gelatin capsules, using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols and the like.

[0061] Tablets can be made by compression or molding, optionally with one or more auxiliary ingredients. Compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), a surfactant or a dispersant. Molded tablets can be prepared by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine.

[0062] Tablets and other solid dosage forms of pharmaceutical compositions, such as dragees, capsules (including dispersible capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the art of pharmaceutical formulation. They may also be formulated to provide sustained or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose, other polymer matrices, liposomes and / or microspheres in different proportions to provide the desired release characteristics. They may be sterilized, for example, by filtering through a bacterial-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifiers and may be such compositions that they release the active ingredient only or preferentially in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, if appropriate, containing one or more of the above-mentioned excipients.

[0063] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art (such as water or other solvents), cyclodextrins and their derivatives, solubilizers and emulsifiers, such as ethanol, isopropanol, 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, tetrahydrofuran methanol, polyethylene glycol and sorbitan fatty acid esters, and mixtures thereof.

[0064] 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.

[0065] Suspensions, in addition to the active compounds, 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.

[0066] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and with any preservatives, buffers, or propellants that may be required.

[0067] Ointments, pastes, creams and gels may contain, in addition to the active compounds, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0068] In addition to the active compound, powders and sprays may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays may additionally contain conventional propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

[0069] Transdermal patches have the added advantage of providing controlled delivery of the compound of the present invention to the body. Such dosage forms can be prepared by dissolving the active compound in an appropriate medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. The rate of this flux can be controlled by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0070] The phrases "parenteral administration" and "administered parenterally" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intraocular (such as intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration may comprise one or more active compounds in combination with one or more pharmaceutically acceptable agents: sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions just before use, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0071] The example of suitable aqueous and non-aqueous carrier that can be used in the pharmaceutical composition of the present invention includes water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters, such as ethyl oleate. The example of suitable aqueous and non-aqueous carrier that can be used in the pharmaceutical composition of the present invention includes water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), 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 using a coating material such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using a surfactant. Suitable fluidity can be maintained, for example, by using a coating material such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using a surfactant.

[0072] These compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. It is possible to ensure that the effects of microorganisms are prevented by including various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, sorbic acid phenol, etc.). It is also desirable to include isotonic agents, such as sugar, sodium chloride, etc. in the compositions. In addition, the absorption extension of injectable drug forms can be achieved by including agents that delay absorption (such as aluminum monostearate and gelatin).

[0073] In some cases, in order to prolong the effect of the drug, it is desirable to slow down the absorption of the subcutaneous or intramuscular injection of the drug. This can be achieved by using a liquid suspension of a crystalline or amorphous substance with low water solubility. The absorption rate of the drug then depends on its dissolution rate, which in turn can depend on crystal size and crystalline form. Alternatively, the absorption of a parenterally administered drug form can be delayed by dissolving or suspending the drug in an oily vehicle.

[0074] Injectable depot forms are prepared by forming a microcapsule matrix of the subject compound in a biodegradable polymer (such as polylactide-polyglycolide). According to the ratio of the drug to the polymer and the properties of the specific polymer adopted, the drug release rate can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot-type injectable formulations are also prepared by embedding the drug in a liposome or microemulsion compatible with human tissue.

[0075] For use in the methods of the invention, the active compound may be administered per se or as a pharmaceutical composition containing, for example, 0.1% to 99.5% (more preferably 0.5% to 90%) active ingredient in combination with a pharmaceutically acceptable carrier.

[0076] The method of introduction can also be provided by a rechargeable or biodegradable device. In recent years, various slow-release polymer devices have been developed and tested in vivo to control the delivery of drugs, including protein biopharmaceuticals. A variety of biocompatible polymers, including hydrogels, including biodegradable and non-degradable polymers, can be used to form implants to continuously release compounds at specific target sites.

[0077] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0078] The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the esters, salts or amides thereof, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0079] A physician or veterinarian with ordinary skills in the art can easily determine and prescribe the therapeutically effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian can start the dosage of the pharmaceutical composition or compound at a dosage lower than the level required to obtain the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. "Therapeutically effective amount" means the concentration of the compound sufficient to cause the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the subject's weight, sex, age and medical history. Other factors affecting the effective amount may include, but are not limited to, the severity of the patient's condition, the condition being treated, the stability of the compound, and, when necessary, 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

[0080] In general, a suitable daily dose of the active compound used in the compositions and methods of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above.

[0081] If desired, the effective daily dose of the active compound can be administered at appropriate intervals throughout the day as 1, 2, 3, 4, 5, 6 or more sub-doses administered separately, optionally in unit dosage form. In certain embodiments of the invention, the active compound can be administered twice or three times a day. In other embodiments, the active compound will be administered once a day.

[0082] The patient receiving such treatment is any animal in need thereof, including primates, particularly humans; and other mammals, such as horses, cattle, pigs, sheep, cats, and dogs; poultry; and pets in general.

[0083] In certain embodiments, the compounds of the invention may be used alone or in combination with another type of therapeutic agent.

