Methods and compositions for preventing or treating food allergy

Abstract

Described herein are methods for preventing or treating food allergy or food intolerance by administering a chaperonin 60.1 related peptide.

Description

Technical Field

[0001] Embodiments of the present invention relate to compositions and methods for preventing or treating food allergies and food intolerances using chaperone 60.1 (Cpn60.1) related peptides. Background Art

[0002] Food allergy is a costly and potentially life-threatening condition. It is a medical condition in which exposure to food triggers a harmful immune response. Food allergy is increasingly recognized as a growing public health burden and has been called the "second wave" of allergy epidemics after asthma. 1 Current evidence suggests that food allergies are common, affecting up to 10% of infants in some countries. 2 Its prevalence has been increasing over the past few decades. This increase in prevalence has preferentially affected industrialized regions, although there is now growing evidence that prevalence is also increasing in rapidly developing countries as economic growth accelerates. 3 In the United States, an estimated 32 million Americans suffer from food allergies, including 5.6 million children under the age of 18. 4，5 Along with the United States, Germany, Italy and Norway reportedly had the highest prevalence of food sensitivities — about 22% of people in each country showed antibodies to some type of food. 6

[0003] Given the dramatic increase in the prevalence of food allergies worldwide, effective prevention strategies have become a public health priority. Several models have emerged surrounding the etiology of food allergies, including the hygiene hypothesis, the dual allergen exposure hypothesis, and the vitamin D hypothesis. Other nutritional interventions, including the use of partially hydrolyzed whey-based formulas for non-breastfed infants, also play an important role. In recent years, there has been a shift from long-term avoidance of food allergens to active introduction of allergens starting at 4 months of age. However, implementation of this strategy at a population level still raises significant logistical issues, including patient selection and the development of food formats suitable for young children. The treatment of food allergies has also undergone significant changes. Concurrently, there has been a greater focus on desensitization and tolerance induction through oral and epithelial immunotherapy. In addition, hypoallergenic infant formulas specifically for the treatment of infants with cow's milk allergy have been reformulated to include the addition of lactose and probiotics to modulate the gut microbiome and early immune responses. These strategies are inadequate for a number of reasons, and strict allergen avoidance remains a key prevention and treatment principle.

[0004] Therefore, there is a need for effective compositions and methods for preventing or treating food allergies and food intolerances. Summary of the invention

[0005] Embodiments of the present invention provide a method for treating or preventing food allergies or food intolerances to food-derived food antigens in a subject by administering a chaperone 60.1 (Cpn60.1)-related peptide to a subject in need thereof. In some embodiments, the Cpn60.1-related peptide is selected from: DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 1) (PIN201104); DGSVVVNKVSELPAGH (SEQID 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 one embodiment, the Cpn60.1-related peptide is SEQ ID NO: 1.

[0006] In some embodiments, the method further comprises, prior to administration of the Cpn60.1-related peptide, diagnosing the subject as having or likely to develop a food allergy or food intolerance, or receiving an assay result diagnosing the subject as having or likely to develop a food allergy or food intolerance.

[0007] In some embodiments, the Cpn60.1-related peptide is administered prior to first exposure to a potentially pathogenic food allergen. In alternative embodiments, the Cpn60.1-related peptide is administered upon the onset of clinical signs of atopic symptoms.

[0008] In some embodiments, the Cpn60.1-related peptides are administered to subjects who have been diagnosed with at least one food allergy or food intolerance to a food antigen. In some embodiments, the food antigens are derived from milk and its products; eggs and its products; meat and its products; fish, molluscs and crustaceans and their products; oils, fats and their products; cereals and their products; beans, seeds, kernels, nuts and their products; vegetables and their products; fruits and their products; mushrooms and their products; sugars, sugar products, chocolate products and candies; and spices and herbs.

[0009] Other implementations are also described and listed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] For the purpose of illustration, certain embodiments of the invention are shown in the accompanying drawings described below. It should be understood, however, that the invention is not limited to the precise arrangements, dimensions and instruments shown. In the drawings:

[0011] Figure 1A schematic diagram showing the two phases associated with allergic inflammatory responses: the early response and the late response. Cpn60.1-related peptides have previously been shown to be highly effective in blocking inflammation associated with the late response. The effects on inflammatory cells lasted for more than 14 days (eosinophils, neutrophils, lymphocytes, and cytokines). Abbreviations: Allergen-induced early asthmatic response (EAR); Allergen-induced late asthmatic response (LAR); Forced expiratory volume in 1 second (FEV1); Inhaled corticosteroids (ICS); Short-acting inhaled beta2-agonists (SABA).

[0012] Figure 2 Shown are the study design and endpoints evaluating the effects of 1140 (80 or 160 μg / kg; three or six doses) three days after the last dose used in the ovalbumin-induced food allergy mouse model.

[0013] Figure 3 The effects of prophylactic and therapeutic administration of '1104 (80 or 160 μg / kg; three or six doses) on the severity of food allergy in an ovalbumin-induced food allergy mouse model as measured by a clinical scoring scale are shown. Data are presented as mean ± SEM; n = 8-16. Comparisons with the OVA vehicle group were performed using one-way analysis of variance (ANOVA) followed by Dunnett's test. *P < 0.05, **P < 0.01, and ***P < 0.001.

[0014] Figure 4 Shows that prophylactic and therapeutic administration of '1104 (80 or 160 μg / kg; three or six doses) on day 28 (i.e., three days after the last dose of '1104) in an ovalbumin-induced food allergy mouse model significantly reduced four indicators of food allergy: OVA-specific immunoglobulin E (IgE) (upper left panel); murine mast cell protease (mMCP-1) (upper right panel); body temperature (lower left panel); and clinical score (lower right panel). Data are expressed as mean ± SEM; n = 8-16. Comparisons with the OVA vehicle group were performed using ANOVA followed by Dunnett's test. *P < 0.05, **P < 0.01, and ***P < 0.001.

[0015] Figure 5Prophylactic and therapeutic administration of '1104 (80 or 160 μg / kg; three or six doses) in an ovalbumin-induced food allergy mouse model is shown to significantly reduce the levels of key Th2 cytokines / chemokines in serum: IL-4 (upper left panel); IL-5 (upper right panel); IL-13 (lower left panel); and eotaxin (lower right panel). Each column represents the mean, and each bar represents the standard error of the mean for n=8-16. OVA+vehicle was compared to each of the other treatment groups using ANOVA followed by Dunnett's test. *P<0.05, **P<0.01 and ***P<0.001.

[0016] Figure 6 Shown are the study design and endpoints evaluating the effects of '1104 (80 or 800 μg / kg; six doses) three and 13 days after the last dose used in the ovalbumin-induced food allergy mouse model.

[0017] Figure 7 The effect of '1104 (80 or 800 μg / kg; six doses) on the severity of food allergy in the ovalbumin-induced food allergy mouse model measured with a clinical scoring scale three days and 13 days after the last dose of '1104 is shown. Data are presented as mean ± SEM; n = 8. Comparisons with the OVA vehicle group were performed using ANOVA followed by Dunnett's test. *P < 0.05, **P < 0.01, and ***P < 0.001.

[0018] Figure 8 It is shown that '1104 (80 or 800 μg / kg; six doses) significantly reduced two indicators of food allergy: clinical symptom score (upper panel) and body temperature (lower panel) at day 28 (i.e., three days after the last dose of '1104) (left panel) and 13 days after the last dose of '1104 (right panel) in the ovalbumin-induced food allergy mouse model. Data are expressed as mean ± SEM; n = 8. Comparison with the OVA vehicle group was performed using ANOVA followed by Dunnett's test. *P < 0.05 and ***P < 0.001.

[0019] Fig. 9It is shown that '1104 (80 or 800 μg / kg; six doses) significantly reduced two indicators of food allergy: OVA-specific IgE (upper panel) and mMCP-1 (lower panel) at day 28 (i.e., three days after the last dose of '1104) (left panel) and 13 days after the last dose of '1104 (right panel) in the ovalbumin-induced food allergy mouse model. Data are expressed as mean ± SEM; n = 8. Comparison with the OVA vehicle group was performed using ANOVA followed by Dunnett's test. *P < 0.05, **P < 0.01, and ***P < 0.001.

[0020] Fig.10 Shown that '1104 (80 or 800 μg / kg; six doses) significantly reduced the levels of key Th2 cytokines / chemokines: IL-4 (upper panel) and IL-5 (lower panel) in the serum at day 28 (i.e., three days after the last dose of '1104) (left panel) and 13 days after the last dose of '1104 (right panel) in the ovalbumin-induced food allergy mouse model. Data are presented as mean ± SEM; n = 8. Comparisons with the OVA vehicle group were performed using ANOVA followed by Dunnett's test. *P < 0.05, **P < 0.01, and ***P < 0.001.

[0021] Fig.11 Shown that '1104 (80 or 800 μg / kg; six doses) significantly reduced the levels of key Th2 cytokines / chemokines in serum at day 28 (i.e., three days after the last dose of '1104) (left panel) and 13 days after the last dose of '1104 (right panel) in the ovalbumin-induced food allergy mouse model: IL-13 (upper panel) and eotaxin (lower panel). Data are presented as mean ± SEM; n = 8. Comparisons with the OVA vehicle group were performed using ANOVA followed by Dunnett's test. **P < 0.01 and ***P < 0.001.

