Methods and compositions for preventing or treating eosinophilic esophagitis

Abstract

Described herein are methods of preventing or treating eosinophilic esophagitis (EoE) by administering a peptide associated with chaperonin 60.1 to a subject suffering from EoE.

Description

Technical Field

[0001] Embodiments of the present invention relate to compositions and methods for preventing or treating eosinophilic esophagitis (EoE) using peptides associated with chaperone protein 60.1 (Cpn60.1). Background Technology

[0002] Eosinophilic esophagitis (EoE) is a chronic, immune-mediated disease in which eosinophils accumulate in the esophagus, leading to inflammation and esophageal dysfunction. It is an inflammatory disease associated with food allergies affecting children and adults. 1,2,3 Over the past two decades, the prevalence of EoE has increased, nearly doubling in both adults and children, and is estimated to be one in 1,500 to 2,000 people. 4 Common symptoms include difficulty swallowing (difficulty swallowing liquids or solids or both, >90%), food impaction (solid food stuck in the esophagus, 50%), dysphagia (pain when swallowing), heartburn (33%), chest pain, asthma (50%), diarrhea, and vomiting. 5,6,7,8 Current treatment options include chronic dietary elimination (including food allergen withdrawal), swallowing topical corticosteroids (not approved for EoE in the US), and esophageal dilation. Currently, only one drug therapy is approved for EoE: DUPIXENT. ® (Dupilumab).

[0003] All current treatments have significant drawbacks. Adhering to an elemental diet that avoids exposure to foods that trigger EoE (esophageal reflux disease) leads to symptom relief in many patients. However, this approach requires disruptive lifestyle and dietary changes, thus placing a heavy burden on patients. While swallowing topical corticosteroids has been reported in clinical trials to induce partial clinical responses and histological remission, these responses are not consistently effective and may be associated with fungal infections and disease relapse after discontinuation. Furthermore, there are concerns about long-term steroid use, particularly in children. Esophageal strictures can be treated with proton pump inhibitors that suppress gastric acid secretion. Endoscopic esophageal dilatation is currently used to treat esophageal strictures and increase esophageal dilatation. However, it is an invasive surgical procedure and can lead to complications such as perforation and bleeding. ® It may cause serious side effects, including, sometimes, severe allergic reactions; eye problems, including eye pain or vision changes such as blurred vision; vascular inflammation, joint pain; eczema; asthma, chronic sinusitis with nasal polyps; and nodular prurigo .

[0004] Therefore, there is a need for effective compositions and methods for the prevention or treatment of eosinophilic esophagitis. Summary of the Invention

[0005] Embodiments of the present invention provide a method for treating or preventing EoE in a subject by administering a peptide associated with chaperone protein 60.1 (Cpn60.1) to the subject in need. In some embodiments, the Cpn60.1-related peptide is selected from: DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 1) ('1104); DGSVVVNKVSELPAGH (SEQ ID NO: 2); GLNVNTLSYGDLAAD (SEQ ID NO: 3); SELPAGHGLNVNLTS (SEQ ID NO: 4); DGSVVVNKVS (SEQ ID NO: 5); ELPAGHGLNV (SEQ ID NO: 6); NTLSYGDLAAD (SEQ ID NO: 7); or functionally equivalent fragments or variants thereof. In some embodiments, the Cpn60.1-related peptide is SEQ ID NO: 1.

[0006] In some embodiments of the invention, administration of the Cpn60.1-related peptide induces a significant reduction in the severity of dysphagia in subjects. In some embodiments, the severity of dysphagia is assessed using a daily dysphagia symptom questionnaire (DSQ) completed by the subjects. In one embodiment, administration of the Cpn60.1-related peptide results in a DSQ reduction of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more.

[0007] In some embodiments of the invention, administration of the Cpn60.1-related peptide induces a significant reduction in esophageal eosinophil counts in esophageal biopsy samples from subjects. In some embodiments, such as when measured in esophageal biopsy samples by high-power field, administration of the Cpn60.1-related peptide results in a 20% or greater reduction in esophageal eosinophil counts relative to baseline. In some embodiments, such as when measured in esophageal biopsy samples by high-power field, administration of the Cpn60.1-related peptide results in a 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% or greater reduction in esophageal eosinophil counts relative to baseline. In alternative embodiments, such as when measured in esophageal biopsy samples by flow cytometry, administration of the Cpn60.1-related peptide results in a 40% or greater reduction in esophageal eosinophil counts relative to baseline. In some implementations, such as those measured by flow cytometry in esophageal biopsy samples, administration of Cpn60.1-related peptide resulted in a 30%-35%, 35%-40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or more reduction in esophageal eosinophil count relative to baseline.

[0008] In some embodiments of the invention, administration of the Cpn60.1-related peptide induces a significant increase in blood regulatory B cells (Breg) in a subject. In alternative embodiments, as measured by flow cytometry, administration of the Cpn60.1-related peptide results in a 100% or greater increase in blood memory Breg cells relative to baseline. In some embodiments, as measured by flow cytometry, administration of the Cpn60.1-related peptide results in a 50%-100%, 100%-150%, 150%-200%, 200%-250%, 250%-260%, 260%-270%, 270%-280%, 280%-290%, 290%-300% or greater increase in blood memory Breg cells relative to baseline. In another alternative embodiment, as measured by flow cytometry, administration of the Cpn60.1-related peptide results in a 15% or greater increase in blood activated Breg cells relative to baseline. In some embodiments, such as those measured by flow cytometry, administration of the Cpn60.1-related peptide resulted in an increase of 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, or more in activated Breg cells in the blood relative to baseline. In other alternative embodiments, such as those measured by flow cytometry, an increase in memory Breg cells or activated Breg cells relative to baseline was detected in esophageal biopsy samples.

[0009] In some embodiments of the invention, administration of the Cpn60.1-related peptide induces a significant increase in regulatory T cells (Tregs) in the subject. In alternative embodiments, such as those measured by flow cytometry in esophageal biopsy samples, administration of the Cpn60.1-related peptide results in an increase of Treg cells of 25% or more relative to baseline. In some embodiments, such as those measured by flow cytometry in esophageal biopsy samples, administration of the Cpn60.1-related peptide results in an increase of Treg cells of 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 45%-50%, 50%-55%, 55%-60% or more relative to baseline.

[0010] In some embodiments of the invention, administration of the Cpn60.1-related peptide induces a significant reduction in one or more leukocytes in a subject. The one or more leukocytes may be mast cells, CD4+ T cells, and / or CD8+ T cells. In some embodiments, such as those measured by flow cytometry in esophageal biopsy samples, administration of the Cpn60.1-related peptide results in a reduction of one or more leukocytes relative to baseline of 10% or more. In some embodiments, such as those measured by flow cytometry in esophageal biopsy samples, administration of the Cpn60.1-related peptide results in a reduction of one or more leukocytes relative to baseline of 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, or more.

[0011] In some embodiments of the invention, administration of the Cpn60.1-related peptide is acute or short-term. In some embodiments, two, three, or more doses are administered over a short period of time, such as 1 day, 3 days, 7 days, 10 days, 14 days, 21 days, 28 days, 56 days, or 112 days. The time between doses administered to the subject can be 3 hours, 1 day, 3 days, 7 days, 14 days, 21 days, 28 days, or 56 days after the previous dose. In alternative embodiments, administration of the Cpn60.1-related peptide is long-term.

[0012] In another aspect, the present invention provides a therapeutically effective amount of a Cpn60.1-related peptide for use in preventing the onset of food allergy or food intolerance to food-derived dietary antigens in subjects in need, or for use in methods of treating food allergy or food intolerance to food-derived dietary antigens.

[0013] This article also describes and lists other implementation methods. Attached Figure Description

[0014] For illustrative purposes, certain embodiments of the invention are shown in the accompanying drawings described below. However, it should be understood that the invention is not limited to the precise arrangements, dimensions, and instruments shown. In the drawings: Figure 1 This diagram illustrates a clinical protocol used in studies involving patients with EoE (Empty Ejaculation). Abbreviation: Dysphagia Symptom Questionnaire (DSQ).

[0015] Figure 2 This is a line graph showing the improvement in patient-reported EoE dysphagia induced by '1104 using the Dysphagia Symptom Questionnaire (DSQ). Patients with EoE were given placebo, 4 mg '1104, or 8 mg '1104 on days 0, 7, and 14. The DSQ was assessed at weeks 2, 4, and 6. Data are the mean reduction in DSQ relative to pre-dose in the placebo group (▲; n=11) and the combined 4 mg and 8 mg groups (●; n=22). Statistical analysis using ANOVA showed the difference between the placebo group and the combined 4 mg + 8 mg '1104 group. * P <0.05;** P <0.005.

[0016] Figure 3 This is a bar graph showing the eosinophil counts measured by high-power field and flow cytometry in patients with EoE. For high-power field histological analysis, results are shown as numerical changes (left) and percentage changes (middle) relative to baseline for patients receiving placebo (n=12), 4 mg '1104 (n=11), or 8 mg '1104 (n=13). For flow cytometry analysis (right), tissue was collected from biopsies of EoE patients receiving placebo (n=7), 4 mg '1104 (n=8), or 8 mg '1104 (n=8), and eos counts are expressed as percentage changes relative to baseline. For the 4 mg and 8 mg treatment groups, unpaired numerical changes in cell counts are also shown. t test P <0.05. Abbreviation: Eosinophils (eos); High power field (hpf).

[0017] Figure 4This is a bar graph showing that '1104 reduced CD4+ and CD8+ cells and increased Treg cells in EoE biopsies, which is the expected effect in controlling inflammation. Tissue was collected from patients who received placebo (n=7), 4 mg '1104 (n=11), or 8 mg '1104 (n=12). Data are presented as percentage changes in CD4+ cells (left), CD8+ cells (middle), and Treg cells (right) relative to baseline. Treg cells are CD3+ CD4+ CD127+ cells. 低 CD25+. For the 8 mg treatment group, unpaired changes in cell count values. t test P <0.05. Abbreviation: Regulatory T cells (Treg).

[0018] Figure 5 This is a bar graph showing that '1104 increased blood Breg cells in EoE patients who received 4 mg '1104 (n=11) or 8 mg '1104 (n=12) compared to EoE patients who received placebo (n=7). The data are presented as percentage changes in memory Breg cells (left panel) and activated Breg cells (right panel) relative to baseline. Memory Bregs are IgD-CD27. 高 CD38 高 CD95+ (gated with CD3-CD19+). Activated Breg is IgD-CD27. 高 CD38 高 (Gated by CD3-CD19+). Unpaired t-test of numerical changes in cell counts in the 4 mg and 8 mg treatment groups in the memory Breg data. P <0.01. Unpaired numerical changes in cell counts in the 4 mg and 8 mg treatment groups in the activated Breg data. t test P <0.001. Abbreviation: Regulatory B cells (Breg).