[0084] The disclosure includes the pharmaceutically acceptable salt of the compound of the present invention for use in the compositions and methods of the present application. In certain embodiments, the salt of the present invention considered includes but is not limited to alkyl, dialkyl, trialkyl or tetraalkyl ammonium salts. In certain embodiments, the salt of the present invention considered includes but is not limited to, L-arginine, benthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino) ethanol, ethanolamine, ethylenediamine, N-methylglucosamine, 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 salt of the present invention considered includes but is not limited to Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, contemplated salts of the 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, (+)-camphoric acid, (+)-camphor-10sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptanoic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, 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, propionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenate.

[0085] Pharmaceutically acceptable acid addition salts may also exist in the form of various solvates, such as solvates with water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates may also be prepared. The source of such solvates may be derived from the crystallization solvent, inherent in the preparation or crystallization solvent, or external to such solvent.

[0086] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0087] Examples of pharmaceutically 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, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0088] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meaning commonly understood by one of ordinary skill in the art to which the present disclosure belongs. It should be understood that the present invention is not limited to the specific methodology, protocols, reagents, etc. described herein and may vary accordingly. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is limited only by the claims. Definitions of commonly used terms in immunology and molecular biology can be found in the following references: The Merck Manual of Diagnosis and Therapy; 9 TheEncyclopedia 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 LaboratoryManual.; 15 Basic Methods in Molecular Biology; 16Laboratory Methods in Enzymology; 17 Current Protocols in Molecular Biology(CPMB); 18 Current Protocols in Protein Science (CPPS); 19 and Current Protocols in Immunology(CPI) 20 .

[0090] In some embodiments of any aspect, the disclosure described herein does not relate to methods for cloning humans, methods for altering the germline genetic characteristics of humans, the use of human embryos for industrial or commercial purposes, or methods for altering the genetic characteristics of animals, and animals produced by such methods, which methods may cause suffering to humans or animals without any substantial medical benefit to humans or animals.

[0091] Additional terms are defined herein in the description of various aspects of the invention.

[0092] Inflammatory bowel disease (IBD)

[0093] 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 (e.g., IBD) are common and major contributors to health burden worldwide.

[0094] IBD relevant to ENS damage is characterized by chronic severe inflammation of the small intestine and / or colon, causing recurrent diarrhea and abdominal pain. Crohn's disease (CD) and ulcerative colitis (UC) are two main clinical pathological subtypes of IBD. Although both are chronic and recurrent inflammatory diseases of the intestine, they can be distinguished by the location of inflammation in the gastrointestinal tract and the nature of histological changes in the intestinal wall. Anatomically, CD can affect the entire gastrointestinal tract from the oral cavity to the anus, but it usually affects the terminal ileum and colon. UC is limited to the rectum, colon and cecum. Under the microscope, CD is transmural and often discontinuous, and UC only affects the intestinal mucosa with a continuous pattern.

[0095] IBD is a very disabling disease due to the fatigue associated with inflammatory symptoms and the chronic pain suffered by patients. The pathogenesis of IBD is only partially understood; various environmental and host (e.g., genetic, epithelial, immune and non-immune) factors are involved. The complex interaction between the immune system, intestinal commensal bacteria / pathogens and host genotype is considered to be the basis for the development of IBD. These recurrent chronic inflammatory conditions appear to be caused by excessively aggressive T cell responses to environmental factors and / or commensal bacteria / pathogen subpopulations that inhabit the terminal ileum and colon of genetically susceptible hosts.

[0096] Currently, there is no cure or effective treatment for patients diagnosed with functional gastrointestinal disorders such as IBD. The main goal of current IBD therapy is to induce clinical remission by focusing on symptoms and then maintain clinical remission over the long term in order to achieve the best achievable quality of life. Due to the limited efficacy of current therapies, new therapies for treating inflammatory and chronic pain in the intestine of subjects with IBD are clinically meaningful.

[0097] Binding immunoglobulin (B I P) and related peptides

[0098] Intracellular BiP is known to be an anti-apoptotic protein with a binding preference for hydrophobic residues exposed by 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 via a KDEL amino acid sequence at the 3' end of the protein that binds to ERD2.

[0099] Natural 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. Present in all nucleated cells, BiP is released from sequestration and upregulated upon activation of the unfolded protein response (UPR). This is triggered by the intracellular accumulation of denatured proteins and nascent polypeptides, which is particularly caused by cellular stress, such as reduced oxygen and / or glucose and increased reactive oxygen species. These conditions are pathological features of chronic inflammation, such as synovial inflammation in patients with rheumatoid arthritis (RA).

[0100] Like other SPs or HSPs with important intracellular functions, these chaperones (including BiP) are expressed on the cell surface and secreted when upregulated, allowing unexpected and apparently distinct extracellular functions. This raises the idea that these highly conserved proteins may provide a link between the innate and adaptive immune systems.