[0022] Fig.12 Shown that '1104 (80 or 800 μg / kg; six doses) significantly reduced the levels of key cytokines / chemokines in serum at day 28 (i.e., three days after the last dose of '1104) (left panel) and 13 days after the last dose of '1104 (right panel): IL-9 (upper panel) and IL-10 (lower panel) in the ovalbumin-induced food allergy mouse model. Data are presented as mean ± SEM; n = 8. Comparisons with the OVA vehicle group were performed using ANOVA followed by Dunnett's test. **P < 0.01 and ***P < 0.001.

[0023] Fig.13 Shown that '1104 (80 or 800 μg / kg; six doses) significantly reduced the levels of key cytokines / chemokines in serum at day 28 (i.e., three days after the last dose of '1104) (left panel) and 13 days after the last dose of '1104 (right panel): IL-17 (upper panel) and CCL-17 (lower panel) in the ovalbumin-induced food allergy mouse model. Data are presented as mean ± SEM; n = 8. Comparisons with the OVA vehicle group were performed using ANOVA followed by Dunnett's test. *P < 0.05 and ***P < 0.001.

[0024] Fig.14 Shown that '1104 (80 or 800 μg / kg; six doses) significantly reduced the levels of key cytokines / chemokines in serum at day 28 (i.e., three days after the last dose of '1104) (left panel) and 13 days after the last dose of '1104 (right panel): CCL-22 (upper panel) and IFN (lower panel) in the ovalbumin-induced food allergy mouse model. Data are presented as mean ± SEM; n = 8. Comparisons with the OVA vehicle group were performed using ANOVA followed by Dunnett's test. *P < 0.05 and ***P < 0.001. DETAILED DESCRIPTION

[0025] The present invention is now described with reference to the accompanying drawings, wherein the same reference numerals are used to refer to the same elements throughout. In the following description, for the purpose of explanation, many specific details are set forth in order to provide a thorough understanding of the present invention. However, it is apparent that the present invention can be practiced without these specific details. In other cases, for ease of describing the present invention, well-known structures and devices are shown in block diagram form. It should be understood that, in order to provide a basic understanding of the present invention, certain aspects, modes, embodiments, variations and features of the present invention are described below with different levels of detail.

[0026] definition

[0027] For convenience, the meanings of some terms and phrases used in the specification, examples and appended claims are provided below. Unless otherwise specified, or implied from the context, the following terms and phrases include the meanings provided below. These definitions are provided to help describe specific embodiments and are not intended to limit the claimed invention, as the scope of the present invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which the present invention belongs. If there is a clear difference between the use of the term in the art and its definition provided herein, the definition provided in the specification shall prevail.

[0028] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, etc.

[0029] As used herein, unless otherwise specified or otherwise apparent from the context (unless such a number would be a possible value of less than 0% or more than 100%), the term "about" or "approximately" with respect to a value or parameter is generally employed to include numbers falling within 5%, 10%, 15%, or 20% ranges in either direction (greater than or less than) of that number. As used herein, reference to "about" or "approximately" a value or parameter includes (and describes) embodiments for that value or parameter. For example, a description referring to "about X" includes a description of "X".

[0030] As used herein, the term "or" means "and / or". The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0031] As used herein, the term "comprising" means that there may be additional elements in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation.

[0032] The term "consisting of refers to the compositions, methods, and respective components thereof as described herein, excluding any elements not recited in this description of the embodiment.

[0033] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel features or functional characteristics of that embodiment of the invention.

[0034] The term "statistically significant" or "significant" refers to statistical significance and generally means a difference of two standard deviations (2SD) or greater.

[0035] As used herein, the term "subject" refers to a mammal, including but not limited to dogs, cats, horses, cattle, pigs, sheep, goats, chickens, rodents, or primates. The subject can be a domestic pet (e.g., dog, cat), agricultural livestock (e.g., cattle, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), but is not limited thereto. The subject includes a human subject. The human subject can be a child, an adult, or an elderly subject. The human subject can be of either gender.

[0036] As used herein, the terms "effective amount" and "therapeutically effective amount" include an amount sufficient to prevent or ameliorate the manifestation of a disease or medical condition, such as an immune condition including eosinophilic esophagitis, hemolytic anemia, thrombocytopenia, thyroiditis, pernicious anemia, Addison's disease, disease), autoimmune diabetes, myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus, atherosclerosis and autoimmune encephalitis, allergic conditions such as eczema, dermatitis, allergic rhinitis, allergic conjunctivitis, allergic airway disease, eosinophilic syndrome, contact dermatitis, food allergies, and respiratory diseases characterized by eosinophilic airway inflammation and airway hyperresponsiveness, such as allergic asthma, intrinsic asthma, allergic bronchopulmonary aspergillosis, eosinophilic pneumonia, allergic bronchitis bronchiectasis, occupational asthma, reactive airway disease syndrome, interstitial lung disease, eosinophilic syndrome or parasitic lung disease. It should be understood that there are many known methods in the art to determine the effective amount for a given application. For example, pharmacological methods for dosage determination can be used in a therapeutic setting. In the case of therapeutic or preventive applications, the amount of the composition administered to the subject will depend on the type and severity of the disease and individual characteristics, such as general health, age, sex, body weight and tolerance to drugs. This also depends on the extent, severity and type of the disease. A skilled artisan will be able to determine the appropriate dosage based on these and other factors. These compositions can also be administered in combination with one or more additional therapeutic compounds.

[0037] As used herein, the terms "treat," "treat," "treat," "alleviate," "alleviate" and "alleviate" refer to (1) therapeutic measures that cure, slow down, alleviate the symptoms of, and / or interrupt the progression of a diagnosed disease or infection, and (2) prophylactic or preventative measures that prevent or slow the development of a disease or infection.

[0038] As used herein, the term "treatment" or "improvement" refers to the therapeutic treatment of a condition when used in reference to a disease, disorder or medical condition, wherein the purpose is to reverse, alleviate, improve, inhibit, slow down or terminate the progression or severity of a symptom or condition. The term "treatment" includes reducing or alleviating at least one harmful effect or symptom of a condition. If one or more symptoms or clinical markers are reduced, the treatment is usually "effective". Alternatively, if the progression of the condition is reduced or stopped, the treatment is "effective". That is, "treatment" includes not only the improvement of symptoms or markers, but also includes stopping or at least slowing down the progression or deterioration of symptoms expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, relief of one or more symptoms, reduction in the degree of defect, stabilization of the state of an immune disorder (i.e., no deterioration), delay or slowing of an immune disorder, and an increase in life expectancy compared to that expected in the absence of treatment.

[0039] As used herein, the term "short-term administration" or "acute administration" means that the therapeutic agent or drug is administered in one dose or daily doses for 2, 3, 4, 5, 6, 7 days or longer. "Short-term administration" can be administered as a preventive measure before potential exposure to one or more pathogenic food allergens or after possible exposure to one or more pathogenic food allergens, or as a therapeutic measure after symptoms appear after exposure to one or more pathogenic food allergens.

[0040] As used herein, the term "long-term administration" means that the therapeutic agent or drug is administered for at least 12 weeks. This includes administering the therapeutic agent or drug so that it is effective for at least 12 weeks or continues to be effective for at least 12 weeks, but does not necessarily mean that the administration itself lasts for 12 weeks, for example, if a sustained release composition or a long-acting therapeutic agent or drug is used. Thus, the subject receives treatment for at least 12 weeks. In many cases, long-term administration lasts for at least 4, 5, 6, 7, 8, 9 months or longer, or for at least 1, 2, 3, 5, 7 or 10 years or longer.

[0041] The administration of the compositions contemplated herein can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, infusion, implantation or transplantation. In a preferred embodiment, the compositions are administered parenterally. As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than intestinal and topical administration, usually administered by injection, and include but are not limited to intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node or site of infection.

[0042] The terms "reduce", "reduced", "reduce" or "inhibit" are all used herein to mean a statistically significant reduction. In some embodiments, "reduce" or "reduce" or "inhibit" generally means a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent), and can include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more. As used herein, "reduce" or "inhibit" does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. For individuals without a given disease, reduction can preferably be reduced to an acceptable level within the normal range.

[0043] The terms "increased", "increase", "enhanced" or "activity" are all used herein to mean an increase in a statistically significant amount. In some embodiments, the terms "increased", "increase", "enhancement" or "activity" may mean an increase of at least 10% compared to a reference level, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including an increase of 100% compared to a reference level or any increase between 10% and 100%, or at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times compared to a reference level, or any increase between 2 times and 10 times or greater. In the case of a marker or symptom, an "increase" is a statistically significant increase in such a level.

[0044] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to refer to a series of amino acid residues that are interconnected by peptide bonds between the α-amino and carboxyl groups of adjacent residues. The terms "protein" and "polypeptide" refer to polymers of amino acids, including modified amino acids (e.g., phosphorylation, saccharification, glycosylation, etc.) and amino acid analogs, regardless of their size or function. "Protein" and "polypeptide" are often used to refer to relatively large polypeptides, while the term "peptide" is often used to refer to small polypeptides, but the use of these terms in the art overlaps. When referring to gene products and fragments thereof, the terms "protein" and "polypeptide" are used interchangeably herein. Therefore, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologues, orthologues, paralogues, fragments and other equivalents, variants, fragments and analogs of the aforementioned substances.

[0045] In the various embodiments described herein, variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservatively substituted variants of any particular polypeptide are further contemplated. With respect to amino acid sequences, one skilled in the art will recognize that a single substitution, deletion, or addition to a nucleic acid, peptide, polypeptide, or protein sequence that changes a single amino acid or a small portion of amino acids in the encoded sequence is a "conservatively modified variant," wherein the change results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles consistent with the present disclosure.