[0019] Figure 6 A table comparing '1104 with other drugs tested in EoE patients. Abbreviation: Antigen-presenting dendritic cells (APDC).

[0020] Figure 7 A schematic diagram is provided illustrating the potential mechanism of action of '1104 in preventing inflammatory cascades.

[0021] Figure 8The results of the EoE diagnostic panel (EDP) are shown, highlighting key genes in '1104-positive reversible EoE patients. The selected genes represent the major EDP gene classes with 5 representative EoE genes (96 genome sets) and were tested in the placebo group (n=7) and the 4 mg and 8 mg treatment groups (n=7), comparing gene expression at screening and at 4 weeks. In columns 3 and 4, light gray shading indicates genes downregulated between screening and V4, and dark gray shading indicates genes upregulated between screening and V4. Differential expression analysis identified 15 genes (bold) that were significantly regulated in patients treated with '1104 compared to the placebo group. Abbreviations: Cathecin-26 (CDH26); Desmosome core protein-1 (DSG1); Seal 10 (CLDN10); Catenin α-like 1 (CTNNAL1); Cell adhesion molecule L1-like (CHL1); Filamentin (FLG); Urolytic protein 1A (UPK1A); Serine peptidase inhibitor Kazal type 7 (SPINK7); Cysteine-rich secretory protein 3 (CRISP3); TNF-α-induced protein 6 (TNFAIP6); Arachidonic acid 15-lipoxygenase (ALOX15); Arginase 1 (ARG1); Matrix metalloproteinase 12 (MMP12); Thymic stromal lymphopoietin (TSLP); Immunoglobulin J (IGJ); Periostrin (POSTN, or osteoblast-specific factor OSF-2); Keratin 23 (KRT23); Type VIII collagen α2 chain (COL8A2); Cathepsin C (CTSC); smooth muscle actin γ2 (ACTG2); anoctamin 1 (ANO1); apolipoprotein B mRNA editing enzyme catalytic subunit 3A (APOBEC3A); Charcot-Leyden crystal galactose lectin (CLC); CC motif chemokine receptor 3 (CCR3); trypsin β2α / β1 (TPSB2 / AB1); carboxypeptidase A3 (CPA3); chymotrypsin 1 (CMA1); CC motif chemokine ligand 26 (CCL26); CC motif chemokine ligand 24 (CCL24); CXC motif chemokine ligand 1 (CXCL1); interleukin 4 (IL-4); interleukin 5 (IL-5); interleukin 13 (IL-13); TNFα-induced protein 3 (TNFAIP3 or A20); programmed death ligand 1 (PD-L1); and CD40 ligand (CD40L).

[0022] Figure 8 Provide additional information on differentially expressed genes in EoE patients treated with '1104 compared to the placebo group. Detailed Implementation

[0023] This subject matter is now described with reference to the accompanying drawings, throughout which similar reference numerals are used to denote similar elements. In the following description, numerous specific details are set forth for purposes of explanation in order to provide a full understanding of the invention. However, it will be apparent that the invention can be practiced without these specific details. In other instances, well-known structures and devices are shown in block diagram form to aid in the description of the invention. It should be understood that certain aspects, modes, embodiments, variations, and features of the invention are described below at various levels of detail to provide a substantial understanding of the invention.

[0024] definition For convenience, the meanings of some terms and phrases used in the specification, embodiments, and appended claims are provided below. Unless otherwise stated or apparent from the context, the following terms and phrases include the meanings provided below. These definitions are provided to aid in describing particular embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. If there is a significant difference between the use of terms in the art and their definitions provided herein, the definitions provided in the specification shall prevail.

[0025] As used in this specification and the appended claims, unless the context clearly specifies otherwise, the singular forms “a / an” and “the” include plural references. For example, a reference to “a cell” includes a combination of two or more such cells, etc.

[0026] As used herein, the term “about” or “approximately” with respect to a value or parameter is generally considered to include numbers that fall within the range of 5%, 10%, 15%, or 20% of the number in either direction (greater than or less than), unless otherwise stated or obvious from the context (unless such a number would be less than 0% or more than 100% of a possible value). As used herein, references to “about” or “approximately” with respect to a value or parameter include (and describe) embodiments relating to that value or parameter. For example, a description of “about X” includes a description of “X”.

[0027] As used herein, the term “or” means “and / or”. The term “and / or” as used 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 "includes" means that other elements may exist in addition to the defined element presented. The use of "includes" indicates inclusion rather than limitation.

[0029] The term "composed of" refers to compositions, methods and their corresponding components as described herein, excluding any elements not described in the description of the embodiments.

[0030] As used herein, the term "consistently of" refers to those elements required for a given embodiment. This term allows for the presence of additional elements that do not materially affect the fundamental, novel, or functional features of that embodiment of the invention.

[0031] The terms “statistically significant” or “significantly” refer to statistical significance and typically mean 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, rodents, or primates. Subjects may be domestic pets (e.g., dogs, cats), agricultural livestock (e.g., cattle, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), but are not limited to these. Subjects include human subjects. Human subjects may be pediatric, adult, or elderly. Human subjects may be of either sex.

[0033] As used herein, the terms "effective amount" and "therapeutic effective amount" include amounts sufficient to prevent or improve the manifestations of a disease or medical condition, such as immune system disorders, including eosinophilic esophagitis (EOE). It should be understood that many methods are known in the art for determining the effective amount for a given application. For example, pharmacological methods for dose determination can be used in a therapeutic context. In the context of therapeutic or prophylactic application, the amount of composition administered to a subject will depend on the type and severity of the disease and individual characteristics such as general health, age, sex, weight, and tolerance to the drug. It will also depend on the extent, severity, and type of the disease. A skilled technician will be able to determine the appropriate dose based on these and other factors. The composition may also be administered in combination with one or more additional therapeutic compounds.

[0034] As used herein, the terms “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” mean 1) therapeutic measures that cure, alleviate, or reduce the symptoms of a diagnosed disease or infection and / or stop the progression of a diagnosed disease or infection, and 2) preventive or preventative measures that prevent or slow the development of a disease or infection.

[0035] As used herein, when referring to a disease, condition, or medical ailment, the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatment of a disease with the aim of reversing, alleviating, improving, suppressing, slowing, or stopping the progression or severity of symptoms or the disease. The term “treatment” includes reducing or alleviating at least one side effect or symptom of the disease. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of the disease is slowed or stopped. That is, “treatment” includes not only improvement of symptoms or markers but also the cessation or at least slowing of the expected progression or worsening of symptoms in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, the relief of one or more symptoms, a reduction in the degree of deficiency, a stable (i.e., non-worsening) state of immune disease, a delay or slowing of immune disease, and an extended lifespan compared to the lifespan expected without treatment. Symptoms of EoE vary from person to person and may include difficulty swallowing, chest pain or heartburn, abdominal pain, vomiting, food getting stuck in the throat due to a narrow throat, and growth retardation or poor weight gain in children.

[0036] As used herein, the term “long-term” administration means a period of time during which a therapeutic agent or drug is administered for at least 12 weeks. This includes administering a therapeutic agent or drug such that it is effective for at least 12 weeks or for a period of time lasting at least 12 weeks, and does not necessarily mean that the administration itself occurs for 12 weeks, for example, if a sustained-release composition or a long-acting therapeutic agent or drug is used. Therefore, the subject receives treatment for a period of 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.

[0037] Without being bound by theory, it should be understood that the Cpn60.1-related peptides of the present invention not only affect the symptoms of diseases or ailments, but also alter the underlying diseases or ailments themselves. Therefore, the application of the peptides of the present invention has long-term effects.

[0038] In some implementations, remission of the disease is maintained without the need for additional doses of Cpn60.1-related peptide. It should be noted that acute treatment may include administering one or more effective doses of Cpn60.1-related peptide molecules during an attack or relapse. In some cases, one or more doses of Cpn60.1-related peptide may be required during remission. However, continuous administration of an effective dose of Cpn60.1-related peptide is not necessary. Preferably, no additional doses are required during remission.

[0039] In some implementations, two, three, or more doses are administered over a short period of time, such as 1 day, 3 days, 7 days, 10 days, 14 days, 21 days, 28 days, 56 days, or 112 days. The time between doses administered to the subject can be 3 hours, 1 day, 3 days, 7 days, 14 days, 21 days, 28 days, or 56 days after the previous dose.

[0040] Remission typically includes the reduction, relief, or elimination of one or more symptoms of EoE. Generally, remission or clinical remission includes a period without EoE-related symptoms or a period of reduced severity and / or number of EoE-related symptoms. EoE-related symptoms include any clinical or laboratory manifestations associated with EoE. Therefore, clinical remission can be measured using, for example, relevant scales or remission indicators well-known to clinicians and researchers in the medical field. Conversely, relapse of the disease can be defined as an increase or appearance of one or more symptoms of EoE.

[0041] The reduction or elimination of one or more symptoms of EoE is typically a significant reduction or elimination of one or more symptoms as identified by a physician. Symptoms of EoE can be measured and quantified using well-known diagnostic tests. Diagnosis of EoE is usually based on symptoms, endoscopic features, and histological findings, with esophagogastric endoscopy (EGD) and biopsy considered the preferred diagnostic tests. EGD is a diagnostic endoscopic procedure that may include visualization of the oropharynx, esophagus, stomach, and proximal duodenum. Common endoscopic features include mucosal edema, loss of vascular pattern, linear grooves, white spots, and concentric rings. Additionally, esophageal function tests can be used to quantify EoE symptoms using scores from the Dysphagia Symptom Questionnaire (DSQ). The DSQ will be described further below.

[0042] As will be discussed below, remission may also be associated with a significant decrease in the number of lymphocytes or a significant increase in the number of regulatory T cells (Tregs) and / or regulatory B cells (Bregs) in human subjects relative to baseline levels or, alternatively, relative to control subjects. Finally, the studies described below also provide a number of genes that can be used as biomarkers for assessing patients with EoE.

[0043] During the asymptomatic or remission phase, patients do not require quantifiable circulating levels of therapeutic Cpn60.1-related peptides. In some implementations, remission is maintained when plasma concentrations of Cpn60.1-related peptides are below the limit of quantification. This limit of quantification can vary depending on the assay method used. Typically, plasma peptide concentrations are undetectable at circulating levels below 40 ng / mL, such as less than 30 ng / mL or less than 20 ng / mL. A typical method for determining plasma peptide concentrations is high-resolution precision mass spectrometry (HRAM) LC-MS / MS.

[0044] In some implementations, remission of EoE disease lasts for at least 7 days, optionally at least 14 days, optionally at least 28 days, optionally at least 6 months or longer after the concentration of peptide molecules in the subject's plasma becomes undetectable.

[0045] In another implementation, remission of EoE disease lasts for a period of at least 7 days, optionally at least 14 days, optionally at least 28 days, or optionally at least 6 months after administration of the final dose of the therapeutic Cpn60.1-related peptide.