[0101] IRL201805 (or 1805) is a modified analog of GRP78 being developed for the treatment of autoimmune diseases. During extensive studies, rhuBiP (IRL201805 lacking the C-terminal KDEL sequence) has been shown to treat and inhibit disease progression in animal models of arthritis. In a collagen-induced arthritis (CIA) model, adoptive cell transfer studies using spleen and lymph node cells from mice treated with rhuBiP / IRL201805 showed that rhuBiP / IRL201805 has therapeutic properties that persist long-term in the absence of rhuBiP / IRL201805 and are associated with changes in immune cell populations.

[0102] Thus, rhuBiP / IRL201805 alters the cellular immune response toward an anti-inflammatory TH2 profile and immune homeostasis. Further studies tracking patient biomarkers have demonstrated increased stabilization and efficacy of regulatory T cells, and a trend toward shifting dendritic cell differentiation toward indoleamine 3,2 oxygenase (IDO)+, a more anti-inflammatory phenotype.

[0103] These data indicate that IRL201805 has potent activity in reducing inflammatory responses associated with rheumatoid arthritis, however, its mode of action suggests potential efficacy in a range of immune-based conditions.

[0104] CD69 is a transmembrane glycoprotein with a C-type lectin domain (CTLD) 21,22,23 , which has recently been identified as a key regulator of intestinal inflammation and a novel target molecule for IBD treatment. 24 CD69 is not expressed at detectable levels on naive leukocytes, but its surface expression is rapidly induced following activation. 25,26 In human disease, CD69 expression is increased on leukocytes at sites of inflammation. 27,28,29 Furthermore, early in vitro studies described CD69 as a pro-inflammatory molecule whose engagement induces intracellular Ca 2+ influx, lymphocyte proliferation and production of pro-inflammatory mediators such as IL-2, tumor necrosis factor- (TNF-) and nitric oxide (NO). 30,31,32,33 CD69 is also required for cell contact-dependent stimulation of macrophages by T cells. 34 However, recent in vivo studies in transgenic mice have shown that CD69 can limit immune responses and suggest a regulatory function for CD69. CD69 has been shown to play a role in leukocyte migration, regulatory T cells, and the function of resident tissue memory T cells. 35 Therefore, it is suggested that stable induction of CD69 expression should lead to decreased migration of lymphocytes to the intestinal LP and decreased CD69 + Generation of Treg cells. 36

[0105] As described below, the present 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 methods for preventing or treating inflammatory bowel disease (IBD).

[0106] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present 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 will be appreciated by those skilled in the relevant art. For example, although the method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially simultaneously. The teachings of the disclosure provided herein may be applied to other procedures or methods as appropriate. The various embodiments described herein may be combined to provide further embodiments. If necessary, aspects of the present disclosure may be modified to provide further embodiments of the present disclosure using the compositions, functions and concepts of the above-mentioned references and applications. Moreover, due to considerations of the equivalence of biological functions, some changes may be made in the protein structure without affecting the biological or chemical effects in kind and amount. According to the detailed description, these and other changes may be made to the present disclosure. It is intended that all such modifications will be included within the scope of the appended claims.

[0107] The specific elements of any of the foregoing embodiments may be combined or substituted for the elements in other embodiments. In addition, although the advantages associated with certain embodiments of the present disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily exhibit such advantages to fall within the scope of the present disclosure.

[0108] The techniques described herein are further illustrated by the following examples, which should in no way be construed as further limiting. 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.

[0109] Example

[0110] The invention, as generally described, will now be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to be limiting of the invention.

[0111] Example 1 Binding immunoglobulin (B I Evaluation of the efficacy of P) analogs in a mouse model of intestinal inflammation

[0112] There are currently more than 50 animal models of human IBD. Although no single model perfectly phenocopies human IBD, many models are useful for studying various aspects of the human disease, including disease onset and progression and wound healing responses.

[0113] In this study, intestinal inflammation was assessed by syngeneic splenic CD4 + CD45RB 高 Elicited by adoptive transfer of T cells into T- and B-cell-deficient recipient mice. 37 CD4 + CD45RB 高 T cell colony mainly contains the original T cell of initial immunity activation, and it can induce chronic inflammation of small intestine and colon.This animal model allows to modify key experimental variables, including innate and adaptive immune cell colony, to answer the biological related questions relevant to the disease pathogenesis.In addition, it provides the accurate initiation of disease onset and the experimental time course of full characterization, and this experimental time course of full characterization allows the clinical characteristics of mouse disease progression to be carried out dynamic study.The intestinal inflammation induced by this method has many characteristics with human IBD, including chronic transmural inflammation of large intestine and small intestine, the pathogenesis driven by cytokines such as TNF and IL-12 and systemic symptoms such as emaciation. 38 Therefore, it is an ideal model system to evaluate the efficacy of BiP analogs such as IRL201805 (SEQ ID NO: 1).

[0114] Materials and methods

[0115] Material

[0116] IRL201805 (SEQ ID NO: 1) was produced by CBC (Bristol, UK) according to GMP standards.

[0117] method

[0118] Experimental Overview

[0119] The male RAG2- / - Mice were randomly assigned to experimental groups and allowed to acclimate for one week. On day 0, spleens were removed from donor C57Bl / 6N male mice and single cell suspensions were generated according to CRL SOP. On day 0, male RAG KO mice were injected intraperitoneally with CD4 + CD45RB 高 T cells (CD45RB only in group 1 低 T cells).