[0046] In some embodiments, the polypeptides described herein (or nucleic acids encoding such polypeptides) may be functional fragments of one of the amino acid sequences described herein. As used herein, a "functional fragment" is a fragment or segment of a peptide that retains at least 50% of the activity of a wild-type reference polypeptide according to an assay described herein below. A functional fragment may comprise conservative substitutions of the sequences disclosed herein.

[0047] In some embodiments, the polypeptides described herein can be variants of sequences described herein. In some embodiments, the variants are conservatively modified variants. For example, conservative substitution variants can be obtained by mutations of natural nucleotide sequences. As referred to herein, "variants" are polypeptides that are substantially homologous to natural or reference polypeptides, but due to one or more deletions, insertions or substitutions, their amino acid sequences are different from the amino acid sequences of natural or reference polypeptides. The DNA sequence encoding the variant polypeptide encompasses sequences that include one or more nucleotide additions, deletions or substitutions compared to a natural or reference DNA sequence, but encodes variant proteins or fragments thereof that retain activity. A variety of PCR-based site-directed mutagenesis methods are known in the art and can be applied by ordinary technicians.

[0048] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, incorporating ribonucleic acid, deoxyribonucleic acid or its analog units. Nucleic acids can be single-stranded or double-stranded. A single-stranded nucleic acid can be a nucleic acid strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, for example, genomic DNA or cDNA. Suitable RNA can include, for example, mRNA.

[0049] In some embodiments of any aspect, the polypeptide, nucleic acid or cell described herein can be engineered. As used herein, "engineered" refers to aspects that have been artificially manipulated. For example, a polypeptide is considered "engineered" when at least one aspect of the polypeptide, such as its sequence, has been artificially manipulated to be different from the aspect when it exists in nature. As is commonly practiced and understood by those skilled in the art, the progeny of an engineered cell are still generally referred to as "engineered", even if the actual manipulation was performed on a previous entity.

[0050] In some embodiments, a nucleic acid encoding a polypeptide as described herein (e.g., an antibody or antibody reagent) is contained in a vector. In some aspects described herein, a nucleic acid sequence encoding a given polypeptide as described herein or any module thereof is operably linked to a vector. A vector may include, but is not limited to, a cloning vector, an expression vector, a plasmid, a phage, a transposon, a cosmid, a chromosome, a virus, a virion, etc.

[0051] As used herein, the term "expression vector" refers to a vector that guides RNA or polypeptides to be expressed from a sequence connected to a transcriptional regulatory sequence on a vector. The expressed sequence is often, but not necessarily, heterologous to the cell. The expression vector may contain additional elements, for example, the expression vector may have two replication systems, thereby allowing it to be maintained in two organisms, such as being maintained in human cells for expression and being maintained in a prokaryotic host for cloning and amplification. The term "expression" refers to a cell process involving the production of RNA and protein and secretion of protein when appropriate, including but not limited to, where applicable, such as transcription, transcription processing, translation and protein folding, modification and processing. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translating mRNA transcribed from a gene. The term "gene" means a nucleic acid sequence that is transcribed (DNA) into RNA in vitro or in vivo when operably linked to an appropriate regulatory sequence. The gene may or may not include regions before and after the coding region, such as a 5' untranslated region (5'UTR) or a "leader" sequence and a 3'UTR or a "trailing" sequence, as well as insertion sequences (introns) between each coding segment (exon).

[0052] In the case of nucleic acids or polypeptides, the term "isolated" or "partially purified" as used herein refers to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide), which is present together with the nucleic acid or polypeptide found in its natural source and / or when expressed by a cell, or when secreted in the case of a secretory polypeptide, it will be present together with the nucleic acid or polypeptide. Chemically synthesized nucleic acids or polypeptides or nucleic acids or polypeptides synthesized using in vitro transcription / translation are considered to be "isolated". The term "purified" or "substantially purified" refers to an isolated nucleic acid or polypeptide, which is at least 95% by weight of the subject nucleic acid or polypeptide, including, for example, at least 96%, at least 97%, at least 98%, at least 99% or more. In some embodiments, the antibodies described herein, their antigen binding portions, or chimeric antigen receptors (CARs) are isolated. In some embodiments, the antibodies, antibody reagents, their antigen binding portions, or CARs described herein are purified.

[0053] As used herein, "engineered" refers to aspects that have been manipulated artificially. For example, when the sequence of an antibody, an antibody reagent, an antigen-binding portion thereof, a CAR, or a bispecific antibody is artificially manipulated to be different from the sequence of the antibody when it exists in nature, the antibody, antibody reagent, antigen-binding portion thereof, CAR, or bispecific antibody is considered to be "engineered". As is commonly practiced and understood by those skilled in the art, the progeny of engineered polynucleotides and / or polypeptides are still generally referred to as "engineered", even if the actual manipulation is performed on a previous entity.

[0054] Pharmaceutical composition

[0055] The compositions and methods of the present invention can be used to treat individuals in need. In certain embodiments, the individual is a mammal, such as a human or non-human mammal. When applied to an animal such as a human, the composition or compound is preferably applied as a pharmaceutical composition, which includes, for example, a compound of the present application and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art, and include, for example, aqueous solutions, such as water or physiologically buffered saline or other solvents or vehicles, such as ethylene glycol, glycerol, oil (such as olive oil) or injectable organic esters. In a preferred embodiment, when such pharmaceutical compositions are used for human administration, particularly for invasive administration routes (i.e., avoiding routes of transport or diffusion through epithelial barriers, such as injection or implantation), the aqueous solution is pyrogen-free, or substantially pyrogen-free. Excipients can be selected, for example, to achieve delayed release of a medicament or selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form, such as tablets, capsules (including dispersed capsules and gelatin capsules), granules, lyophiles for reconstruction, powders, solutions, syrups, suppositories, injections, etc. The composition may also be present in a transdermal delivery system, such as a skin patch. The composition may also be present in a solution suitable for topical administration, such as a lotion, cream or ointment.

[0056] A pharmaceutically acceptable carrier may contain a physiologically acceptable agent that, for example, acts to stabilize a compound (such as a compound of the present invention), increase its solubility, or increase its absorption. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins or other stabilizers or excipients. The selection of a pharmaceutically acceptable carrier including a physiologically acceptable agent depends, for example, on the route of administration of the composition. The formulation or pharmaceutical composition may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) may also be a liposome or other polymer matrix, into which, for example, a compound of the present invention may be incorporated. For example, liposomes comprising phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively easy to prepare and administer.

[0057] The phrase "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, and with a reasonable benefit / risk ratio commensurate.

[0058] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; (4) astragalus powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution solution); (19) ethanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0059] The pharmaceutical composition (preparation) can be administered to a subject by any of a variety of routes of administration, including, for example, oral administration (e.g., drenches in the form of aqueous or non-aqueous solutions or suspensions, tablets, capsules (including dispersible capsules and gelatin capsules), pills, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (e.g., as a patch applied to the skin); and topically (e.g., as a cream, ointment or spray applied to the skin). The compound can also be formulated for inhalation. In certain embodiments, the compound can simply be dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable therefor can be found in, for example, U.S. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896 and the patents cited therein.

[0060] The formulation can be conveniently present in unit dosage form and can be prepared by any method well known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated, the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally the amount of the compound that produces the therapeutic effect. Typically, one hundred percent, the range of this amount will be about 1% to about 99% of the active ingredient, preferably about 5% to about 70%, and most preferably about 10% to about 30%.

[0061] The methods for preparing these formulations or compositions include the step of bringing the active compound (such as a compound of the present invention) into association with a carrier and optionally one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0062] Formulations of the present invention suitable for oral administration may be in the form of capsules (including dispersible capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), lyophiles, powders, granules, or as a solution or suspension in an aqueous liquid or a non-aqueous liquid, or as an oil-in-water or water-in-oil emulsion, or as an elixir or syrup, or as a pastille (using an inert base such as gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, etc., each containing a predetermined amount of the compound of the present invention as the active ingredient. The composition or compound may also be administered in the form of a bolus, electuary or paste.

[0063] To prepare solid dosage forms for oral administration (capsules (including dispersible capsules and gelatin capsules), tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders such as, for example, carboxymethyl cellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and / or gum arabic; (3) wetting agents such as Such as glycerol; (4) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retardants, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (8) absorbents, such as kaolin and bentonite; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof; (10) complexing agents, such as modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including dispersible capsules and gelatin capsules), tablets and pills, the pharmaceutical composition may also contain a buffer. Similar types of solid compositions can also be used as fillers in soft-filled and hard-filled gelatin capsules, using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols and the like.

[0064] Tablets can be made by compression or molding, optionally with one or more auxiliary ingredients. Compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), a surfactant or a dispersant. Molded tablets can be prepared by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine.

[0065] Tablets and other solid dosage forms of pharmaceutical compositions, such as dragees, capsules (including dispersible capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the art of pharmaceutical formulation. They may also be formulated to provide sustained or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose, other polymer matrices, liposomes and / or microspheres in different proportions to provide the desired release characteristics. They may be sterilized, for example, by filtering through a bacterial-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifiers and may be such compositions that they release the active ingredient only or preferentially in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, if appropriate, containing one or more of the above-mentioned excipients.

[0066] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art (such as water or other solvents), cyclodextrins and their derivatives, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuran methanol, polyethylene glycol and sorbitan fatty acid esters, and mixtures thereof.

[0067] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0068] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0069] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and with any preservatives, buffers, or propellants that may be required.