[0046] The compositions considered herein may be administered in any suitable 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 “parenterally” and “externally administered” refer to administration methods other than enteral and local administration, typically by injection, and include, but are not limited to, intravascular, intravenous, intramuscular, intra-arterial, intrasheath, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions. In one embodiment, the compositions considered herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

[0047] The terms “decrease,” “reduced,” “reduction,” or “inhibit” are used herein to mean a reduction of a statistically significant amount. In some embodiments, “reduce,” “reduction,” or “decrease” 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 may include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, “decrease” or “inhibit” does not cover complete inhibition or reduction compared to a reference level. “Complete inhibition” is 100% inhibition compared to a reference level. The reduction can preferably be reduced to a level that is acceptable within the normal range for individuals who do not have the given condition.

[0048] The terms “increased,” “increase,” “enhance,” or “activate” are used herein to mean an increase in a statistically significant amount. In some implementations, the terms “increased,” “increase,” “enhance,” or “activate” may mean an increase of at least 10% compared to a reference level, such as 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 100%, or any increase between 10% and 100%, or an increase of at least about 2, or at least about 3, or at least about 4, or at least about 5, or at least about 10, or any increase between 2 and 10, or greater, compared to a reference level. In the context of a biomarker or symptom, “increased” means a statistically significant increase in this level.

[0049] Definition of biopharmaceuticals: As used herein, the terms “protein” and “polypeptide” are used interchangeably to refer to a series of amino acid residues linked together 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., phosphorylated amino acids, glycosylated amino acids, 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; however, there is overlap in the use of these terms in the art. 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, homologs, orthologs, paralogs, the aforementioned fragments and other equivalents, variants, fragments, and analogs.

[0050] In the various embodiments described herein, further consideration is given to variants (naturally occurring or otherwise), alleles, homologs, conservedly modified variants, and / or conservedly substituted variants of any particular polypeptide described. Regarding amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter a single amino acid or a small percentage of amino acids in the encoded sequence are “conservedly modified variants,” wherein said alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservedly modified variants are attached to, and do not exclude, polymorphic variants, interspecies homologs, and alleles consistent with this disclosure.

[0051] In some embodiments, the polypeptide (or nucleic acid encoding such polypeptide) described herein may be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a segment or region of a peptide that retains at least 50% of the activity of a wild-type reference polypeptide according to the assays described below. A functional fragment may contain conserved substitutions of the sequences disclosed herein.

[0052] In some embodiments, the polypeptide described herein may be a variant of the sequence described herein. In some embodiments, the variant is a variant with conserved modifications. For example, conserved substitution variants can be obtained by mutation of a native nucleotide sequence. As referred to herein, a “variant” is a polypeptide that is substantially homologous to a native or reference polypeptide, but whose amino acid sequence differs from that of a native or reference polypeptide due to one or more deletions, insertions, or substitutions. The DNA sequence encoding the variant polypeptide includes sequences containing one or more additions, deletions, or substitutions of nucleotides compared to a native or reference DNA sequence, but encoding a variant protein or fragment thereof that retains its activity. Various PCR-based site-specific mutagenesis methods are known in the art and can be applied by those skilled in the art.

[0053] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymer molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid, or the like. Nucleic acids can be single-stranded or double-stranded. A single-stranded nucleic acid can be a single strand of denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid 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 may include, for example, genomic DNA or cDNA. Suitable RNA may include, for example, mRNA.

[0054] In some embodiments of any aspect, peptides, nucleic acids, or cells as described herein may be engineered. As used herein, “engineered” means an aspect that has been artificially manipulated. For example, a peptide is considered “engineered” when at least one aspect of it (e.g., its sequence) has been artificially manipulated to differ from its aspect as it exists in nature. By convention and as those skilled in the art will understand, progeny of engineered cells are often still referred to as “engineered” even if the actual manipulation was performed on the previous entity.

[0055] In some embodiments, a nucleic acid (e.g., an antibody or antibody reagent) encoding a polypeptide as described herein is contained in a vector. In some aspects described herein, a nucleic acid sequence encoding a given polypeptide or any module thereof as described herein is operatively ligated to a vector. Vectors may include, but are not limited to, cloning vectors, expression vectors, plasmids, bacteriophages, transposons, granules, chromosomes, viruses, viral particles, etc.

[0056] As used herein, the term "expression vector" refers to a vector that directs the expression of RNA or polypeptide from a sequence linked to a transcriptional regulatory sequence on the vector. The expressed sequence is typically, but not necessarily, heterologous to the cell. Expression vectors may contain additional elements; for example, an expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example, for expression in human cells and for cloning and amplification in a prokaryotic host. The term "expression" refers to the cellular process involved in the production of RNA and proteins, and, where appropriate, the secretion of proteins, including, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing, where appropriate. "Expression products" include RNA transcribed from a gene and polypeptides obtained through the translation of mRNA transcribed from a gene. The term "gene" refers to a nucleic acid sequence that is transcribed (DNA) into RNA in vitro or in vivo when operatively linked to a suitable regulatory sequence. Genes may or may not include regions before and after the coding region, such as a 5' untranslated (5'UTR) or "leader" sequence and a 3' UTR or "tail" sequence, as well as insertion sequences (introns) between the individual coding segments (exons).

[0057] As used herein, the terms “isolated” or “partially purified” in the case of nucleic acids or peptides refer to nucleic acids or peptides that are present with nucleic acids or peptides found in their natural sources and / or that will be present with nucleic acids or peptides when expressed by cells or secreted in the case of secreted peptides (e.g., nucleic acids or peptides). Chemically synthesized nucleic acids or peptides or nucleic acids or peptides synthesized using in vitro transcription / translation are considered “isolated”. The terms “purified” or “substantially purified” refer to isolated nucleic acids or peptides for at least 95% by weight (including, for example, at least 96% by weight, at least 97% by weight, at least 98% by weight, at least 99% by weight or more) of the subject nucleic acid or peptide. In some embodiments, the antibodies, their antigen-binding moieties, or chimeric antigen receptors (CARs) described herein are isolated. In some embodiments, the antibodies, antibody reagents, their antigen-binding moieties, or CARs described herein are purified.

[0058] As used herein, “engineered” refers to aspects that have been artificially manipulated. For example, an antibody, antibody reagent, its antigen-binding moiety, CAR, or bispecific antibody is considered “engineered” when its sequence is artificially manipulated to differ from the antibody sequence it has in nature. By convention and as those skilled in the art will understand, progeny and copies of engineered polynucleotides and / or peptides are often still referred to as “engineered” even if the actual manipulation is performed on a prior entity.

[0059] Pharmaceutical Composition The compositions and methods of the present invention can be used to treat individuals in need. In some embodiments, the individual is a mammal, such as a human or a non-human mammal. When administered to an animal such as a human, the composition or compound is preferably administered in the form of a pharmaceutical composition comprising, for example, a compound of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline, or other solvents or mediators such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are used for human administration, particularly for invasive routes of administration (i.e., routes that avoid transport or diffusion across the epithelial barrier, such as injection or implantation), the aqueous solution is pyrogen-free or substantially pyrogen-free. Excipients may be selected (e.g.) to achieve delayed release of the agent or selective targeting of one or more cells, tissues, or organs. The pharmaceutical compositions may be in the form of dosage units such as tablets, capsules (including spray capsules and gelatin capsules), granules, lyophilized formulations for reconstitution, powders, solutions, syrups, suppositories, injections, etc. The composition may also be present in transdermal delivery systems (e.g., skin patches). The composition may also be present in solutions suitable for topical application (e.g., lotions, creams, or ointments).

[0060] Pharmaceutically acceptable carriers may contain physiologically acceptable agents, such as those used to stabilize compounds (like those of the present invention), increase the solubility of said compounds, or increase the absorption of said compounds. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins or other stabilizers or excipients. The choice of pharmaceutically acceptable carriers (including physiologically acceptable agents) depends on, for example, the route of administration of the composition. Formulations or pharmaceutical compositions may be self-emulsifying drug delivery systems or self-microemulsifying drug delivery systems. Pharmaceutical compositions (formulations) may also be liposomes or other polymer matrices in which compounds, such as those of the present invention, may be contained. For example, liposomes containing phospholipids or other lipids are relatively simple to manufacture and administer, being non-toxic, physiologically acceptable, and metabolizable carriers.

[0061] The phrase “pharmaceutically acceptable” is used in this document to refer to compounds, materials, compositions, and / or dosage forms that are suitable for contact with human and animal tissues to the extent of reasonable medical judgment without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio.

[0062] As used herein, the phrase “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be “acceptable” in the sense that it is compatible with other components of the formulation and does not harm 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 carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered astragalus gum; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) di Alcohols, 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; (19) ethanol; (20) phosphate buffer solution; and (21) other non-toxic and compatible substances used in pharmaceutical preparations.

[0063] Pharmaceutical compositions (formulations) may be administered to a subject via any of a number of routes of administration, including, for example, oral (e.g., as an infusion, tablet, capsule (including spray capsules and gelatin capsules), granules, powder, pellets, or paste for application to the tongue); absorption through the oral mucosa (e.g., sublingual); subcutaneous; transdermal (e.g., as a patch applied to the skin); and topical (e.g., as a cream, ointment, or spray applied to the skin). Compounds may also be formulated for inhalation. In some embodiments, the compound may simply be dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable for said routes of administration can be found, for example, in 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 referenced therein.

[0064] The formulation can be conveniently present in a single 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 and the specific route 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. Generally, in one hundred parts, this amount will be in the range of about 1% to about 99% of the active ingredient, preferably about 5% to about 70%, and most preferably about 10% to about 30% of the active ingredient.

[0065] Methods for preparing these formulations or compositions include the step of associating an active compound (such as the compound of the present invention) with a carrier and optionally one or more auxiliary components. Generally, formulations are prepared by uniformly and tightly associating the compound of the present invention with a liquid carrier or a finely dispersed solid carrier, or both, and then (if necessary) shaping the product.

[0066] Formulations of the present invention suitable for oral administration may be in the following forms: capsules (including spray capsules and gelatin capsules), sachets, pills, tablets, lozenges (using a flavored base, typically sucrose and gum arabic or astragalus gum), lyophilized forms, powders, granules, or as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as soft lozenges (using an inert base, such as gelatin and glycerin or sucrose and gum arabic), and / or as mouthwashes, each containing a predetermined amount of the compound of the present invention as an active ingredient. The compositions or compounds may also be administered as large pills, electuals, or pastes.