[0120] Animals were monitored daily for clinical signs of colitis, including weight loss, loose stools, and / or diarrhea, from day 21 until the end of the experiment on day 49. On day 49, animals were sacrificed, colons were dissected, and colon weight and length were measured. Colons from half of the animals in each group were transferred to tissue fixative, then processed for paraffin embedding, and stored for histopathological analysis. Lamina propria from half of the animals in each group were processed for flow cytometry.

[0121] Administration schedule

[0122] Treatments were administered according to the schedule in Table 1 below. n=10 for all groups.

[0123] Table 1

[0124]

[0125] Abbreviations: NA: not applicable, IP: intraperitoneal, IV: intravenous, TBC: to be confirmed, CRL: Charles River protocol.

[0126] Morbidity and mortality

[0127] Morbidity and mortality were assessed daily. Animals were monitored for clinical signs, including abnormal posture (e.g., hunched back), abnormal coat condition (e.g., piloerection), color changes in hairless areas (including cyanosis or jaundice), presence of masses and / or swelling, abnormal respiration, abnormal movements, and decreased activity. Cause of death was recorded whenever confirmed. Survival was recorded.

[0128] weight

[0129] Animals were weighed at the beginning of the study (day 0) and regularly during the first three weeks. All animals were weighed daily from day 21 until the end of the study. Data were analyzed and graphed.

[0130] Clinical scoring

[0131] 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 following scoring system is provided in Table 2.

[0132] Table 2

[0133]

[0134] Flow cytometry

[0135] Single cell suspensions were prepared from colonic lamina propria cells isolated from half of the animals in each group using the manufacturer's protocol (Miltenyi Biotec). Cells were harvested, restimulated with PMA / ionomycin, and stained with the following panel containing vital dyes and surface and intracellular markers: CD44, CD45, CD3, CD4, IL-17, CD25, and FOXP3. Determine the counts and percentages of each population.

[0136] Gross Pathology

[0137] At termination, the colon was dissected out and photographed to allow length measurements to be made on digital images using Image J software. The colon was also weighed.

[0138] Histopathology

[0139] At the time of termination, the colon of half of the animals in each group was transferred to a tissue fixative and then paraffin-embedded in a "Swiss roll" format. These samples were sliced ​​and stained with hematoxylin and eosin (H&E), and up to two slices of each animal were scored by a qualified histopathologist according to a semiquantitative scoring system. As described in Table 3, the mucosal thickness, mucosal ulceration, lamina propria mononuclear cell infiltration, lamina propria granulocyte infiltration, and crypt abscess / expansion / deformation of the slices were scored. The overall severity score was calculated by adding the scores of each standard.

[0140] Table 3

[0141]

[0142] result

[0143] In this study, IRL201805 caused a significant reduction in clinical signs of colitis, including weight loss, loose stools and / or diarrhea, and severity scores based on sections of mucosal thickness, mucosal ulceration, lamina propria mononuclear cell infiltration, lamina propria granulocyte infiltration, and crypt abscess / dilation / deformation scores.

[0144] These results suggest that administering a binding immunoglobulin (BiP) or functional analog to a subject at risk for or suffering from IBD is an effective method for preventing or treating IBD. The BiP or functional analog useful in the methods of the invention include, but are not limited to, a polypeptide having an amino acid sequence selected from SEQ ID NO: 1-5.

[0145] Example 2 Binding immunoglobulin (B I Evaluation of the efficacy of P) analogs in a dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) mouse model

[0146] Perhaps the most widely used mouse model of colitis employs dextran sulfate sodium (DSS), a chemical colitis agent with anticoagulant properties, to induce 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 murine colitis, which closely resembles human UC, 39Caused by administration of 40-50 kDa DSS in drinking water. The mechanism by which DSS induces intestinal inflammation is unclear, but it may be the result of damage to the epithelial monolayer lining the large intestine, allowing pro-inflammatory intestinal contents (e.g., bacteria and their products) to spread into underlying tissues. The DSS colitis model is very popular in IBD research due to its rapidity, simplicity, reproducibility, and controllability. By varying the concentration and frequency of administration of DSS, acute, chronic, and recurrent intestinal inflammation models can be obtained.

[0147] In this study, intestinal inflammation is a DDS murine model of UC that will be used to evaluate the efficacy of BiP analogs such as IRL201805 (SEQ ID NO: 1).

[0148] Materials and methods

[0149] Material

[0150] IRL201805 (SEQ ID NO: 1) was produced by CBC (Bristol, UK) according to GMP standards.

[0151] method

[0152] Experimental Overview

[0153] Adult C57bl / 6 mice were randomly assigned to experimental groups and allowed to acclimate for one week. Starting from day 2, treatment was administered according to the following administration schedule. On day 0, drinking water was replaced with an aqueous solution of 5% dextran sulfate sodium (DSS) (excluding Group 1, which always maintained normal drinking water). Animals had free access to a 5% DSS solution. From day 0 to the end of the experiment on day 7, animals were monitored daily for clinical signs of colitis, including: weight loss, loose stools and / or diarrhea, and the presence of occult or visible blood in the stool.