[0070] Ointments, pastes, creams and gels may contain, in addition to the active compounds, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0071] In addition to the active compound, powders and sprays may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays may additionally contain conventional propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

[0072] Transdermal patches have the added advantage of providing controlled delivery of the compound of the present invention to the body. Such dosage forms can be prepared by dissolving the active compound in an appropriate medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. The rate of this flux can be controlled by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0073] The phrases "parenteral administration" and "administered parenterally" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intraocular (such as intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration may comprise one or more active compounds in combination with one or more pharmaceutically acceptable agents: sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions just before use, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0074] The example of suitable aqueous and non-aqueous carrier that can be used in the pharmaceutical composition of the present invention includes water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters, such as ethyl oleate. The example of suitable aqueous and non-aqueous carrier that can be used in the pharmaceutical composition of the present invention includes water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters, such as ethyl oleate. Suitable fluidity can be maintained, for example, by using a coating material such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using a surfactant. Suitable fluidity can be maintained, for example, by using a coating material such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using a surfactant.

[0075] These compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. It is possible to ensure that the effects of microorganisms are prevented by including various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, sorbic acid phenol, etc.). It is also desirable to include isotonic agents, such as sugar, sodium chloride, etc. in the compositions. In addition, the absorption extension of injectable drug forms can be achieved by including agents that delay absorption (such as aluminum monostearate and gelatin).

[0076] In some cases, in order to prolong the effect of the drug, it is desirable to slow down the absorption of the subcutaneous or intramuscular injection of the drug. This can be achieved by using a liquid suspension of a crystalline or amorphous substance with low water solubility. The absorption rate of the drug then depends on its dissolution rate, which in turn can depend on crystal size and crystalline form. Alternatively, the absorption of a parenterally administered drug form can be delayed by dissolving or suspending the drug in an oily vehicle.

[0077] Injectable depot forms are prepared by forming a microcapsule matrix of the subject compound in a biodegradable polymer (such as polylactide-polyglycolide). According to the ratio of the drug to the polymer and the properties of the specific polymer adopted, the drug release rate can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot-type injectable formulations are also prepared by embedding the drug in a liposome or microemulsion compatible with human tissue.

[0078] For use in the methods of the invention, the active compound may be administered per se or as a pharmaceutical composition containing, for example, 0.1% to 99.5% (more preferably 0.5% to 90%) active ingredient in combination with a pharmaceutically acceptable carrier.

[0079] The method of introduction can also be provided by a rechargeable or biodegradable device. In recent years, various slow-release polymer devices have been developed and tested in vivo to control the delivery of drugs, including protein biopharmaceuticals. A variety of biocompatible polymers, including hydrogels, including biodegradable and non-degradable polymers, can be used to form implants to continuously release compounds at specific target sites.

[0080] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0081] The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the esters, salts or amides thereof, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0082] A physician or veterinarian with ordinary skills in the art can easily determine and prescribe the therapeutically effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian can start the dosage of the pharmaceutical composition or compound at a dosage lower than the level required to obtain the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. "Therapeutically effective amount" means the concentration of the compound sufficient to cause the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the subject's weight, sex, age and medical history. Other factors affecting the effective amount may include, but are not limited to, the severity of the patient's condition, the condition being treated, the stability of the compound, and, when necessary, another type of therapeutic agent administered together with the compound of the present invention. A larger total dose can be delivered by multiple administrations of the agent. Methods for determining efficacy and dosage are known to those skilled in the art. See, for example, Isselbacher et al. (1996). 7

[0083] In general, a suitable daily dose of the active compound used in the compositions and methods of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above.

[0084] If desired, the effective daily dose of the active compound can be administered at appropriate intervals throughout the day as 1, 2, 3, 4, 5, 6 or more sub-doses administered separately, optionally in unit dosage form. In certain embodiments of the invention, the active compound can be administered twice or three times a day. In other embodiments, the active compound will be administered once a day.

[0085] The patient receiving such treatment is any animal in need thereof, including primates, particularly humans; and other mammals, such as horses, cattle, pigs, sheep, cats, and dogs; poultry; and pets in general.

[0086] In certain embodiments, the compounds of the invention may be used alone or in combination with another type of therapeutic agent.

[0087] The disclosure includes the pharmaceutically acceptable salt of the compound of the present invention for use in the compositions and methods of the present application. In certain embodiments, the salt of the present invention considered includes but is not limited to alkyl, dialkyl, trialkyl or tetraalkyl ammonium salts. In certain embodiments, the salt of the present invention considered includes but is not limited to, L-arginine, benthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino) ethanol, ethanolamine, ethylenediamine, N-methylglucosamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl) morpholine, piperazine, potassium, 1-(2-hydroxyethyl) pyrrolidine, sodium, triethanolamine, tromethamine and zinc salts. In certain embodiments, the salt of the present invention considered includes but is not limited to Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptanoic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenate.

[0088] Pharmaceutically acceptable acid addition salts may also exist in the form of various solvates, such as solvates with water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates may also be prepared. The source of such solvates may be derived from the crystallization solvent, inherent in the preparation or crystallization solvent, or external to such solvent.

[0089] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0090] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0091] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meaning commonly understood by one of ordinary skill in the art to which the present disclosure belongs. It should be understood that the present invention is not limited to the specific methodology, protocols, reagents, etc. described herein and may vary accordingly. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is limited only by the claims. Definitions of commonly used terms in immunology and molecular biology can be found in the following references: The Merck Manual of Diagnosis and Therapy; 8 TheEncyclopedia of Molecular Cell Biology and Molecular Medicine; 9 Molecular Biology and Biotechnology: a Comprehensive Desk Reference; 10 Immunology; 11 Janeway’s Immunobiology; 12 Lewin's Genes XI; 13 Molecular Cloning:A LaboratoryManual.; 14 Basic Methods in Molecular Biology; 15Laboratory Methods in Enzymology; 16 Current Protocols in Molecular Biology(CPMB); 17 Current Protocols in Protein Science (CPPS); 18 and Current Protocols in Immunology (CPI). 19

[0093] In some embodiments of any aspect, the disclosure described herein does not relate to methods for cloning humans, methods for altering the germline genetic characteristics of humans, the use of human embryos for industrial or commercial purposes, or methods for altering the genetic characteristics of animals, and animals produced by such methods, which methods may cause suffering to humans or animals without any substantial medical benefit to humans or animals.

[0094] Additional terms are defined herein in the description of various aspects of the invention.

[0095] Food Allergies

[0096] Allergy is an immune disorder in which an individual develops an immune response to substances that are usually harmless in themselves (called allergens), resulting in a hypersensitivity reaction. The main antibody involved in allergic reactions is immunoglobulin E (IgE). IgE plays an important role in type I hypersensitivity reactions, 20 Type I hypersensitivity reactions manifest in a variety of allergic diseases, such as allergic asthma, most types of sinusitis, allergic rhinitis, food allergies, and certain types of chronic urticaria and atopic dermatitis. IgE also plays a key role in reactions to allergens, such as anaphylactic reactions to drugs, bee stings, and antigen preparations used in desensitization immunotherapy.

[0097] The constant region (Fc region) of IgE is able to bind to specific receptors on cells, which can release histamine or other inflammatory mediators, cytokines and / or proteases to surrounding tissues. Histamine-releasing cells are mainly mast cells and basophils. The release of histamine begins when cell-bound IgE comes into contact with allergens and cross-links. Histamine is mainly stored in mast cells and basophils and is the main cause of allergic diseases. It has been recorded that during allergic reactions and experimental allergic reactions in the skin, nasal cavity and airways, plasma or tissue histamine levels increase. For example, histamine released from the nasal cavity, eyes and sinuses can stimulate sneezing, runny nose and itchy eyes; histamine released from the lungs can cause narrowing and swelling of the airway lining and secretion of thick mucus; histamine released from the skin can cause rashes and hives; and histamine released from the digestive system can cause stomach cramps and diarrhea. Typical allergens come from plant pollens (e.g., ryegrass, ragweed, timothy grass, birch pollen), mold spores, drugs (e.g., penicillin, sulfonamides, salicylates, and local anesthetics), foods (e.g., nuts, seafood, eggs, peas, beans, peanuts and other legumes, milk), insect products (e.g., bee stings, wasp stings, cockroach mycorrhiza, dust mites), and animal hair and dander.

[0098] A food allergy is an abnormal immune response to a food. Its signs and symptoms can range from mild to severe and can include itching, swelling of the tongue, vomiting, diarrhea, hives, difficulty breathing or low blood pressure, and in severe cases, anaphylaxis. This usually occurs within minutes to hours of exposure. Although sensitivity levels vary from country to country, the most common food allergies are to milk, eggs, peanuts, tree nuts, seafood, shellfish, soy and wheat. One of the most common food allergies is to peanuts, a member of the legume family. Peanut allergies can be serious, but children with peanut allergies sometimes outgrow the allergy as they grow older. Tree nuts, including cashews, Brazil nuts, hazelnuts, macadamia nuts, pecans, pistachios, pine nuts, coconut and walnuts, are also common allergens. Sufferers may be sensitive to a specific tree nut or to a variety of different tree nuts. In addition, seeds, including sesame seeds and poppy seeds, contain oils that contain proteins that may cause allergic reactions.