[0067] To prepare solid dosage forms (capsules (including spray capsules and gelatin capsules), tablets, pills, sugar tablets, powders, granules, etc.) for oral administration, the active ingredient is mixed with one or two pharmaceutically acceptable carriers (such as sodium citrate or dicalcium phosphate) and / or any of the following: (1) fillers or enriching agents, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or gum arabic; (3) humectants, etc. Examples of active ingredients include: (4) disintegrants such as agar, calcium carbonate, potato or cassava starch, alginic acid, certain silicates, and sodium carbonate; (5) solution delay agents such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents such as, 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) colorants. In the case of capsules (including spray capsules and gelatin capsules), tablets, and pills, the pharmaceutical composition may also contain a buffer. Similar types of solid compositions may also be used as fillers in soft-filled and hard-filled gelatin capsules using such excipients such as lactose or milk sugar and high molecular weight polyethylene glycol.

[0068] Tablets can be prepared by compression or molding, optionally together with one or more excipients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropyl methylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or croscarmellose sodium), surfactants, or dispersants. Molded tablets can be prepared by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine.

[0069] Solid dosage forms of tablets and other pharmaceutical compositions, such as sugar-coated pills, capsules (including spray capsules and gelatin capsules), pellets, and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings or other coatings well known in the field of pharmaceutical formulation. They may also be formulated to provide slow or controlled release of the active ingredient contained therein using, for example, different proportions of hydroxypropyl methylcellulose, other polymer matrices, liposomes, and / or microspheres to provide the desired release characteristics. They may be sterilized, for example, by filtering through a bacterial-retaining filter or by incorporating immediately before use with a sterilizing agent in the form of a sterile solid composition soluble in sterile water or some other sterile injectable medium. These compositions may also optionally contain light-blocking agents and may have a composition in which they optionally release the active ingredient in a delayed manner only or preferably in a portion of the gastrointestinal tract. Examples of encapsulation compositions that may be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form and suitably contain one or more of the excipients described above.

[0070] Liquid dosage forms suitable for oral administration include pharmaceutically acceptable emulsions, lyophilized formulations for reconstitution, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms may also contain inert diluents commonly used in the art, such as, for example, 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-butanediol, oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuranol, fatty acid esters of polyethylene glycol and sorbitol, and mixtures thereof.

[0071] In addition to inert diluents, oral compositions may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, coloring agents, aroma agents and preservatives.

[0072] In addition to the active composition, the suspension may also contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar and astragalus gum, and mixtures thereof.

[0073] Dosage forms for topical or transdermal application include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalers. Active compounds may be mixed under sterile conditions with pharmaceutically acceptable carriers and any preservatives, buffers, or propellants that may be required.

[0074] In addition to active compounds, ointments, pastes, creams and gels may also contain excipients such as animal and vegetable fats, oils, waxes, paraffin waxes, starches, astragalus gums, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0075] In addition to the active compound, powders and aerosols may also contain excipients such as lactose, talc, silica, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures of these substances. Aerosols may also contain conventional propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons (such as butane and propane).

[0076] Transdermal patches offer the additional advantage of controlled delivery of the compounds of the present invention into the body. Such dosage forms can be prepared by dissolving or dispersing the active compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of this flux can be controlled by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or gel.

[0077] As used herein, the phrases “parenteral administration” and “extraterrestrial administration” refer to administration methods other than enteral and local administration, typically by injection, and include, but are not limited to, intravenous, intraocular (e.g., intravitreal), intramuscular, intra-arterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds and one or more pharmaceutically acceptable sterile isotonic or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders reconstituted into sterile injectable solutions or dispersions immediately before use. These compositions may contain antioxidants, buffers, antibacterial agents, solutes that make the formulation isotonic with the blood of the intended recipient, or suspending agents or thickeners.

[0078] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Suitable flowability can be maintained, for example, by using a coating material such as lecithin, in the case of a dispersion by maintaining a desired particle size, and by using a surfactant.

[0079] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Antimicrobial activity can be ensured by including various antimicrobial and antifungal agents, such as parabens, chlorobutanol, and phenolic sorbic acid. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the composition. Furthermore, prolonged absorption of injectable drug forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0080] In some cases, to prolong the effect of a drug, it is desirable to slow the absorption of subcutaneously or intramuscularly injected drugs. This can be achieved by using liquid suspensions of crystalline or amorphous materials with low water solubility. The absorption rate of the drug depends on its dissolution rate, which in turn depends on the crystal size and crystal form. Alternatively, delayed absorption of parenteral drug forms can be achieved by dissolving or suspending the drug in an oil-based medium.

[0081] Injectable reservoir formulations are prepared by forming a microencapsulated matrix of the subject compound within a biodegradable polymer such as poly(lactide-polyglycolic acid). The rate of drug release can be controlled depending on the drug-to-polymer ratio and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable reservoir formulations are also prepared by encapsulating the drug in tissue-compatible liposomes or microemulsions.

[0082] For use in the method of the present invention, the active compound may be administered either on its own or as a pharmaceutical composition containing, for example, 0.1% to 99.5% (more preferably 0.5% to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0083] The introduced methods can also be provided by rechargeable or biodegradable devices. Various sustained-release polymer devices have been developed in recent years and tested in vivo for controlled delivery of drugs, including protein biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), encompassing both biodegradable and non-degradable polymers, can be used to form implants for the sustained release of compounds at specific target sites.

[0084] The actual dose level of the active ingredient in a pharmaceutical composition can be altered to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response for a specific patient, composition, and administration method without being toxic to the patient.

[0085] The chosen dose level will depend on a variety of factors, including the activity of the specific compound or combination of compounds or its esters, salts or amides used, the route of administration, the time of administration, the excretion rate of the specific compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific compound used, the age, sex, weight, disease, general health and prior medical history of the patient being treated, and similar factors well known in the medical field.

[0086] Physicians or veterinarians with ordinary skills in the art can readily determine and prescribe a therapeutically effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian may start with a dose of the pharmaceutical composition or compound below the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. A “therapeutically effective amount” refers to the concentration of the compound sufficient to elicit the desired therapeutic effect. It should generally be understood that the effective amount of a compound will vary depending on 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 (if necessary) another type of therapeutic agent administered with the compound of the present invention. A larger total dose can be delivered by multiple administrations. Methods for determining efficacy and dosage are known to those skilled in the art. See, for example, Isselbacher et al. (1996). 9 Generally, the appropriate daily dose of the active compound used in the compositions and methods of the present invention will be the amount of the compound at which the lowest effective therapeutic effect is achieved. This effective dose will generally depend on the factors described above.

[0087] If desired, the effective daily dose of the active compound may optionally be administered in one, two, three, four, five, six, or more sub-dose units at appropriate time intervals throughout the day, in unit dosage form. In some embodiments of the invention, the active compound may be administered two or three times daily. In other embodiments, the active compound will be administered once daily.

[0088] Patients receiving this treatment are any animals in need, including primates, especially humans; other mammals such as horses, cattle, pigs, sheep, cats and dogs; poultry; and pets in general.

[0089] In some embodiments, the compounds of the present invention may be used alone or in combination with another type of therapeutic agent.

[0090] This disclosure includes the use of pharmaceutically acceptable salts of the compounds of the present invention in the compositions and methods of the present invention. In some embodiments, the salts of the present invention contemplated include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkylammonium salts. In some embodiments, the salts of the present invention contemplated include, but are not limited to, L-arginine, phenethylbenzylamine, benzoxin, betaine, calcium hydroxide, choline, tannin, 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 some embodiments, the salts of the present invention contemplated include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts. In some embodiments, the salts of the present invention considered include, but are not limited to, 1-hydroxy-2-naphthylcarboxylic 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-10-sulfonic acid, capric acid, caproic acid, hexanoic acid, caprylic acid, octanoic acid, carbonic acid, cinnamic acid, citric acid, and cyclamic acid. (acid), dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactosidic acid, gentian acid, d-glucoheponic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutamate, glycerophosphate, 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, dihydroxynaphthalic acid, phosphoric acid, propionic acid, L-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, L-tartaric acid, thiocyanate, p-toluenesulfonic acid, trifluoroacetic acid, and undecanoic acid salt.

[0091] Pharmaceutically acceptable acid addition salts can also exist as various solvates such as those with water, methanol, ethanol, and dimethylformamide. Mixtures of such solvates can also be prepared. These solvates can originate from the solvent used for crystallization, are inherent in the solvent used for preparation or crystallization, or are insoluble in such solvents.

[0092] Wetting agents, emulsifiers and lubricants (such as sodium lauryl sulfate and magnesium stearate), as well as colorants, release agents, coating agents, sweeteners, flavorings and aromas, preservatives and antioxidants may also be present in the composition.

[0093] 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 palmitic acid ascorbate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0094] Unless otherwise defined herein, scientific and technical terms used in connection with this application will have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains. It should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, and therefore is subject to variation. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the claims. Definitions of commonly used terms in immunology and molecular biology can be found in *The Merck Manual of Diagnosis and Therapy*; 10 The Encyclopedia of Molecular Cell Biology and Molecular Medicine; 11 Molecular Biology and Biotechnology: a Comprehensive Desk Reference; 12 Immunology; 13 Janeway's Immunobiology; 14 Lewin's Genes XI; 15 Molecular Cloning: A Laboratory Manual.; 16 Basic Methods in Molecular Biology; 17 Laboratory Methods in Enzymology; 18 Current Protocols in Molecular Biology (CPMB); 19 Current Protocols in ProteinScience (CPPS); 20 And Current Protocols in Immunology (CPI). 21 In any of the embodiments described herein, the disclosure does not relate to methods for cloning humans, methods for modifying the germline genetic characteristics of humans, the use of human embryos for industrial or commercial purposes, or methods for modifying the genetic characteristics of animals that may cause them suffering and have no substantial medical benefit to humans or animals, and animals produced by such methods.

[0095] Other terms are defined herein within the description of various aspects of the invention.