[0154] Blood samples were collected on day -2, day 3 (life stage), and day 7 (terminal stage), processed into serum, and stored for analysis.

[0155] On the 7th day, the animals were killed, the colon was dissected out, and the colon length was measured. One colon sample (distal end) was taken from each animal and fixed for histopathological analysis. Before analyzing cytokines, another colon sample (proximal end) was taken at termination and cultured for 48 hours in the presence or absence of LPS. The remaining supernatant was stored for analysis.

[0156] Administration schedule

[0157] Treatments were administered according to the schedule in Table 1 below. Unless otherwise stated, n=10 for all groups.

[0158] Table 1

[0159]

[0160] Abbreviations: TBC: not applicable, NA: not applicable, IV: intravenous, PO: oral.

[0161] Morbidity and mortality

[0162] Morbidity and mortality were assessed daily. Animals were monitored for clinical signs, including abnormal posture (e.g., hunched back), abnormal coat condition (e.g., piloerection), color changes in hairless areas (including cyanosis or jaundice), presence of masses and / or swelling, abnormal respiration, abnormal movements, and decreased activity. Cause of death was recorded whenever confirmed. Survival was recorded.

[0163] weight

[0164] All animals were weighed daily from day 1 until the end of the study. Data were analyzed and graphed.

[0165] Clinical scoring

[0166] 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, and the presence of occult or visible blood in the stool. The following scoring system is provided in Table 2.

[0167] Table 2

[0168]

[0169] Gross Pathology

[0170] At termination, the colon was dissected out and photographed to allow length measurements to be made on digital images using Image J software. The colon was also weighed.

[0171] Measurement of cytokine levels in colon grafts

[0172] Colon samples collected on day 7 were cultured for 48 h in the presence or absence of LPS prior to analysis of cytokine content by Luminex. The following cytokines were analyzed by multiplex (8-plex) Luminex: sTNFRII, sIL-6R, IL-6, IL-10, IL-1b, TNF-a, The samples were analyzed in a single replicate using multiplex xMAP bead technology, which utilizes microspheres as a solid support for sandwich immunoassays and measures multiple cytokines (Luminex) in the same sample.

[0173] Histopathology

[0174] At the time of termination, the colon of half of the animals in each group was transferred to a tissue fixative and then paraffin-embedded in a "Swiss roll" format. These samples were sliced ​​and stained with hematoxylin and eosin (H&E), and up to two slices of each animal were scored by a qualified histopathologist according to a semiquantitative scoring system. As described in Table 3, the mucosal thickness, mucosal ulceration, lamina propria mononuclear cell infiltration, lamina propria granulocyte infiltration, and crypt abscess / expansion / deformation of the slices were scored. The overall severity score was calculated by adding the scores of each standard.

[0175] Table 3

[0176]

[0177] result

[0178] In this study, IRL201805 caused a significant reduction in clinical signs of colitis, including weight loss, loose stools and / or diarrhea, and severity scores based on sections of mucosal thickness, mucosal ulceration, lamina propria mononuclear cell infiltration, lamina propria granulocyte infiltration, and crypt abscess / dilation / deformation scores.

[0179] These results indicate that administering a binding immunoglobulin (BiP) or a functional analog to a subject at risk for or suffering from IBD is an effective method for preventing or treating IBD. The BiP or functional analog that can be used in the method of the present invention includes, but is not limited to, a polypeptide having an amino acid sequence selected from SEQ ID NO: 1-5,

[0180] Example 3 Binding immunoglobulin (B I P) Evaluation of the effect of analogs on CD69 in colon punch biopsies obtained from IBD patients

[0181] In this study, punch biopsies (2 mm) of inflamed and noninflamed colonic tissue were performed. 2 ) were obtained from each consenting IBD patient with hospital ethics approval, e.g. Figure 6 A- Figure 6 E. Tissue biopsies were then incubated with or without IRL201805 (20 μg / mL) for 12-14 hours and assessed for CD69+ / CD39+ regulatory T cells (Tregs).

[0182] As mentioned above, CD69 is known to have multiple biological functions. In regulatory T cells (Tregs), CD69 is believed to promote CD39 surface expression, FoxP3 transcription, and STAT5 phosphorylation, leading to STAT3 inhibition. CD39 is an integral membrane protein that binds to Ca 2+ and Mg 2+It phosphohydrolyzes ATP and less efficiently ADP in a dependent manner to produce AMP. The resulting adenosine is a potent activator of tolerogenic dendritic cells (DCs). CD39 becomes catalytically active after localization on the cell surface.

[0183] like Figure 7 As shown, exposure of primary immune cells within inflamed colon tissue to IRL201805 induced a 20% increase in double-positive CD69+ / CD39+ Tregs compared to the same treatment of non-inflamed tissue from the same patient. In this study, IRL201805 was also confirmed by flow cytometry to produce increased p-STAT5 in immune cell subsets (data not shown). In previously unpublished data, BiP was also shown to inhibit STAT3 phosphorylation in human lymphocytes. These data suggest that BiP-related peptides only enhance CD69 activity and the resulting cascade in inflamed tissues of IBD patients, but not in non-inflamed tissues.