[0099] Diagnosis is usually based on medical history, elimination diets, skin prick testing, blood tests for food-specific IgE antibodies, or oral food challenges. For skin prick testing, a small plate with a protruding needle is used. The allergen is placed on the plate or directly on the skin. The plate is then placed on the skin, pricking the skin and allowing the allergen to enter the body. If hives appear, the person is considered positive for an allergy. This test is only for IgE antibodies. Allergic reactions caused by other antibodies cannot be detected by skin prick testing. Patch testing is used to determine if a specific substance causes allergic inflammation of the skin. It can test for delayed food reactions. A blood test is another way to test for allergies; however, it has the same disadvantages and can only test for IgE allergens, not all possible allergens. A food challenge tests for allergens other than those caused by IgE allergens. The allergen is given to the person in pill form so the person can directly ingest the allergen. The person is observed for signs and symptoms. The problem with a food challenge is that it must be done in a hospital under close supervision because of the possibility of anaphylaxis. For tests involving the subject's own reactions, it is not possible to administer multiple different tests to a subject in a short period of time. In addition, these types of tests are expensive and invasive. Other diagnostic tools to evaluate eosinophil or non-IgE antibody-mediated reactions include endoscopy, colonoscopy, and biopsy.

[0100] Chaperone 60.1 related peptide

[0101] Before describing the compositions and methods of the present invention, it should be understood that the present invention is not limited to the specific compositions, methods and experimental conditions described, as such compositions, methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, as the scope of the present invention is limited only by the appended claims.

[0102] Compositions useful in the methods of the present invention include, but are not limited to:

[0103] DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD(SEQ ID NO:1)(PIN201104);

[0104] DGSVVVNKVSELPAGH(SEQ ID NO:2);GLNVNTLSYGDLAAD(SEQ ID NO:3);

[0105] SELPAGHGLNVNLTS(SEQ ID NO:4); DGSVVVNKVS(SEQ ID NO:5);

[0106] ELPAGHGLNV(SEQ ID NO:6);NTLSYGDLAAD(SEQ ID NO:7);

[0107] or functionally equivalent fragments or variants thereof.

[0108] Other compositions that can be used in the methods of the present invention include, but are not limited to, U.S. Published Patent Application No. 20040132163 21 and U.S. Patent 11,098,090 22 ; 9,320,791 23 ; 9,085,632 24 Cpn60.1-related peptides disclosed in.

[0109] In some aspects, the Cpn60.1 related peptides are composed 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 agonist or ligand of the present invention is composed of amino acids with the standard structure NH2-C(H)(R)-COOH, where R represents a single amino acid side chain. In some aspects, the agonist or ligand is composed of amino acid residues that are naturally occurring amino acids. In some aspects, naturally occurring amino acids include one of the twenty standard amino acids present in naturally occurring peptides and proteins. In some such aspects, the agonist or ligand consists of at least one naturally occurring amino acid residue that 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").

[0110] In other aspects, the Cpn60.1-related peptide used in the method of the present invention is composed of at least one amino acid residue that is a non-natural or synthetic amino acid. In some such aspects, the non-natural or synthetic amino group is a chemically modified amino acid, and the chemically modified amino acid includes but is not limited to amino acids modified by methylation, amidation, acetylation, protective groups, and / or replaced with other chemical groups that can change the physicochemical properties of the peptide. In other aspects, the non-natural or synthetic amino group is a chemically modified amino acid modified with one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.).

[0111] 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 (chirality of L-amino acids). 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 (chirality of D-amino acids).

[0112] In certain aspects, the agonists or ligands of the invention are about 50aa in length; 49aa in length; 48aa in length; 47aa in length; 46aa in length; 45aa in length; 44aa in length; 43aa in length; 42aa in length; 41aa in length; 40aa in length; 39aa in length; 39aa in length; 38aa in length; 37aa in length; 36aa in length; 35aa in length; 34aa in length; 33aa in length; 32aa in length; 31aa in length; 30aa in length; The length is 29aa; the length is 28aa; the length is 27aa; the length is 26aa; the length is 25aa; the length is 24aa; the length is 23aa; the length is 22aa; the length is 21aa; or the length is 20aa; the length is 19aa; the length is 18aa; the length is 17aa; the length is 16aa; the length is 15aa; the length is 14aa; the length is 13aa; the length is 12aa; the length is 11aa; the length is 10aa; the length is 9aa; the length is 8aa; the length is 7aa; the length is 6aa; or the length is 5aa.

[0113] In certain aspects, the compositions of the invention are administered to a patient by any suitable route known and / or employed by those skilled in the art. In some aspects, the compositions of the invention are administered orally (PO), intravenously (IV), intramuscularly (IM), intraarterially, intramedullary, intrathecally, subcutaneously (SQ), intraventricularly, transdermally, interdermally, intradermally, rectally (PR), vaginally, intraperitoneally (IP), intragastrically (IG), topically (e.g., by powders, ointments, creams, gels, lotions, and / or drops), mucosally, intranasally, buccally, intestinally, intravitreally, sublingually, by intratracheal instillation, bronchial instillation, and / or inhalation, as an oral spray, nasal spray, aerosol, and / or by a portal vein catheter.

[0114] In certain aspects, the Cpn60.1-related peptides and / or pharmaceutical compositions thereof used in the methods of the present invention can be administered intravenously, for example, by intravenous infusion. In other aspects, the Cpn60.1-related peptides and / or pharmaceutical compositions thereof used in the methods of the present invention can be administered by intramuscular injection. In more aspects, the Cpn60.1-related peptides and / or pharmaceutical compositions thereof used in the methods of the present invention can be administered by intratumoral injection. In certain aspects, the Cpn60.1-related peptides and / or pharmaceutical compositions thereof used in the methods of the present invention can be administered by subcutaneous injection. In other aspects, the Cpn60.1-related peptides and / or pharmaceutical compositions thereof used in the methods of the present invention can be administered by a portal vein catheter. In more aspects, the present invention encompasses the delivery of the Cpn60.1-related peptides and / or pharmaceutical compositions thereof used in the methods of the present invention by any appropriate route, taking into account possible advances in the field of drug delivery.

[0115] In some aspects, the Cpn60.1 related peptides and / or pharmaceutical compositions thereof used in the methods of the present invention can be administered at a dosage level sufficient to deliver about 0.001 mg / kg to 100 mg / kg, about 0.01 mg / kg to 50 mg / kg, about 0.1 mg / kg to 40 mg / kg, about 0.5 mg / kg to 30 mg / kg, about 0.01 mg / kg to 10 mg / kg, about 0.1 mg / kg to 10 mg / kg, or about 1 mg / kg to 25 mg / kg of patient weight / day to obtain the desired therapeutic effect. In some aspects, the delivery of the desired dosage can be more than three times a day, three times a day, twice a day, once a day, once every other day, once every two 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 aspects, the desired dose can be delivered by multiple administrations (e.g., twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times or more administrations). In some aspects, the desired dose can be delivered by one or more administrations during an initial time period, and then no dose is administered for a period of time.

[0116] In other aspects, the Cpn60.1-related peptides used in the methods of the present invention can be used for prophylactic administration. In more aspects, prophylactic administration relates to systems and methods for preventing, inhibiting the progression of food allergies and / or delaying the appearance of food allergies in individuals susceptible to food allergies and / or showing symptoms of food allergies.

[0117] In some aspects, the Cpn60.1-related peptides used in the methods of the present invention are administered to target cells in vivo. In other aspects, the Cpn60.1-related peptides used in the methods of the present invention are administered to target cells in vitro. In other aspects, the Cpn60.1-related peptides used in the methods of the present invention are administered to target cells in vitro, and then the target cells are reintroduced into the organism. In some such aspects, the target cells are cultured in vitro into a plurality of daughter cells, and then the daughter cells are reintroduced into the organism. In more aspects, the organism is a human. In other aspects, the organism is a human patient. In some aspects, the target cell originally originates from the organism into which it is reintroduced. In other aspects, the target cell originally originates from a different organism into which it is reintroduced.

[0118] In certain aspects, the Cpn60.1-related peptides and / or pharmaceutical compositions thereof used in the methods of the present invention are used in combination therapy to treat or reduce the risk of food allergies. In this regard, administration can be combined with one or more additional therapeutic agents. As used herein, the phrases "combination therapy," "in combination with," "combination," and the like refer to the use of more than one drug or treatment at the same time to increase a response. In certain aspects, the Cpn60.1-related peptides and / or pharmaceutical compositions thereof used in the methods of the present invention are administered simultaneously with, before, or after one or more other desired treatments or medical procedures. In certain aspects, the Cpn60.1-related peptides and / or pharmaceutical compositions thereof used in the methods of the present invention are administered in combination in a single composition or separately in different compositions.

[0119] In certain aspects, the specific combination of therapies employed in the combination regimen will generally take into account the compatibility of the desired treatment and / or procedure, as well as the desired therapeutic effect to be achieved. In other aspects, the therapies employed may achieve the desired effect for the same purpose (e.g., the Cpn60.1-related peptides used in the methods of the present invention that can be used to treat, prevent and / or delay the emergence of food allergies may be administered simultaneously with another therapeutic agent that can also be used to treat, prevent and / or delay the emergence of food allergies), or they may achieve different effects. In other aspects, the combination of therapies employed may achieve the same or substantially similar desired effect for the same disease, condition or illness; the same or substantially similar desired effect may be achieved for one or more different diseases, conditions or illnesses; different desired effects may be achieved for the same disease, condition or illness; or different expected effects may be achieved for one or more different diseases, conditions or illnesses.

[0120] In another aspect, the Cpn60.1-related peptides used in the methods of the present invention are delivered as a pharmaceutical composition in combination with one or more additional components that can improve the bioavailability of the Cpn60.1-related peptides used in the methods of the present invention, reduce and / or alter their metabolism, inhibit their excretion, and / or alter their distribution in the body.