[0096] Eosinophilic esophagitis (EoE) Eosinophilic esophagitis (EoE) is a chronic immune-mediated disorder characterized by high levels of eosinophils in the esophageal mucosa. Diagnosis requires ≥15 eosinophils per high-power field (eos / hpf) and esophageal dysfunction. 22,23 Patients with this condition exhibit a range of symptoms, including dysphagia / difficulty swallowing. The clinical presentation of eosinophilic esophagitis varies with age. Young children often present with nonspecific symptoms such as feeding difficulties, vomiting, abdominal pain, inability to establish normal eating habits (e.g., inability to progress to liquids / soft solids), and occasional growth retardation. Adolescents and adults present with typical symptoms, including dysphagia and food impaction; EoE is now one of the most common causes of dysphagia and food impaction in the United States. 24 Some patients may also experience symptoms similar to gastroesophageal reflux, such as heartburn, upper abdominal pain, indigestion, and chest pain. Patients with EoE often develop coping mechanisms to compensate for their symptoms, such as eating slowly, chewing thoroughly, taking small bites, cutting food into small pieces, using different sauces to lubricate food, drinking liquids after most meals, and avoiding textured foods such as meat and bread. 25 Diagnosis of esophageal eosinophilia (EoE) is based on symptoms, endoscopic features, and histological findings, with esophagogastric and duodenal endoscopy (EGD) and biopsy considered the preferred diagnostic tests. EGD is a diagnostic endoscopic procedure that includes visualization of the oropharynx, esophagus, stomach, and proximal duodenum. Common endoscopic features include mucosal edema, loss of vascular pattern, linear grooves, white spots, and concentric rings. Because the esophageal mucosa may appear normal in some patients, esophageal mucosal biopsy must be performed in all cases to avoid missed diagnoses. Due to the patchy nature of EoE, multiple biopsies from both the proximal and distal esophagus are recommended. Biopsies from patients with EoE show ≥15 intraepithelial eosinophils per high-power field; other findings include basal cell hyperplasia, elongated vascular papillae, eosinophilic microabscesses, and lamina propria fibrosis. It is important to consider other conditions that can cause esophageal eosinophilia, such as gastroesophageal reflux disease (GERD). 26 Laboratory and radiological examinations often have limited utility in evaluating patients with esophageal endoscopic esophageal refractory (EoE). Upper gastrointestinal contrast studies are not sensitive enough to detect subtle changes in EoE, but can help identify alternative pathologies or complications of EoE, such as esophageal stricture and small-diameter esophagus. EndoFLIP (Endoluminal functional luminal imaging) is currently under investigation. ® The role of an endoscopic tool (a type of endoscopy) is to assess the dilatational nature of the esophagus, thereby identifying early fibrous stenosis. 27 Due to the invasive nature of EGD required for diagnosis and disease monitoring, there is a need to find non-invasive biomarkers and minimally invasive clinic procedures.

[0097] Chaperone protein 60.1 related peptide Before describing the compositions and methods of the present invention, it should be understood that the invention is not limited to the specific compositions, methods, and experimental conditions described, as such compositions, methods, and conditions can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention is limited only by the appended claims.

[0098] Compositions that can be used in the methods of the present invention include, but are not limited to: DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 1) ('1104); DGSVVVNKVSELPAGH (SEQ ID NO: 2);GLNVNTLSYGDLAAD (SEQ ID NO: 3); SELPAGHGLNVNLTS (SEQ ID NO: 4);DGSVVVNKVS (SEQ ID NO: 5); ELPAGHGLNV (SEQ ID NO: 6); NTLSYGDLAAD (SEQ ID NO: 7); Or its functionally equivalent fragments or variants.

[0099] Cpn60.1-related peptide '1104' is also known as PIN201104 and IRL201104. Other compositions that can be used in the methods of this invention include, but are not limited to, U.S. Patent Application Publication No. 20040132163. 28 and U.S. Patent No. 11,098,090; 29 9,320,791; 30 Cpn60.1-related peptide disclosed in 9,085,632. 31 In some aspects, Cpn60.1-related peptides consist of amino acid residues. As used herein, the term "amino acid residue" is used interchangeably with the term "amino acid" or "aa" and refers to an amino acid that is part of a peptide or protein. In some such aspects, the agonists or ligands of the present invention consist of amino acids having the standard structure NH2—C(H)(R)—COOH, where R represents a single amino acid side chain. In some aspects, the agonists or ligands consist of amino acid residues of naturally occurring amino acids. In some aspects, naturally occurring amino acids include one of the twenty standard amino acids found in naturally occurring peptides and proteins. In some of these aspects, the agonist or ligand consists of at least one naturally occurring amino acid residue, which is alanine (“A”), arginine (“R”), asparagine (“N”), aspartic acid (“D”), cysteine ​​(“C”), glutamine (“Q”), glutamic acid (“E”), glycine (“G”), histidine (“H”), isoleucine (“I”), leucine (“L”), lysine (“K”), methionine (“M”), phenylalanine (“F”), proline (“P”), serine (“S”), threonine (“T”), tryptophan (“W”), tyrosine (“Y”), or valine (“V”).

[0100] In other aspects, the Cpn60.1-related peptide used in the methods of the present invention consists of amino acid residues of at least one non-natural or synthetic amino acid. In some such aspects, the non-natural or synthetic amino group is a chemically modified amino acid, including but not limited to amino acids that have been modified by methylation, amidation, acetylation, protecting groups, and / or substitution with other chemical groups that can alter the physiological and chemical properties of the peptide. In other aspects, the non-natural or synthetic amino group is a chemically modified amino acid that has been modified with one or more chemical entities (e.g., methyl, acetate, acetyl, phosphate, formyl moiety, isoprene-like group, sulfate group, polyethylene glycol moiety, lipid moiety, carbohydrate moiety, biotin moiety, etc.).

[0101] In other aspects, the Cpn60.1-related peptide used in the method of the present invention consists of at least one amino acid having an L-configuration (the chirality of an L-amino acid). In another aspect, the Cpn60.1-related peptide used in the method of the present invention consists of at least one amino acid having a D-configuration (the chirality of a D-amino acid).

[0102] In some aspects, the agonist or ligand of the present invention has a length of about 50 aa; a length of 49 aa; a length of 48 aa; a length of 47 aa; a length of 46 aa; a length of 45 aa; a length of 44 aa; a length of 43 aa; a length of 42 aa; a length of 41 aa; a length of 40 aa; a length of 39 aa; a length of 39 aa; a length of 38 aa; a length of 37 aa; a length of 36 aa; a length of 35 aa; a length of 34 aa; a length of 33 aa; a length of 32 aa; a length of 31 aa; a length of 30 aa; a length of 29 aa; a length of 28 aa; a length of 27 aa; a length of 26 aa; a length of 25 aa; a length of 24 aa; a length of 23 aa; a length of 22 aa; a length of 21 aa; or a length of 20 aa; a length of 19 aa. aa; length 18 aa; length 17 aa; length 16 aa; length 15 aa; length 14 aa; length 13 aa; length 12 aa; length 11 aa; length 10 aa; length 9 aa; length 8 aa; length 7 aa; length 6 aa; or length 5 aa.

[0103] In some aspects, the compositions of the present invention are administered to a patient via any suitable route known to and / or employed by those skilled in the art. In some aspects, the compositions of the present invention are administered orally (PO), intravenously (IV), intramuscularly (IM), intraarterially, intramedullaryly, intrathecally, subcutaneously (SQ), intraventricularly, percutaneously, intradermally, rectically (PR), vaginally, intraperitoneally (IP), intragastrically (IG), topically (e.g., by powder, ointment, cream, gel, lotion, and / or drops), via mucosa, intranasally, buccally, intraenterically, intravitreally, sublingually, via endotracheal instillation, intrabronchial instillation, and / or inhalation, as oral sprays, nasal sprays, aerosols, and / or via portal vein catheters.

[0104] In some aspects, the Cpn60.1-related peptide and / or its pharmaceutical composition used in the methods of the present invention may be administered intravenously, for example, by intravenous infusion. In other aspects, the Cpn60.1-related peptide and / or its pharmaceutical composition used in the methods of the present invention may be administered by intramuscular injection. In still other aspects, the Cpn60.1-related peptide and / or its pharmaceutical composition used in the methods of the present invention may be administered by intratumoral injection. In some aspects, the Cpn60.1-related peptide and / or its pharmaceutical composition used in the methods of the present invention may be administered by subcutaneous injection. In still other aspects, the Cpn60.1-related peptide and / or its pharmaceutical composition used in the methods of the present invention may be administered via a portal vein catheter. In still other aspects, the present invention covers the delivery of the Cpn60.1-related peptide and / or its pharmaceutical composition used in the methods of the present invention by any suitable route, taking into account possible advancements in the field of drug delivery.

[0105] In some aspects, the Cpn60.1-related peptide and / or its pharmaceutical composition used in the methods of the present invention can be administered at dose levels sufficient to deliver from about 0.001 mg / kg to 100 mg / kg, from about 0.01 mg / kg to 50 mg / kg, from about 0.1 mg / kg to 40 mg / kg, from about 0.5 mg / kg to 30 mg / kg, from about 0.01 mg / kg to 10 mg / kg, from about 0.1 mg / kg to 10 mg / kg, or from about 1 mg / kg to 25 mg / kg of patient body weight per day to achieve the desired therapeutic effect. In some aspects, the desired dose can be delivered more than three times a day, three times a day, twice a day, once a day, every other day, every three days, once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every six months, or once every twelve months. In some respects, the required dose may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). In other respects, the required dose may be delivered using one or more administrations during an initial time period, followed by a period of no administration.

[0106] In other respects, the Cpn60.1-related peptide used in the methods of the present invention can be used for prophylactic applications. In further respects, prophylactic applications relate to systems and methods for preventing EoE, inhibiting the progression of EoE, and / or delaying the onset of EoE in individuals susceptible to EoE and / or exhibiting EoE symptoms.

[0107] In some aspects, the Cpn60.1-related peptide used in the method of the present invention is administered to target cells in vivo. In other aspects, the Cpn60.1-related peptide used in the method of the present invention is administered to target cells in vitro. In still other aspects, the Cpn60.1-related peptide used in the method of the present invention is administered to target cells in vitro, and then the target cells are reintroduced into an organism. In some such aspects, the target cells are cultured in vitro into multiple progeny cells before being reintroduced into an organism. In many other aspects, the organism is a human. In other aspects, the organism is a human patient. In some aspects, the target cells were originally derived from the organism in which they were reintroduced. In other aspects, the target cells were originally derived from a different organism in which they were reintroduced.

[0108] In some aspects, the Cpn60.1-related peptide and / or its pharmaceutical composition used in the methods of the present invention are used in combination therapy to treat EoE or reduce the risk of EoE. In such aspects, administration may be combined with one or more other therapeutic agents. As used herein, the phrases “combination therapy,” “in combination with,” “combination,” etc., refer to the simultaneous use of more than one drug or treatment to increase response. In some aspects, the Cpn60.1-related peptide and / or its pharmaceutical composition used in the methods of the present invention are administered simultaneously, before, or after one or more other desired therapeutic agents or medical procedures. In some aspects, the Cpn60.1-related peptide and / or its pharmaceutical composition used in the methods of the present invention are administered together in a single composition or individually in different compositions.

[0109] In some respects, the specific combination of therapies employed in a combination regimen will generally take into account the compatibility of the desired therapeutic agents and / or procedures and the desired therapeutic effect to be achieved. In other respects, the therapies employed may achieve the desired effect for the same purpose (e.g., a Cpn60.1-related peptide used in the method of the present invention, which can be used to treat, prevent, and / or delay the onset of EoE, may be administered concurrently with another therapeutic agent, which can also be used to treat, prevent, and / or delay the onset of EoE), or they may achieve different effects. In other respects, the combination of therapies employed may achieve the same or substantially similar desired effect against the same disease, ailment, or condition; may achieve the same or substantially similar desired effect against one or more different diseases, ailments, or conditions; may achieve different desired effects against the same disease, ailment, or condition; or may achieve different desired effects against one or more different diseases, ailments, or conditions.