[0184] Example 4 Binding immunoglobulin (B I P) Evaluation of the effect of analogs on T cell activation in PBMC

[0185] CD69 and CD154 (CD40L) surface markers

[0186] T cell activation can be monitored by detecting increased expression of several surface markers, such as CD69 and CD154 (CD40L). Expression of CD69 is usually transient on most T cells, especially T effector cells, and decreases within 24 hours.

[0187] In this study, PBMCs were isolated from two healthy donors. PBMCs were then incubated with IRL201805 (20 μg / mL) at 37°C for 20-72 hours. Flow cytometry was used to measure mean fluorescence intensity and evaluate the surface expression of CD69 and CD39 on Tregs from PBMCs.

[0188] like Figure 8 As shown, IRL201805 treatment induced sustained expression of CD69 on CD154- cells and was maintained for at least 72 hours. These results are indicative of T cell receptor (TCR) activated Treg cells.

[0189] JAK-STAT signaling pathway

[0190] Janus kinase (JAK) / signal transduction and activator of transcription (STAT) (JAK / STAT) pathways coordinate and control many cytokines and differentiation pathways, which control and regulate immune responses. Signal transduction via the JAK-STAT signal transduction pathway involves the rapid transmission of signals from the cell membrane to the nucleus, followed by a highly organized response and the controlled downregulation and attenuation of the initial signal thereafter. Suppression of cytokine signaling (SOCS) proteins are the main drivers of signal attenuation. They are induced by cytokine exposure (via STAT) and then act as negative feedback inhibitors to shut down the signal transduction cascade. SOCS3 is an effective regulator of cytokine signaling in monocytes / macrophages and T cells.

[0191] In this study, the effect of IRL201805 on the JAK-STAT signaling pathway was evaluated at the transcriptional and protein levels. PBMCs were isolated from healthy subjects and treated with 20 μg / mL IRL201805 for 24 h.

[0192] RNAseq analysis was used to assess gene transcript levels of key signaling genes involved in CD69 activation. Fig.9A As shown, IRL201805 treatment significantly increased gene transcript levels for JAK3 and SOCS3, with an approximately 10-fold increase measured 24 hours after treatment. IRL201805 did not affect STAT5A or STAT5B transcript levels in monocytes from three healthy individuals.

[0193] PBMC cell lysates were immunoblotted with recombinant anti-STAT5 (phospho Y694) antibody [E208] (ab32364) (Abcam, ab32364) to assess p-STAT5 levels in untreated and IRL1805-treated PBMCs. Fig. 9B As shown, PBMCs isolated from healthy subjects and treated with 20 μg / mL IRL201805 for 24 hours showed increased levels of p-STAT5 compared to untreated PBMCs.

[0194] FOXP3 transcription and STAT5 phosphorylation

[0195] In Tregs, CD69 is believed to promote CD39 surface expression, FoxP3 transcription, and STAT5 phosphorylation, leading to STAT3 inhibition. This study evaluated the effect of IRL1805 on p-STAT5. IRL201805 produced increased p-STAT5 in immune cell subsets. Treatment of PBMCs with IRL201805 resulted in changes in the p-STAT5 status of CD4 / CD8 T cells and a 3-fold increase in FoxP3 transcription levels (see Table 4).

[0196] Table 4

[0197] deal with <![CDATA[%CD4 +ve ]]> <![CDATA[CD25 高 / CD127 低 Treg]]> %FoxP3+Treg <![CDATA[%CD8 +ve ]]> Unprocessed 10 1.8 0.3 0.3 PHA-treated 4.4 4.3 0.6 4.2 1805 Processed 7.1 3.2 0.9 3.2

[0198] CD69-related ligands

[0199] Fig.10 A provides a schematic diagram of CD69-related ligands. CD69 surface expression increases upon activation of APC-derived MHC class II TCR. Engagement of the TCR induces modest activation of the metabolic sensor mTOR, which is augmented by CD28 and IL-2 signaling, which can promote T cell commitment to the generation of TH1, TH2, and TH17 effector cells. Membrane SiP1 / SiPR1 interactions 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 and activate JAK3 / STAT5 phosphorylation. Phosphorylated STAT5 can translocate to the nucleus and promote the differentiation of FoxP3 Treg, inhibit STAT3 activation and prevent Th17 cell differentiation.

[0200] Gal-1 and S100A8 / A9 are known potential ligands that bind to CD69. 43 To examine the expression of the Treg phenotype and influence the ability of T cells to maintain the Treg phenotype, this study investigated the effect 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 the levels of Gal-1 and S100A8 / A9 proteins.

[0201] The abundance of Gal-1 and S100A8 / A9 proteins on the cell surface was detected by tandem mass tag (TMT) quantitative proteomics. Fig.10 As shown in B, the expression of Gal-1 and S100A8 / A9 on the cell surface was not changed by 1805.