[0121] In certain aspects, the combination therapy may involve the administration of multiple Cpn60.1-related peptides according to the present invention. In other aspects, the combination therapy may involve the administration of multiple Cpn60.1-related peptides that treat, prevent, improve, achieve relief of food allergies and / or reduce the risk of food allergies. In more aspects, the combination therapy may be a variety of Cpn60.1-related peptides that treat, prevent, improve, achieve relief of multiple food allergies and / or reduce the risk of multiple food allergies.

[0122] In certain aspects, the Cpn60.1-related peptide used in the method of the present invention is combined with at least one pharmaceutically acceptable excipient to form a pharmaceutical composition. As used herein, "pharmaceutical composition" refers to a preparation containing an active ingredient, and optionally a pharmaceutically acceptable carrier, diluent or excipient. The term "active ingredient" can refer interchangeably to "effective ingredient" and refers to any agent that can cause a desired effect after administration. Examples of active ingredients include, but are not limited to, compounds, drugs, therapeutic agents, small molecules, and the like.

[0123] In certain aspects of the invention, the active ingredient is a Cpn60.1 related peptide as disclosed herein. In specific aspects, the active ingredient is PIN201104, or a derivative thereof. In certain aspects, the active ingredient is PIN201360, PIN201361, PIN201362, PIN201116, PIN201105, or a derivative thereof. In other aspects, the active ingredient is a peptide described in WO2009 / 106819, or a derivative thereof.

[0124] In some aspects, the pharmaceutical composition can be used in medicine or drug manufacturing. In other aspects, the pharmaceutical composition can be used in one or more of the therapeutic administrations disclosed herein, for example, can be used for individuals suffering from autoimmune diseases. In other aspects, the pharmaceutical composition is formulated for administration to human patients.

[0125] In some aspects, the pharmaceutical composition is a sterile injection form (e.g., a form suitable for subcutaneous injection or intravenous infusion). In more aspects, the pharmaceutical composition is a liquid dosage form suitable for injection. In other aspects, the pharmaceutical composition is in a powder (e.g., freeze-dried and / or sterilized powder) form, optionally in a powder form under vacuum, and the powder is reconstituted with an aqueous diluent (e.g., water; buffer; saline solution, etc.) before injection. In 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 a form that can be refrigerated and / or frozen. In other aspects, the pharmaceutical composition is a form that cannot be refrigerated and / or frozen. In some aspects, the pharmaceutical composition is a reconstituted solution and / or liquid dosage form, and the reconstituted solution and / or liquid dosage form can be stored for a period of time (e.g., 2 hours, 12 hours, 24 hours, 2 days, 5 days, 7 days, 10 days, 2 weeks, one month, two months or longer) after reconstitution.

[0126] In certain aspects, the method for preparing the pharmaceutical composition comprises combining the active ingredient (e.g., the Cpn60.1-related peptide used in the method of the present invention) with one or more pharmaceutically acceptable excipients, and then molding and / or packaging the product into the desired single-dose or multi-dose units. The pharmaceutical composition according to the present invention can be prepared, packaged in bulk, packaged into a single unit dose, and / or packaged into multiple single unit doses. As used herein, "unit dose" refers to a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dose administered to the subject and / or a convenient fraction of such a dose, for example, half or one-third of such a dose. The relative amounts of the active ingredient, pharmaceutically acceptable excipients and / or any additional ingredients in the pharmaceutical composition according to the present invention may vary according to the identity, size and / or condition of the subject being treated and / or according to the route of administration of the composition. In certain aspects, for example, the composition may contain about 0.1% to 100% (w / w) of the active ingredient.

[0127] In another aspect, the present invention includes a kit that can be used to perform the method of the present invention. The components included in the kit depend on multiple factors, including the specific administration (e.g., the specific administration route adopted or the specific disease, condition or illness treated). In certain aspects, the present invention provides a kit for administering a Cpn60.1-related peptide according to the present invention to treat a disease, condition or illness disclosed herein. In some such aspects, the kit also includes instructions for administration. In certain aspects, the kit is used to administer Cpn60.1-related peptides to treat patients with food allergies. In certain aspects, the kit contains one or more Cpn60.1-related peptides. In certain aspects, the kit includes a pharmaceutical composition containing a certain number of unit doses of Cpn60.1-related peptides. In other aspects, the kit used according to the present invention includes instructions (e.g., for administration, storage, etc.), buffers and / or other reagents. In some such aspects, the kit includes (i) at least one Cpn60.1-related peptide, (ii) a syringe, a needle, an applicator, etc., for administering at least one Cpn60.1-related peptide to a patient, and (iii) instructions for use. In other aspects, the test kit includes a treatment schedule for specifying when to use a unit dose. In more aspects, a placebo dose is included, the form of the placebo is similar to or different from the dosage of the pharmaceutical composition. In some aspects, the test kit includes one or more containers, so that some independent components or reagents can be accommodated separately. In some aspects, the test kit can include a device for packaging a single container in a relatively closed space for commercial sale, for example, a plastic box, wherein instructions, packaging materials such as Styrofoam (Styrofoam) or the like can be packaged.

[0128] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise form disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure as will be appreciated by those skilled in the relevant art. For example, although the method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially simultaneously. The teachings of the disclosure provided herein may be applied to other procedures or methods as appropriate. The various embodiments described herein may be combined to provide further embodiments. If necessary, aspects of the present disclosure may be modified to provide further embodiments of the present disclosure using the compositions, functions and concepts of the above-mentioned references and applications. Moreover, due to considerations of the equivalence of biological functions, some changes may be made in the protein structure without affecting the biological or chemical effects in kind and amount. According to the detailed description, these and other changes may be made to the present disclosure. It is intended that all such modifications will be included within the scope of the appended claims.

[0129] The specific elements of any of the foregoing embodiments may be combined or substituted for the elements in other embodiments. In addition, although the advantages associated with certain embodiments of the present disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily exhibit such advantages to fall within the scope of the present disclosure.

[0130] The techniques described herein are further illustrated by the following examples, which should in no way be construed as further limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below.

[0131] Example

[0132] The invention, as generally described, will now be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to be limiting of the invention.

[0133] Example 1 Effectiveness of CPN60.1-related peptides in an animal model of ovalbumin-induced food allergy

[0134] It has been demonstrated that chaperone 60.1 (Cpn60.1) related peptides, such as PIN201104 ("'1104"), are very effective in blocking inflammation associated with the late phase response. See, Figure 1. However, previous work has not investigated the effects of Cpn60.1-related peptides on early responses, which are characterized by acute allergic reactions in which mast cells and basophils play an important role. For some indications, such as food allergy, early therapeutic effectiveness is critical. This study evaluated whether 1104 has an effect on early responses in addition to its known effects on late responses. A mouse model of food allergy has been developed that exhibits progressive and severe ovalbumin (OVA)-induced anaphylaxis.

[0135] Allergens, whether from food, air pollution, dust mites, pet dander, or mold, can induce hypersensitivity reactions mediated by IgE antibodies. The consequence of allergic reactions is an asthmatic response characterized by airflow obstruction and bronchospasm. In allergic reactions, helper T cells induce the expression of multiple cytokines, including TNF-α, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-13 (1-4). Of these cytokines, IL-4 and IL-13 play key roles in allergic reactions. IL-13 has a unique role in mucus production, while IL-4 has been found to be the major driver of IgE and IgG1 synthesis by B cells. In addition, IL-5 leads to blood and tissue eosinophilia, which is a classic feature of allergic reactions. 25，26

[0136] Ovalbumin (OVA) and house dust mite (HDM) are antigens widely used to induce allergic reactions. 27，28，29，30 For example, both OVA-induced asthma model and HDM-induced asthma model can induce airway inflammation, leading to airflow obstruction and airway remodeling (AR).

[0137] Many chemokines have been implicated in allergic reactions, including CCL11, CCL17, and CCL22, all of which have been found to be upregulated in bronchoalveolar lavage fluid from patients with asthma. In murine models, inhibition of CCL11 has been shown to reduce eosinophil recruitment and reduce airway hyperresponsiveness. In fact, plasma levels of CCL11 were higher in samples from patients with asthma than in healthy volunteers. Chemokines CCL17 and CCL22 bind to CCR4, which is primarily expressed by Th2 cells and plays an important role in T cell recruitment in mice and humans. CCL17 and CCL22 have been implicated in a variety of allergic reactions, including skin allergies and food allergies. In addition, Th1 / Th2 cytokines IL-4, IL-5, IL-13, and INF-γ have been reported to be upregulated. 31

[0138] Food Allergy Mouse Model

[0139] In this study, a mouse model of food allergy that exhibits a progressive and severe anaphylactic response to ovalbumin (OVA) has been developed. Figure 2 are shown and summarized in Table 1.

[0140] Table 1 – Food allergy models

[0141]

[0142] The clinical scale used for scoring of allergic reactions is provided in Table 2.

[0143] Table 2 – Clinical Rating Scale*

[0144] Rating Scale

[0145] 0 No symptoms

[0146] 1. Scratching and rubbing around the nose and head

[0147] 2 Puffiness around the eyes and mouth, diarrhea, piloerection, decreased activity, and / or decreased activity with increased respiratory rate

[0148] 3. Wheezing, dyspnea, perioral and caudal cyanosis

[0149] 4 No movement or tremor or spasm after the sting

[0150] 5 Death

[0151] *This clinical rating scale is a validated allergic reaction scale commonly used to assess symptom severity in murine food allergy studies. 32，33

[0152] Animals: Male BalbC mice (weighing 20-30 g when received, Charles Rivers, UK) were used in this study.