[0110] In another aspect, the Cpn60.1-related peptide used in the method of the present invention is combined with one or more additional components as a delivery of the pharmaceutical composition, said additional components being able to improve the bioavailability of the Cpn60.1-related peptide used in the method of the present invention, reduce and / or alter its metabolism, inhibit its excretion, and / or alter its distribution in vivo.

[0111] In some aspects, the combination therapy may involve the administration of multiple Cpn60.1-related peptides according to the invention. In other aspects, the combination therapy may involve the administration of multiple Cpn60.1-related peptides for treating, preventing, improving EoE, achieving EoE remission, and / or reducing the risk of EoE. In still other aspects, the combination therapy may be multiple Cpn60.1-related peptides for treating, preventing, improving EoE, achieving EoE remission, and / or reducing the risk of EoE.

[0112] In some aspects, the Cpn60.1-related peptide used in the methods of this invention is combined with at least one pharmaceutically acceptable excipient in the form of a pharmaceutical composition. As used herein, a "pharmaceutical composition" means a formulation containing an active ingredient, and optionally a pharmaceutically acceptable carrier, diluent, or excipient. The term "active ingredient" is interchangeable with "effective ingredient" and means any agent capable of inducing the desired effect upon administration. Examples of active ingredients include, but are not limited to, chemical compounds, pharmaceuticals, therapeutic agents, small molecules, etc.

[0113] In some aspects of the invention, the active ingredient is a Cpn60.1-related peptide as disclosed herein. In certain aspects, the active ingredient is PIN201104 or a derivative thereof. In some aspects, the active ingredient is PIN201360, PIN201361, PIN201362, PIN201116, PIN201105 or a derivative thereof. In other aspects, the active ingredient is a peptide or a derivative thereof described in WO2009 / 106819.

[0114] In some respects, the pharmaceutical composition may be used in the manufacture of medicine or pharmaceuticals. In other respects, the pharmaceutical composition may be used for one or more therapeutic applications disclosed herein, such as for individuals suffering from autoimmune diseases. Furthermore, the pharmaceutical composition is formulated for administration to human patients.

[0115] In some aspects, the pharmaceutical composition is in a sterile, injectable form (e.g., a form suitable for subcutaneous or intravenous infusion). In many other aspects, the pharmaceutical composition is in a liquid dosage form suitable for injection. In other aspects, the pharmaceutical composition is in powder form (e.g., lyophilized and / or sterilized), optionally reconstituted under vacuum with an aqueous diluent (e.g., water; buffer solution; saline solution, etc.) prior to injection. In still other aspects, the pharmaceutical composition is diluted and / or reconstituted in an aqueous diluent (e.g., water, sodium chloride solution, sodium acetate solution, benzyl alcohol solution, phosphate-buffered saline, etc.). In some aspects, the pharmaceutical composition is in a form that can be refrigerated and / or frozen. In other aspects, the pharmaceutical composition is in a form that cannot be refrigerated and / or frozen. In some aspects, the pharmaceutical composition is a reconstituted solution and / or liquid dosage form that can be stored for a period of time after reconstitution (e.g., 2 hours, 12 hours, 24 hours, 2 days, 5 days, 7 days, 10 days, 2 weeks, 1 month, 2 months, or longer).

[0116] In some aspects, methods of preparing a pharmaceutical composition include associating an active ingredient (e.g., a Cpn60.1-related peptide used in the methods of the present invention) with one or more pharmaceutically acceptable excipients, and then shaping and / or packaging the product into desired single-dose or multi-dose units. Pharmaceutical compositions according to the invention may be prepared, packaged in bulk, packaged as a single unit dose, and / or packaged as multiple single unit doses. As used herein, a “unit dose” refers to a discrete amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dose to be administered to a subject and / or a convenient portion of such a dose, such as half or one-third of such a dose. The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any other components in a pharmaceutical composition according to the invention may vary depending on the identity, size, and / or disease of the subject being treated and / or depending on the route of administration of the composition. In some aspects, for example, the composition may contain between about 0.1% and 100% (w / w) of the active ingredient.

[0117] In another aspect, the present invention includes a kit for use in carrying out the methods of the invention. The components contained in the kit depend on various factors, including the specific application (e.g., the specific route of administration to be used, or the specific disease, ailment, or condition to be treated). In some aspects, the present invention provides a kit for administering a Cpn60.1-related peptide according to the invention to treat the diseases, ailments, or conditions disclosed herein. In some such aspects, the kit also includes instructions for administration. In some aspects, the kit is used for administering the Cpn60.1-related peptide to treat a patient suffering from EoE. In some aspects, the kit contains one or more Cpn60.1-related peptides. In some aspects, the kit comprises a plurality of unit doses of a pharmaceutical composition containing the Cpn60.1-related peptide. In other aspects, the kit used according to the invention includes instructions (e.g., for administration, for storage, etc.), buffers, and / or other reagents. In some aspects, the pillbox includes (i) at least one Cpn60.1-related peptide; (ii) a syringe, needle, applicator, etc., for administering said at least one Cpn60.1-related peptide to a patient; and (iii) instructions for use. In other aspects, the pillbox includes a treatment regimen specifying when to administer a unit dose. In still other aspects, it includes a placebo dose, which may be similar to or different from the dose of the pharmaceutical composition. In some aspects, the pillbox includes one or more containers such that certain individual components or reagents can be contained separately. In some aspects, the pillbox may include means for sealing individual containers in a relatively tight enclosure for commercial sale, such as a plastic box, instructions for use, or packaging materials such as polystyrene foam, into which the pillbox may be sealed.

[0118] The description of embodiments of this disclosure is not intended to be exhaustive or to limit this disclosure to the exact form disclosed. While specific embodiments and examples of this disclosure are described herein for illustrative purposes, various equivalent modifications can be made within the scope of this disclosure, as will be recognized by those skilled in the art. For example, although 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 can be suitably applied to other procedures or methods. The various embodiments described herein can be combined to provide other embodiments. If desired, aspects of this disclosure may be modified to employ the compositions, functions, and concepts of the foregoing references and applications to provide other embodiments of this disclosure. Furthermore, due to considerations of biological functional equivalence, some changes may be made to the protein structure without affecting the biological or chemical action in kind or quantity. These and other changes may be made to this disclosure according to specific embodiments. All such modifications are intended to be included within the scope of the appended claims.

[0119] Specific elements of any of the foregoing embodiments may be combined with or replace elements in other embodiments. Furthermore, while advantages associated with certain embodiments of this disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments must exhibit such advantages to fall within the scope of this disclosure.

[0120] The techniques described herein are further illustrated by the following examples, which should not be construed as further limitations. Although similar or equivalent methods and materials to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

[0121] Example The invention has now been generally described, and will be more readily understood by referring to the following embodiments, which are included only for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention.

[0122] Example 1: Evaluation of the efficacy of CPN60.1-related peptide in adult patients with dysphagia using a dysphagia symptom questionnaire. A multicenter, double-blind, placebo-controlled phase 2a study was conducted in the United States to evaluate the role of IRL201104 ('1104) (a 31-amino acid linear peptide derived from the mycobacterial chaperone protein Cpn60.1) in adult patients with EoE. Figure 1 A schematic diagram of a clinical protocol for studies in patients with EoE is provided.

[0123] Patients with EoE received placebo, 4 mg '1104, or 8 mg '1104 on days 0, 7, and 14. From the screening period to the end of the study, patients with EoE completed the Daily Dysphagia Symptom Questionnaire (DSQ). The DSQ is a patient-reported outcome measure that assesses the frequency and severity of dysphagia in patients with EoE. The DSQ has been used in clinical studies to evaluate the effects of pharmacological therapy on dysphagia symptoms in adolescents and adults with EoE. 32,33,34 The psychometric properties of the DSQ were also evaluated in patients aged 11–40 years with EoE, using data from a 12-week, phase 2, randomized, placebo-controlled trial of budesonide oral suspension. This psychometric analysis demonstrates that the DSQ is an effective and reliable measure of dysphagia in patients with EoE, thus supporting its use in clinical trials. 35 result Patients receiving '1104' at days 0, 7, and 14 reported significantly greater improvement in EoE dysphagia compared to those receiving placebo. Figure 2 As shown, in 4 weeks ( p =0.032) and 6 weeks ( p At 0.003, the median DSQ reduction was significantly greater in the '1104 treatment group compared to the placebo group.

[0124] These data suggest that Cpn60.1-related peptides, such as '1104, can effectively improve the symptoms of EoE.

[0125] Example 2: Effect of CPN60.1-related peptides on leukocytes in esophageal biopsies from adult patients with end-stage renal disease (EOE). As discussed above, EoE is an allergic disease characterized by the infiltration of leukocytes (particularly eosinophils) in the esophagus. Eosinophils play a major role in the inflammatory response and are used to diagnose EoE based on histological analysis of biopsy tissue. However, other cell types, including mast cells, B cells, and T cells, are also found in the esophagus during active EoE. Chronic EoE often leads to esophageal remodeling, making swallowing difficult and even causing food impaction.

[0126] In the aforementioned multicenter, double-blind, placebo-controlled phase 2a study, endoscopic examination and esophageal biopsy were performed on day 0 and day 28 in every adult EoE patient (see [link to study]). Figure 1 The aim of this study was to determine the number of eosinophils, mast cells, basophils, B cells, CD4 T cells, CD8 T cells, and CD4+ cells in esophageal biopsy samples collected from EoE patients participating in a clinical trial. + CD25 +The prevalence of Treg cells, as well as memory and activated Breg cells.

[0127] Material The materials used in this study included: mouse anti-human IgD BB515 (BD Biosciences, catalog number 565243); mouse anti-human CD86 PE (BD Biosciences, catalog number 555658); mouse anti-human CD27 PerCP-Cy5.5 (BD Biosciences, catalog number 560612); mouse anti-human CD21 PE-Cy7 (BD Biosciences, catalog number 561374); mouse anti-human CD95 APC (BD Biosciences, catalog number 558814); mouse anti-human CD38 APC-H7 (BD Biosciences, catalog number 653314); mouse anti-human CD24 BV421 (BD Biosciences, catalog number 562789); mouse anti-human CD19 V500 (BD Biosciences, catalog number 561121); blank matrix (BioIVT, Hicksville, NY); SPHERO™ Ultra Rainbow Calibration Particle Kit (Gentaur Nederland BV, Eersel, NL, catalog number URCP-38-2K); and UUltraComp Bead Kit (BD Biosciences, catalog number 01-2222-42).

[0128] method The proposal is adapted from Wen et al. (2017). 36 In this study, additional cellular targets (B, T, Treg, and Breg) were added.