[0202] However, 1805 treatment increased the phosphorylation of S100A9 by 58% ( Fig.10 C). Mass spectrometry analysis identified an increase in trimethyl post-translational modification of threonine 113 on S100A9 after 1805 treatment, a modification known to promote active secretion of S100A9 from myeloid cells.

[0203] Surface CD154 and CD69 activation markers on T lymphocytes from PBMCs

[0204] CD69 is commonly detected by flow cytometry as the most common activated cell marker on lymphocytes, and its appearance on the cell surface serves as a biomarker for MHC-TCR interactions between APCs and T cells. Both CD4 and CD8 T cells express CD69 under steady-state conditions, indicating that many of these cells are constantly exposed to antigenic attack.

[0205] 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 at 37°C for 20 hours in the presence or absence of 20 μg / mL IRL201805.

[0206] like Fig.11 As shown, the basal level of cell surface CD69 expression was highest on CD8 T cells (42%, Fig.11 C), followed by Treg (24%, Fig.11 A) and Teff (15%, Fig.11 B). These results demonstrate that various T cell subsets display CD69 on their cell surface. However, 1805 only produced a statistically significant 2-fold increase on the surface of Treg cells (p=0.008). These results indicate that Tregs exposed to 1805 are selectively activated.

[0207] IRL201805-Possible Mode of Action

[0208] Not bound by a specific mode of action, Fig.12 A schematic diagram of the possible mode of action of BiP-related peptides such as IRL201805 on CD69-related ligands is provided. Within 2-20 hours after APC presents the 1805 peptide to the TCR, the CD69 receptor is expressed on the membrane of activated regulatory T cells. The CD69 receptor binds to ligands (e.g., S100A8 / A9 or Gal-1) on the surface of APC. The cytoplasmic tail of CD69 associates with Jak3 and Stat5 proteins, triggering the phosphorylation of Stat5 and its translocation to the nucleus, where it can activate the transcription factor FoxP3, stimulating the differentiation of regulatory T cells. 44 Engagement of CD69 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 the differentiation of regulatory T cells and tolerogenic APCs. CD69 can inhibit the Th17 differentiation pathway through at least two mechanisms: CD69-activated Stat5 directly inhibits the translocation of Stat3 to the nucleus, and indirectly antagonizes Stat3-mediated RORt activation via FoxP3 activation.

[0209] in conclusion

[0210] CD69 has recently been identified as a key regulator of intestinal inflammation and a novel target molecule for IBD treatment. 45 Recent in vivo studies in transgenic mice have shown that CD69 can limit immune responses and suggest a regulatory function for CD69. CD69 has been shown to play a role in leukocyte migration, regulatory T cells, and the function of resident tissue memory T cells. Therefore, it is suggested that stable induction of CD69 expression should lead to reduced migration of lymphocytes to the intestinal LP and CD69 + Generation of Treg cells.

[0211] The present disclosure provides evidence that BiP-related polypeptides produce stable induction of CD69 expression only in inflamed colon tissue and induce activation of several CD69 activation markers and CD69-related ligands, thereby providing methods for modulating inflammatory responses associated with intestinal inflammation and methods for preventing or treating inflammatory bowel disease (IBD).

[0212] References:

[0213] 1GBD 2017Inflammatory Bowel Disease Collaborators(2020). "The global, regional, and national burden of inflammatory bowel disease in 195 countries and territories, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017." Lancet Gastroenterol Hepatol. 5:17-30.

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[0215] 3 Previously described as SEQ ID NO:3 in PCT application,WO 2019 / 239126.

[0216] 4 Previously described as SEQ ID NO:4 in PCT application,WO 2019 / 239126.

[0217] 5 Previously described as SEQ ID NO:1 in PCT application,WO2000 / 21995.

[0218] 6 Previously described as SEQ ID NO:2 in PCT application,WO2000 / 21995.

[0219] 7 Previously described as SEQ ID NO:5 in PCT application,WO 2019 / 239126.

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[0223] 11 MOLECULAR BIOLOGY AND BIOTECHNOLOGY:A COMPREHENSIVE DESKREFERENCE,(1995).Robert A.Meyers(ed.),published by VCH Publishers,Inc.(ISBN1-56081-569-8).

[0224] 12 IMMUNOLOGY,(2006).Werner Luttmann,published by Elsevier.

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[0227] 15 Michael Richard Green and Joseph Sambrook,(2012).MOLECULARCLONING:A LABORATORY MANUAL,4th ed.,Cold Spring Harbor Laboratory Press,ColdSpring Harbor,N.Y.,USA(ISBN 1936113414).

[0228] 16 Davis et al.,(2012).BASIC METHODS IN MOLECULAR BIOLOGY,ElsevierScience Publishing,Inc.,New York,USA(ISBN 044460149X).

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[0231] 19 CURRENT PROTOCOLS IN PROTEIN SCIENCE(CPPS),(2005).John E.Coligan(ed.),John Wiley and Sons,Inc.

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[0233] 21 Llera,A.S.,et al.(2001).“Crystal structure of the C-type lectin-like domain from the human hematopoietic cell receptor CD69”J.Biol.Chem.276(10):7312-7319.