[0153] Animal Welfare: After receiving the animals from the supplier, the animals were acclimated for 7 days before the experimental procedures. Upon receipt, the mice were housed in four cages according to body weight (animal weight was evenly distributed to each cage by the animal technician) with a 12-h light and dark cycle. Room temperature and humidity were maintained between 17-24°C and 40%-70%, respectively. Environmental enrichment was provided in all cages. The mice had free access to standard chow and free access to water from bottles.

[0154] Animals were housed in four cages as outlined below, and the health of all animals was checked daily. Throughout the study, the following guidelines were used to evaluate controlled animals for nonspecific or unexpected adverse reactions involving procedures or test compound administration.

[0155] Weight loss greater than 20% of the individual's highest body weight measurement.

[0156] Pronounced piloerection and other signs of dehydration, such as doming of the skin.

[0157] ·Unresponsive to activity and provocation.

[0158] ·Persistent hunching (stiffness).

[0159] Pain – Continuous sounding.

[0160] ·Continuous and heavy discharge from the eyes and nose.

[0161] Difficulty breathing.

[0162] Persistent tremors.

[0163] ·Continued spasms.

[0164] Animals showing two or more of any limiting clinical signs in categories comparable to the protocol severity limits will be removed from the study and sacrificed by the methods of Schedule 1 (cervical dislocation). When animals reach the limit of either or both of the first two signs, regardless of any additional signs, they will be removed from the study and sacrificed at the institution by the methods of Schedule 1. However, no animals were removed during this study.

[0165] OVA sensitization and challenge: On day 0, mice were sensitized intraperitoneally with 25 μL of 100 μg ovalbumin adsorbed on 1 mg aluminum hydroxide adjuvant (Imject Alum-aluminum content 40 mg / mL). Mice were then challenged with 5 mg OVA in 200 μL or 200 μL PBS by oral gavage on days 14, 16, 18, 21, 23, 25 and 28 (Groups 1-5).

[0166] Body temperature and allergic reaction assessment: After each challenge, animals were monitored for 60 min and rectal temperature was recorded at 0, 15, 30 and 60 min after oral gavage of PBS or OVA. Each mouse was assigned a score of 0-5 according to the symptoms of allergic reactions. The clinical scale for allergic reaction scoring is provided in Table 2 above.

[0167] Animal treatment with '1104 or vehicle: Mice were weighed and placed in a hot box for 5 minutes, then placed in a full body restraint and received intravenous administration (5 mL / kg) of '1104 (80 μg / kg or 800 μg / kg) or vehicle via the tail vein 15 minutes prior to OVA challenge on days 14, 16, 18, 21, 23, and 25.

[0168] Blood sample collection: One hour after the last OVA challenge on day 28 or day 38, terminal blood samples were collected by cardiac puncture and placed in serum tubes. Each serum sample was kept at room temperature for 45 minutes to allow the serum sample to clot, and then centrifuged (2000×g, 15 minutes, at 4°C), and the resulting supernatant was extracted from the centrifuged sample, and the supernatant was divided into aliquots and stored at -80°C for analysis.

[0169] Tissue collection: Immediately after the terminal blood sample was collected, the animals were euthanized with an overdose of pentobarbital. The abdomen was opened and the small intestine was dissected from each animal and rinsed with PBS. The tissue was then divided into the duodenum, jejunum, and ileum. Each segment was cut longitudinally and rolled on a wooden stick to form a roll. Each tissue roll was then sectioned and one section was placed in 10% formalin for 48 hours and then transferred to 70% ethanol for future histopathological examination. The second section was placed in a sterile Eppendorf tube, quickly frozen, and then stored at -80°C for future biomarker analysis.

[0170] After harvesting the small intestine, mesenteric lymph nodes were dissected from each animal. Following dissection, the lymph nodes were placed in sterile Eppendorf tubes, snap frozen, and then stored at -80°C for future analysis.

[0171] OVA-specific IgE and mMCP1 ELISA assay: According to the manufacturer's instructions, ELISA kits (from AssayGenie and Invitrogen, respectively) were used to evaluate the OVA-specific IgE and mMCP1 concentrations of serum supernatants. A microplate reader (SpectraMax 340PC) was used to measure the optical density at 450 nM. SoftMax Pro v.6.4 (Molecular Devices) was used to determine the concentration of IgE. Data were reported as mean ± SEM (standard error of the mean) of OVA-specific IgE (pg / mL) or mMCP1 concentration (pg / mL).

[0172] Cytokine / Chemokine Assay: Serum samples (all groups) were measured for cytokine / chemokine concentrations using a magnetic multiplex assay (see below for details on biomarkers to be assessed) according to the manufacturer's instructions. Levels were measured using the Magpix system (Luminex Corp.).

[0173] A microplate reader (SpectraMax 340PC) was used to measure the optical density at 450 nM. Mouse cytokines / chemokines magnetic multiplex (Biotechne): IL-4, IL-5, IL-13, and eotaxin. Data are reported as mean ± SEM (standard error of the mean) of cytokines / chemokines (pg / mL).

[0174] Data analysis: Statistical analysis of intergroup differences was performed by one-way analysis of variance (ANOVA). When the means of different treatment levels were significantly different, comparisons with the vehicle group were performed using Dunnett's test. p < 0.05 was considered statistically significant.

[0175] result

[0176] Clinical scoring: This model provides a progressive model of food allergy. Figure 3 As shown, an increase in reactions over time was observed in all OVA-exposed mice. Animals were clinically scored on days 21, 23, 25, and 28. As expected, mice challenged every other day with 5 mg OVA (intragusively) from day 14 to 28 (—■—) showed a statistically significant progressive increase in the severity of anaphylactic reactions compared to PBS-treated animals (—●—). By day 28, some animals in the OVA / vehicle experimental group had a score of 4 on the Anaphylaxis Symptom Scale. On day 28, mice exposed to the three doses of OVA showed a statistically significant increase in the severity of anaphylactic reactions compared to the vehicle-exposed group. Treatment of animals with '1104 (80 μg / kg) significantly reduced the severity of allergic reactions (—■—), while treatment with six doses (—▲—) of '1104 (80 μg / kg) significantly reduced the severity of allergic reactions compared to the vehicle group exposed to OVA on days 23, 25, and 28 (—■—). On days 25 and 28, treatment with three higher doses of '1104 (160 μg / kg) (—●—) significantly reduced the severity of allergic reactions compared to the vehicle group exposed to OVA (—■—), which was similar to the group receiving six lower doses of 80 μg / kg. Interestingly, the results of the last day confirmed the hypothesis that Cpn60.1-related peptides have short pharmacokinetics (PK) and long pharmacodynamics (PD), because the serum pharmacokinetics of '1104 is approximately 15 minutes, and the last OVA challenge is 3 days after the last '1104 dose.

[0177] Biomarker analysis was performed on day 28, where the most significant differences between the vehicle and '1104 treated groups were observed. OVA-specific IgE, mMCP, body temperature, and clinical scores were recorded. Figure 4 As shown, prophylactic and therapeutic administration of '1104 (80 or 160 μg / kg; three or six doses) on day 28 (i.e., three days after the last '1104 dose) significantly reduced all four biomarkers of food allergy: OVA-specific immunoglobulin E (IgE) (upper left panel); murine mast cell protease (mMCP-1) (upper right panel); body temperature (lower left panel); and clinical score (lower right panel).

[0178] Key Th2 Cytokines / Chemokines: Serum levels of key Th2 cytokines / chemokines were also measured on day 28: IL-4, IL-5, IL-13, and eotaxin. As expected, mice challenged every other day with 5 mg OVA (intragus) from day 14 to 28 showed statistically significant increases in the levels of these key Th2 cytokines / chemokines compared to PBS treated animals. See, Figure 5 : IL-4 (upper left panel); IL-5 (upper right panel); IL-13 (lower left panel); and eotaxin (lower right panel). Confirming the biomarker results described above, six doses of 80 μg / kg and three doses of 160 μg / kg of '1104 significantly reduced the levels of key Th2 cytokines / chemokines in the ovalbumin-induced food allergy mouse model, consistent with the expected result that '1104 reduces the severity of allergic reactions in these animals.

[0179] in conclusion

[0180] The results of this study using a mouse model of food allergy suggest that Cpn60.1-related peptides are effective for the prevention and treatment of food allergy and represent a novel non-allergen specific therapy for food allergy. This study provides the first evidence that Cpn60.1-related peptides have effects on early responses in addition to previously known effects on early responses.

[0181] Example 2 Long-term effectiveness of CPN60.1-related peptides in an animal model of ovalbumin-induced food allergy

[0182] As shown in Example 1, Cpn60.1-related peptides have been shown to be effective in preventing and treating food allergies. This study evaluated the effectiveness of a higher dose of '1104 (800 μg / kg) in the animal model of ovalbumin (OVA)-induced food allergy described above, as well as the long-term effectiveness of '1104 in preventing food allergies over a longer period of time after administration of '1104.

[0183] Materials and methods

[0184] Study design: The design and endpoints are as follows Figure 6 are shown and summarized in Table 3.

[0185] Table 3 – Study design

[0186]

[0187] Animals: As described in Example 1.

[0188] Animal welfare: As described in Example 1.

[0189] OVA sensitization and challenge: For animals in groups 1-5, ss was as described in Example 1. Animals in groups 6-10 were rested for 10 days and then received an additional challenge on day 38.

[0190] Body temperature and allergic reaction assessment: as described in Example 1.

[0191] Animal treatment with '1104 or vehicle: Mice were weighed and placed in a hot box for 5 minutes, then placed in a full body restraint and received intravenous administration (5 mL / kg) of '1104 (80 μg / kg or 800 μg / kg) or vehicle via the tail vein 15 minutes prior to OVA challenge on days 14, 16, 18, 21, 23, and 25.