[0129] Biopsy material processing: Esophageal biopsy samples were placed in RPMI medium and processed on the same day. The biopsy tissue was incubated in a 37°C water bath for 15 minutes, followed by washing with PBS. The precipitate was resuspended in 0.5 mL RPMI containing 10% FBS, and collagenase A (Sigma / Roche) was added at a final concentration of 2 mg / mL. Enzymatic digestion was performed in a 37°C water bath for 30 minutes. Cells were washed with PBS and passed through a 30 μm cell filter. At this stage, a visible, well-defined, translucent precipitate was obtained, which was resuspended in 250 μL FACS staining buffer (PBS, 2 mM EDTA, 0.5% BSA).

[0130] Flow cytometry: The suspension was aliquoted into three tubes, two containing approximately 100 μL and one containing approximately 50 μL (unstained control). Cells were stained against granulocytes with CD45-APC, Siglec-8-PE, CD11b-PE-Cy7, CD117-BV421, and FcεRI-FITC, or against lymphocytes with CD3-BV605, CD4-PE-Cy7, CD8-FITC, CD25-PE, CD127-APC, and CD19-BV421. All antibodies were purchased from Biolegend and BD Biosciences. Cells were stained in the dark at 4°C for 30 minutes. Cells were washed and the pellet was resuspended in 200 μL of 1% formaldehyde solution. Data were acquired using an Attune NxT flow cytometer (Thermo Fisher).

[0131] Protocol for flow cytometry data analysis: FlowJo software (Treestar and Stanford University, Palo Alto, CA, USA) was used to analyze samples. Phenotypic evaluation was performed on B cells (CD3-CD19+), CD4+ T cells (CD3+CD4+CD8-), CD8+ T cells (CD3+CD8+CD4-), Tregs (CD3+CD4+CD25+), eosinophils (CD45+SIGLEC8highCD11b+), mast cells (CD45+FcεRI+SIGLEC8+CD117 high), and basophils (CD45+FcεRI+SIGLEC8+CD117 low).

[0132] Flow cytometry analysis of Breg cells: To measure Breg, human peripheral blood mononuclear cell (PBMC) samples (i.e., blood samples) were analyzed using a flow cytometer (FACSCanto II) equipped to measure BB515, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-H7, BV421, and V500.

[0133] For data acquisition, BD FACSDiva flow cytometry software was used. For data processing, DeNovo FCSExpress 6 Flow Clinical Edition was used. Cross-validation was performed on flow cytometers USFC01, USFC02, USFC03, USFC04, USFC05, and USFC06 for use in the study.

[0134] The stability of the flow cytometer is ensured by using setup beads that exhibit stable fluorescence intensity over time. The setup beads were measured each day of analysis. The BD Flow Cytometer Setup and Tracking (CS&T) beads are used in conjunction with BD FACSCanto acquisition software.

[0135] If applicable, SPHERO™ Ultra Rainbow fluorescent particles were used to calculate MESF values. The Rainbow particles contain a known amount of fluorescent molecules. Rainbow particle tubes were measured prior to sample acquisition without compensation. Rainbow particles were acquired as part of each run, and the MFI values ​​generated by flow cytometry were plotted against MESF units to generate a calibration curve for samples with fluorescence as a reportable value. The calibration curve was used to calculate the MESF of the samples via interpolation of the MFI generated by FACSCanto II.

[0136] Use BD Comp beads to determine the compensation settings. Obtain the compensation settings (percentage) before each run.

[0137] Hematology Analyzer: Before cell staining, a white blood cell count (WBC count) is performed on the Sysmex XS-1000i hematology analyzer to dilute the sample to the target concentration.

[0138] White blood cell (WBC) count: A WBC count is performed before staining the cells to adjust to 10 x 10⁻⁶. 6 Cell concentration per cell / mL. These figures are not reported but are available in the raw data.

[0139] Quality Control (QC) Samples: To validate antibody staining, one QC sample is included in each run. The QC sample consists of peripheral blood mononuclear cells (PBMCs) and is stored in liquid nitrogen. During each staining process, the QC sample is thawed and stained simultaneously with the bioanalytical sample. The QC sample is used to confirm that all antibodies are present as expected and that the study sample has been properly processed.

[0140] Statistical analysis: Data are expressed as mean ± SEM. Unpaired or paired data were used. t The results were interpreted using the GraphPad Prism 9 test. A p-value < 0.05 was considered statistically significant between the indicated groups.

[0141] result In esophageal biopsies from patients who received 4 mg or 8 mg of '1104, there was a significant decrease in eosinophils (see [link to original text]). Figure 3Mast cell reduction was observed in patients who received a 4 mg dose of '1104' (data not shown), while 8 mg '1104 treatment significantly reduced the number of CD4+ and CD8+ T cells in esophageal tissue and enhanced Treg cells (see [link to treatment]). Figure 4 ).

[0142] Regarding Breg cells, compared to EoE patients who received placebo, '1104 increased blood memory Breg cells and activated Breg cells in EoE patients who received 4 mg or 8 mg of '1104. Figure 5 ).

[0143] discuss Studies of targeted biologics have failed to show a significant improvement in dysphagia compared to placebo, even with a decrease in peak eosinophil count. For example, although Nucala (mepolilimab), Cinqair (relizumab), Fasenra (benazolizumab), Xolair (omalizumab), decrekumab, lirentelimab, and cendakimab have all been shown to reduce eosinophil counts in clinical trials in patients with end-stage regurgitation (ESR) (see [link to relevant documentation]). Figure 6 However, these biologics were not effective in treating EoE. This suggests that factors other than eosinophils are involved in esophageal remodeling and dysfunction in adult EoE.

[0144] Compared to previous targeted biologics, the data from this study suggest that Cpn60.1-related peptides such as '1104 have a broad mechanism of action in reducing eosinophils and other EoE cell types (see [link to study]). Figure 6 ).

[0145] Importantly, this study also demonstrated that Cpn60.1-related peptides, such as '1104, significantly increased Treg and Breg cells in the esophageal tissue of EoE patients. Regulatory T cells (Tregs) are a specialized subset of T cells that suppress immune responses, thereby maintaining homeostasis and self-tolerance. Treg cells have been shown to inhibit T cell proliferation and cytokine production, playing a crucial role in preventing autoimmunity. Regulatory B cells (Bregs) also suppress immune responses. Bregs help maintain tolerance, thereby limiting persistent immune responses and restoring immune homeostasis. Figure 6 As shown, previous targeted biologics have not shown an increase in Treg and Breg cells in the esophageal tissue of EoE patients.

[0146] Not limited to a specific mechanism of action, Figure 7A schematic diagram is provided illustrating the potential mechanism of action of Cpn60.1-related peptides such as '1104 in preventing inflammatory cascades such as EoE. Cpn60.1-related peptides such as '1104 have a broad mechanism of action, acting upstream by (1) binding to monocytes maturing into dendritic cells; and (2) modifying "regulatory" signals to generate activated regulatory dendritic cells, T regulatory cells, and B regulatory cells to regulate multiple cell types to prevent inflammatory cascades. Tregs and Bregs then protect the immune system from overdrive by controlling the proliferation of T effector cells. Due to the long lifespan of Tregs and Bregs, Cpn60.1-related peptides such as '1104 may provide long-term remission of EoE.

[0147] Example 3: Genomic mantle in adult EOE patients The aim of this study was to investigate changes in RNA expression of EoE-related genes in esophageal biopsy samples from patients treated with '1104, which were collected as part of the Phase 2A clinical trial described in Example 1 above.

[0148] A total of 65 samples were tested, from screening (baseline) and week 4 (see [link]). Figure 1 The sample consisted of paired biopsies collected from each patient at time V4 (hereinafter referred to as V4). The expected sample quantity was 36 pairs of biopsies, for a total of 72 samples. Reasons for missing samples included incomplete subject IDs on the samples; collection time not provided on the shipping manifest; samples being missing upon receipt; and failure to collect at the clinical site.

[0149] RNA was extracted from the samples using TRI reagents and tested on the Nanostring nCounter platform using a custom code set designed to detect a set of 110 target genes. Laboratory work and analysis were performed blinded.

[0150] Material The materials used in this study included: TRI reagent (Invitrogen, catalog number AM9738); chloroform (VWR, catalog number UN1888); 75% ethanol (VWR, catalog number UN1170); isopropanol (Acros Organics, catalog number 67-63-02100608 NA); RNase-free water (QIAGEN, catalog number 1105260); 1% Distel solution (Tristel Solutions, catalog number 5060171913825); universal human RNA (ThermoFisher, catalog number QS0639); nCounter Master kit (NanoString, catalog number NAA-AKIT-048); nCounter reagent plate (NanoString, catalog number NAA-AKIT-048); nCounter kit (NanoString, catalog number NAA-AKIT-048); nCounter reporter gene code set (NanoString, custom product); and nCounter capture code set (NanoString NA, custom product).

[0151] The equipment used in this study included: a temperature-controlled microcentrifuge (Eppendorf, model 521-1542); a microcentrifuge with an 8-row tube adapter (VWR, model 5212845); a handheld tissue homogenizer (Benchmark, model HOM3064); a NanoDrop One (Thermo Scientific, model ND-ONE-W); a thermal cycler (Bio-Rad, model T100); an nCounter preparation station (Nanostring, model 100500 prep); and an nCounter analyzer (Nanostring, model Analyser).

[0152] method Sample Collection and Transport: EoE samples for testing were collected during screening and at week 4 (V4). A total of 36 patients were treated with 4 mg IRL201104, 8 mg IRL201104, or placebo. Following endoscopy, four biopsies were collected from the area of ​​most severe esophageal inflammation in each patient. One biopsy was placed in an RNAlater, one was flash-frozen, and two were formalin-fixed and paraffin-embedded. Biopsies suspended in the RNAlater were transported on dry ice for analysis.

[0153] Samples were inspected according to the procedures outlined in the Standard Operating Procedure (SOP). Of the 72 expected samples, 65 were received, and 63 of these met the acceptance criteria. Samples were stored at -20°C until RNA extraction.

[0154] Sample processing and analysis: RNA was extracted from EoE biopsy samples using TRI reagent, and the RNA eluent was tested on a NanoDrop One instrument to determine RNA concentration and quality. The RNA eluent was then normalized to 20.0 ng / µL and tested on the Nanostring nCounter platform. RNA eluent concentrations below 20 ng / µL were tested for purity.

[0155] The nCounter code set used in this study is a set of 110 genes associated with EoE, designed to include Wen et al. (2013). 37 The text describes 96 gene characteristics in detail, as well as some housekeeping genes.

[0156] After RNA extraction, the eluent was stored on ice for all steps of the test to minimize RNA degradation, and when not actively processed, all eluent was stored in a Human Biological Sample (HBS) -80°C freezer.