[0234] 22 Ziegler,S.F.,et al.(1994).“The mouse CD69 gene:structure,expression,and mapping to the NK gene complex.”J.Immunology 152(3):1228-1236.

[0235] 23 Hamann,J.,et al.(1993).“Expression cloning of the early activationantigen CD69,a type II integral membrane protein with a C-type lectindomain.”J.Immunology 150(11):4920-4927.

[0236] 24 Radulovic,K.and Niess,J.H.(2015).“CD69 Is the Crucial Regulator ofIntestinal Inflammation:A New Target Molecule for IBD Treatment?”J.ofImmunology Res.Vol.2015,Article ID 497056.

[0237] 25 Testi,R.et al.(1994).“The CD69 receptor:a multipurpose cell-surface trigger for hematopoietic cells.”Immunology Today,15(10):479-483.

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[0240] 28 Li,L.,et al.(2011).“Identification of M.tuberculosis-specificth1cells expressing CD69 generated in vivo in pleural fluid cells frompatients with tuberculous pleurisy.”PLoS ONE,6(8):Article ID e23700.

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[0246] 34 Mcinnes,I.B.,et al.(1997).“Interleukin-15 mediates T cell-dependent regulation of tumor necrosis factor-production in rheumatoidarthritis.”Nature Medicine,3(2):189-195.

[0247] 35 Radulovic,K.and Niess,J.H.(2015).“CD69 Is the Crucial Regulator ofIntestinal Inflammation:A New Target Molecule for IBD Treatment?”J.ofImmunology Res.Vol.2015,Article ID 497056.

[0248] 36 Id.

[0249] 37 Powrie,F.,et al.(1993).“Phenotypically distinct subsets of CD4+Tcells induce or protect from chronic intestinal inflammation in C.B-17scidmice.”Int Immunol.,5(11):1461-1471.

[0250] 38 Ostanin D.V.,et al.(2009).“T cell transfer model of chroniccolitis:concepts,considerations,and tricks of the trade.”Am.J.Physiol.Gastrointest.Liver Physiol.,296(2):135-146.

[0251] 39 Okayasu I.,et al.(1990).“A novel method in the induction ofreliable experimental acute and chronic ulcerative colitis in mice.”Gastroenterology,98:694-702.

[0252] 40 Liu,G.,et al.(2010).“The S1P(1)-mTOR axis directs the reciprocaldifferentiation of T(H)1 and T(reg)cells.”Nat.Immunol.11(11):1047-1056.

[0253] 41 de la Fuente,H.,et al.(2014).“The leukocyte activation receptorCD69 controls T cell differentiation through its interaction with galectin-1.”Mol.Cell Biol.34:2479-2487.

[0254] 42 Lin,C.-R.,et al.(2015).“Glycosylation-dependent interactionbetween CD69 and S100A8 / S100A9 complex is required for regulatory T-celldifferentiation.”FASEB J.,29(12):5006-5017.

[0255] 43 Gorabi,A.M.,et al.(2020).“The pivotal role of CD69 inautoimmunity.”J.Autoimmunity,111:102453.

[0256] 44 Cibrian,et al.(2017).European Journal of Immunology,47:923-1086.

[0257] 45 Radulovic,K.and Niess,J.H.(2015).“CD69 Is the Crucial Regulator ofIntestinal Inflammation:A New Target Molecule for IBD Treatment?”J.ofImmunology Res.Vol.2015,Article ID 497056.

[0258] All patents and other publications cited throughout this application, including references, issued patents, published patent applications, and co-pending patent applications, are expressly incorporated herein by reference for the purpose of describing and disclosing methods, such as those described in such publications, which can be used in conjunction with the techniques 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 virtue of prior invention or for any other reason. All statements regarding 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 correctness of the dates or contents of these documents.

[0259] The foregoing written description is considered sufficient to enable those skilled in the art to practice aspects and embodiments of the present invention. The scope of aspects and embodiments of the present invention is not limited by the examples provided, as these examples are intended to be a single illustration of one aspect, and other functionally equivalent embodiments are also within the scope of the present disclosure. Various modifications other than those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and purposes described herein are not necessarily covered by each embodiment. Those skilled in the art will recognize that, or simply using routine experiments, many equivalents of the specific embodiments described herein can be determined. Such equivalents are intended to be covered by the following claims.

Claims

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

2. The method of claim 1, wherein the 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 . The method according to claim 2 , wherein the BiP or a functional analogue thereof has the following amino acid sequence: SEQ ID NO:

1. 4 .

4. The method of 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 enteritis.

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

6. A therapeutically effective amount of a binding immunoglobulin (BiP) or a functional analog thereof for use as claimed in claim 5, wherein the 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. The therapeutically effective amount of a binding immunoglobulin (BiP) or a functional analogue thereof for use as claimed in claim 6, wherein the BiP or a functional analogue thereof has the following amino acid sequence: SEQ ID NO:

1.

8. A therapeutically effective amount of a binding immunoglobulin (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 enteritis.

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