[0157] Data Processing: Of the 70 samples received, 42 passed the system quality control (QC) and normalization QC in the nSolver software. Ten samples failed due to binding density QC marking, and 28 samples failed due to a large normalization factor, both of which are symptoms of insufficient RNA on the machine. Upon review of the raw counts, the six samples that failed the binding density QC marking were added back along with the samples that passed QC, bringing the total to 48.

[0158] result Cluster analysis: The expression levels of all genes in the group were visualized using a heatmap, where normalized counts were logarithmically transformed and scaled using z-scores represented on a color scale. Hierarchical clustering grouped samples based on the similarity of their expression profiles across a set of genes. Samples were divided into two main clusters with different gene expression profiles. Interestingly, the right-hand cluster (cluster 2) primarily contained the treated samples; however, no significant clustering was observed for screening or V4 visits. The genes were also divided into two distinct clusters, with most genes downregulated in cluster 2 and the remainder upregulated (data not shown). A heatmap of non-clustered gene expression was also visualized, showing changes in gene expression within and between groups, with samples separated by treatment and visits (data not shown). Principal component analysis (PCA) plots were also visualized for changes in gene expression in the treatment and visits (data not shown). No significant clustering was observed in either the treatment or visit groups.

[0159] Differential expression analysis: Differential expression analysis was performed using the 'limma' R package to compare gene expression between placebo and treated samples at screening and V4 visit. Gene expression between placebo and treated samples was also compared at V4. In any of the comparisons studied, there were no genes with significantly differentially expressed adjusted p-values ​​<0.05 and log2FC>1 or <-1.

[0160] Identifying responsive patients: As shown in the heatmap with hierarchical clustering, the samples were grouped into two distinct clusters with different gene expression profiles (data not shown).

[0161] Patients who changed cluster groups between screening and V4 visits were identified. Three patients were transferred from cluster 2 at screening to cluster 1 at V4, two of whom were placebo and one was treated. Three patients were transferred from cluster 1 at screening to cluster 2 at V4, all of whom were treated with '1104 (data not shown). Since samples from cluster 2 showed downregulation of most EoE-related genes in the group, patients in cluster 1 at screening and cluster 4 at V4 were most likely to respond to treatment. Differential expression analysis was performed only on these three potentially responding patients to investigate changes in gene expression between screening and V4. As a result, 15 significantly differentially expressed genes were identified between screening and V4 in these patients.

[0162] Compared with placebo EoE patients, the 15 genes identified as significantly differentially expressed in EoE patients treated with '1104 were: CDH26; DSG1; FLG; TNFAIP6; MMP12; POSTN; CTSC; ACTG2; ANO1; APOBEC3A; CLC; TPSB2 / AB1; CPA3; CCL26 / eosinophil chemokine 1; and CCL24 / eosinophil chemokine 2 (in Figure 8 (Genes shown in bold in the second column). Furthermore, preclinical studies have identified three genes important for the mode of action (MOA) of Cpn60.1-related peptides such as '1104: A20 / TNFAIP3; PD-L1; and CD40L. These genes are included in the genomic kit of this invention and have been found to be upregulated in EoE patients receiving '1104 treatment (see [link to genomic kit]). Figure 8 (The bottom three lines).

[0163] Differentially expressed genes can be used as biomarkers. Additional information about these differentially expressed genes is provided below. Figure 9 In this context, the term "biomarker" refers to a measurable entity of the present invention that has been identified as differentially regulated in the EoE. Biomarkers may include, but are not limited to, those shown in the embodiments and figures.

[0164] EoE score calculation: In Wen et al. (2013) 38 In this study, CT values ​​from RT-qPCR analysis of EoE-related genes were used to calculate the EoE score. In this scoring system, samples from EoE patients had lower scores than healthy patients, and these scores could be used to classify patients as either EoE or healthy.

[0165] In this study, genes were grouped into different clusters with up- or down-regulated profiles, and the EoE score calculation was adapted to provide a similar type of scoring system applicable to gene expression data. EoE scores were calculated for each sample and compared between the visit and treatment groups. In both placebo and treated samples, there was no significant difference in EoE scores at V4 compared to screening (paired t-test, placebo p = 0.6478, treated p = 0.9843). For the three responsive treated patients, post-treatment EoE scores at V4 were significantly increased (paired t-test, p = 0.0366), indicating disease improvement in these patients.

[0166] Gene expression profiling was performed on 65 samples using the Nanostring nCounter system and EoE diagnostic features. Thirteen patients had paired, QC-compliant data at both the screening and V4 time points. In these patients, differential expression analysis did not identify any genes significantly differentially expressed between screening and V4 in either placebo or '1104-treated patients. Hierarchical clustering identified three '1104-treated patients who exhibited altered gene expression between screening and V4. Differential expression analysis identified 15 genes significantly regulated between screening and V4 in three responsive patients. Changes in the expression levels of these genes were visualized using paired plots and fold change plots. An EoE score was calculated for each sample, with higher scores indicating improvement in disease status. A significant increase in EoE score was observed between screening and V4 in responsive patients, suggesting potential improvement in EoE. In conclusion, the data suggest that '1104 may effectively improve disease status in some patients.

[0167] discuss This study identified 15 genes and three genes associated with the mode of action of Cpn60.1-related peptide as significantly differentially expressed in EoE patients treated with '1104 compared to placebo-treated EoE patients. These genes can be used as biomarkers for diagnostic or therapeutic assessment purposes. Furthermore, the significant increase in EoE score observed between screening and V4 in responsive '1104-treated patients indicates an improvement in EoE in these patients.

[0168] References: All patents and other publications (including references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing methods that may be used in conjunction with the techniques described herein, such as those in such publications. These publications are provided solely for their disclosure prior to the filing date of this application. In this regard, nothing should be construed as an admission that the inventor was not entitled to prior disclosure by virtue of a prior invention or for any other reason. All statements regarding the dates or contents of these documents are based on information available to the applicant and do not constitute any admission of the accuracy of the dates or contents of these documents.

[0169] The foregoing written description is considered sufficient to enable those skilled in the art to practice various aspects and embodiments of the invention. The scope of the aspects and embodiments of the invention is not limited to the examples provided, as the examples are intended as a single illustration of one aspect, and other functionally equivalent embodiments are within the scope of this disclosure. Various modifications beyond 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 every embodiment. Those skilled in the art will recognize, or be able to determine, many equivalents of the specific embodiments described herein using only conventional experimentation. Such equivalents are intended to be covered by the following claims.

Claims

1. A method for treating eosinophilic esophagitis (EoE) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide related to chaperone protein 60.1 (Cpn60.1).

2. The method of claim 1, wherein the Cpn60.1 -related peptide is selected from the group consisting of: DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 1; '1104); DGSVVVNKVSELPAGH (SEQ ID NO: 2); GLNVNTLSYGDLAAD (SEQ ID NO: 3); SELPAGHGLNVNLTS (SEQ ID NO: 4); DGSVVVNKVS (SEQ ID NO: 5); ELPAGHGLNV (SEQ ID NO: 6); NTLSYGDLAAD (SEQ ID NO: 7); and or a functionally equivalent fragment or variant thereof.

3. The method of claim 2, wherein the Cpn60.1 -related peptide is SEQ ID NO:

1.

4. The method of any one of claims 1-3, wherein administration of the Cpn60.1 -related peptide induces a significant reduction in the severity of dysphagia in the subject.

5. The method of any one of claims 1-4, wherein administration of the Cpn60.1 -related peptide induces a significant reduction in esophageal eosinophil counts in the subject.

6. The method of any one of claims 1-5, wherein administration of the Cpn60.1 -related peptide induces a significant increase in regulatory B (Breg) cells in the subject.

7. The method of any one of claims 1-6, wherein administration of the Cpn60.1 -related peptide induces a significant increase in regulatory T (Treg) cells in the subject.

8. The method of any one of claims 1-7, wherein administration of the Cpn60.1 -related peptide induces a significant reduction in one or more leukocytes in the subject.

9. The method of claim 8, wherein the one or more leukocytes is selected from the group consisting of: mast cells, CD4+ T cells, and CD8+ T cells.

10. The method of any one of claims 1-9, wherein administration of the Cpn60.1 -related peptide is acute or short-term.

11. The method of any one of claims 1-9, wherein administration of the Cpn60.1 -related peptide is long-term.

12. A therapeutically effective amount of a peptide related to Cpn60.1 for use in a method of preventing the onset of a food allergy or food intolerance to a dietary antigen derived from food or for treating a food allergy or food intolerance to a dietary antigen derived from food in a subject in need thereof.

13. The therapeutically effective amount of a Cpn60.1 -related peptide for use according to claim 12, wherein the Cpn60.1 -related peptide is selected from the group consisting of: DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 1 ; '1104); DGSVVVNKVSELPAGH (SEQ ID NO: 2); GLNVNTLSYGDLAAD (SEQ ID NO: 3); SELPAGHGLNVNLTS (SEQ ID NO: 4); DGSVVVNKVS (SEQ ID NO: 5); ELPAGHGLNV (SEQ ID NO: 6); NTLSYGDLAAD (SEQ ID NO: 7); and or a functional equivalent fragment or variant thereof.

14. The therapeutically effective amount of a Cpn60.1 -related peptide for use according to claim 12, wherein the Cpn60.1 -related peptide is SEQ ID NO:

1.

15. The therapeutically effective amount of a Cpn60.1 -related peptide for use according to any one of claims 12-14, wherein administration of the Cpn60.1 -related peptide induces a significant reduction in the severity of dysphagia in the subject.

16. The therapeutically effective amount of a Cpn60.1 -related peptide for use according to any one of claims 12-15, wherein administration of the Cpn60.1 -related peptide induces a significant reduction in esophageal eosinophil counts in the subject.

17. The therapeutically effective amount of a Cpn60.1 -related peptide for use according to any one of claims 12-16, wherein administration of the Cpn60.1 -related peptide induces a significant increase in regulatory B (Breg) cells in the subject.

18. The therapeutically effective amount of a Cpn60.1 -related peptide for use according to any one of claims 12-17, wherein administration of the Cpn60.1 -related peptide induces a significant increase in regulatory T (Treg) cells in the subject.

19. The therapeutically effective amount of a Cpn60.1 -related peptide for use according to any one of claims 12-18, wherein administration of the Cpn60.1 -related peptide induces a significant reduction in one or more leukocytes in the subject.

20. The therapeutically effective amount of a Cpn60.1 -related peptide for use according to claim 19, wherein the one or more leukocytes is selected from the group consisting of: mast cells, CD4+ T cells, and CD8+ T cells.

21. The therapeutically effective amount of a Cpn60.1 -related peptide for use according to any one of claims 12-20, wherein administration of the Cpn60.1 -related peptide is acute or short-term.

22. The therapeutically effective amount of a Cpn60.1 -related peptide for use according to any one of claims 12-21, wherein administration of the Cpn60.1 -related peptide is long-term.

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