[0192] Blood sample collection: One hour after the last OVA challenge on day 28 or day 38, terminal blood samples were collected by cardiac puncture and placed in serum tubes. Each serum sample was kept at room temperature for 45 minutes to allow the serum sample to clot, and then centrifuged (2000×g, 15 minutes, at 4°C), and the resulting supernatant was extracted from the centrifuged sample, and the supernatant was divided into aliquots and stored at -80°C for analysis.

[0193] Tissue collection: As described in Example 1.

[0194] OVA-specific IgE and mMCP1 ELISA assays: as described in Example 1.

[0195] Cytokine / Chemokine Assay: Serum samples (all groups) were measured for cytokine / chemokine concentrations using a magnetic multiplex assay (see below for details on biomarkers to be assessed) according to the manufacturer's instructions. Levels were measured using the Magpix system (Luminex Corp.).

[0196] According to the manufacturer's instructions, commercial ELISA kits (Biotechne, UK) are used to measure IL-9 and CCL-17. Microplate reader (SpectraMax 340PC) is used to measure the optical density at 450nM. The concentration of IL-9 and CCL-17 will be determined using SoftMax Pro v.6.4 (Molecular Devices). Mouse cytokine / chemokine magnetic multiplex (Biotechne): IL-4, IL-5, IL-10, IL-13, IL-17A, eotaxin, CCL-22, IFN-γ, TNFα. Data are reported as mean ± SEM (standard error of the mean) of cytokines / chemokines (pg / mL).

[0197] Data analysis: As described in Example 1.

[0198] result

[0199] Clinical scoring: This model provides a progressive model of food allergy. Figure 7 As shown, an increase in reactions over time was observed in all mice exposed to OVA. Animals were clinically scored on days 21, 23, 25, and 28. As expected, treatment of animals with six doses (—▲—) of '1104 (80 μg / kg) significantly reduced the severity of allergic reactions on days 23, 25, and 28 compared to the vehicle group exposed to OVA (—■—). Treatment with a higher dose of '1104 (800 μg / kg) (—●—) significantly reduced the severity of allergic reactions on days 23, 25, and 28 compared to the vehicle group exposed to OVA (—■—). Interestingly, 13 days after the last dose of '1104, two doses of '1104 (80 and 800 μg / kg) showed long-term effectiveness by significantly reducing the severity of allergic reactions compared to the vehicle group exposed to OVA. These results again confirmed that Cpn60.1-related peptides have short pharmacokinetics (PK) and long pharmacodynamics (PD).

[0200] Biomarker analysis: Biomarker analysis was performed on day 28 and again on day 38. Consistent with the results in Example 1, on day 28, '1104 (80 and 800 μg / kg; six doses) significantly improved symptom scores and body temperature ( Figure 8 , left panel). Interestingly, these beneficial effects were maintained at day 38 without further administration of '1104 ( Figure 8 , right panel).

[0201] OVA-specific IgE and mMCP1 levels on days 28 and 38: Biomarker analysis was performed on serum samples obtained on day 28 from OVA-sensitized mice (day 0) that were challenged with OVA and vehicle or '1104 (80 or 800 μg / kg, intravenous) by oral gavage on days 14, 16, 18, 21, 23, and 25. Samples were collected 1 h after the last OVA challenge on day 28. On day 28 (i.e., three days after the last dose of '1104), both doses of '1104 significantly reduced OVA-specific IgE levels ( Fig. 9 , upper left panel) and mMCP-1 levels ( Fig. 9 , lower left panel) Clinical scores. These data confirm the results of Example 1.

[0202] In another group of mice, biomarker analysis was performed on serum samples obtained on day 38 from OVA-sensitized mice (day 0) that were challenged with OVA and vehicle or '1104 (80 or 800 μg / kg, intravenous) by oral gavage on days 14, 16, 18, 21, 23, and 25. Animals in the group sampled on day 38 also received an additional OVA challenge on day 38. Samples were collected 1 h after the last OVA challenge on day 38. Again, both doses of '1104 reduced OVA-specific IgE ( Fig. 9 , upper right panel) and mMCP-1 levels ( Fig. 9 , lower right panel), but only the higher doses reached statistical significance. Interestingly, these data suggest that despite a short half-life (approximately 10-15 min across species), '1104 exhibits long-term effects in this food allergy model, supporting both a preventive and therapeutic role for food allergy.

[0203] Key Cytokines / Chemokines: Serum levels of key Th2 cytokines / chemokines were measured on days 28 and 38. As expected, mice challenged every other day with 5 mg OVA (intragusively) from day 14 to 28 showed statistically significant increases in the levels of these key cytokines / chemokines compared to PBS-treated animals. See, Figure 10-14 .

[0204] Confirming the biomarker results described above, six doses of 80 μg / kg and 800 μg / kg of '1104 significantly reduced the levels of key cytokines / chemokines in the ovalbumin-induced food allergy mouse model, consistent with the expected result that '1104 reduces the severity of allergic reactions in these animals. Key cytokines / chemokines include IL-4 ( Fig.10, upper panel); IL-5 ( Fig.10 , lower panel); IL-9 ( Fig.12 , upper panel); IL-10 ( Fig.12 , lower panel); IL-13 ( Fig.11 , upper panel); IL-17 ( Fig.13 , upper panel); CCL-17 ( Fig.13 , lower panel); CCL-22( Fig.14 , upper panel); IFN( Fig.14 , lower panel); and eotaxin ( Fig.11 , lower panel). Significant reductions in key Th2 cytokine / chemokine levels were observed on day 28, ie three days after the last dose of '1104 (left panel) and on day 28, ie 13 days after the last dose of '1104 (right panel).

[0205] Interestingly, IL-9 has been reported to play an important role in the development of IgE-mediated food allergy. 34 For example, IL-9 has been reported to be a key cytokine that promotes mast cell expansion and has been shown to be derived from peTh2 cells in food allergy. 35 Therefore, it is considered a potential target for the treatment of food allergies. The above results show that '1104 significantly reduced IL-9 in the animal model of food allergy ( Fig.12 , upper panel) supports the use of Cpn60.1-related peptides for the prevention and treatment of food allergies.

[0206] in conclusion

[0207] The results of this study using a food allergy mouse model indicate that Cpn60.1-related peptides are effective for the prevention and treatment of food allergies. Cpn60.1-related peptides reduce early anaphylactic allergic reactions in an ovalbumin-driven food allergy model. The effects after prophylactic and therapeutic administration were consistent in terms of "clinical" symptoms (anaphylactic reaction scores and body temperature) and related serum biomarkers (i.e., mMCP-1, OVA-IgE, IL-4, IL-5, IL-13, and eotaxin). Although the plasma half-life is short (approximately 15 min), Cpn60.1-related peptides show long pharmacodynamic effects (13 days) in this early allergic reaction model, which is consistent with the effects observed in the late allergic reaction model. Therefore, these studies support the use of Cpn60.1-related peptides as an alternative non-allergen-specific therapy for the treatment of food allergies.

[0208] References

[0209] _____________

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[0247] Xie, J., et al. (2012). "Elevated Antigen-Driven IL-9Responses AreProminent in Peanut Allergic Humans." PLoS ONE 7(10):e45377.

[0248] 35 Berin, MC (2023). "Targeting type 2immunity and the future of foodallergy treatment." J.Exp.Med., 220(4):e20221104.

[0249] All patents and other publications cited throughout this application, including references, issued patents, published patent applications, and co-pending patent applications, are expressly incorporated herein by reference for the purpose of describing and disclosing methods, such as those described in such publications, which can be used in conjunction with the techniques described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements regarding dates or representations regarding the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates or contents of these documents.

[0250] The foregoing written description is considered sufficient to enable those skilled in the art to practice aspects and embodiments of the present invention. The scope of aspects and embodiments of the present invention is not limited by the examples provided, as these examples are intended to be a single illustration of one aspect, and other functionally equivalent embodiments are also within the scope of the present disclosure. Various modifications other than those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and purposes described herein are not necessarily covered by each embodiment. Those skilled in the art will recognize that, or simply using routine experiments, many equivalents of the specific embodiments described herein can be determined. Such equivalents are intended to be covered by the following claims.

[0251] sequence

[0252] DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 1; '1104);

[0253] DGSVVVNKVSELPAGH(SEQ ID NO:2);

[0254] GLNVNTLSYGDLAAD(SEQ ID NO:3);

[0255] SELPAGHGLNVNLTS(SEQ ID NO:4);

[0256] DGSVVVNKVS (SEQ ID NO: 5);

[0257] ELPAGHGLNV (SEQ ID NO: 6);

[0258] NTLSYGDLAAD (SEQ ID NO:7)

Claims

1. A method for treating or preventing food allergy or food intolerance to a food antigen derived from food in a subject in need thereof, the method comprising administering a chaperone 60.1 (Cpn60.1) related peptide to the subject.

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 functionally equivalent fragments or variants 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 to 3, further comprising, prior to administration, diagnosing the subject as having or likely to develop a food allergy or food intolerance, or receiving an assay result diagnosing the subject as having or likely to develop a food allergy or food intolerance.

5. The method of any one of claims 1 to 4, wherein the Cpn60.1-related peptide is administered prior to first exposure to a potentially pathogenic food allergen, or upon the onset of clinical signs of atopic symptoms.

6. The method of any one of claims 1 to 5, wherein the subject has been diagnosed with at least one food allergy or food intolerance.

7. The method according to any one of claims 1 to 6, wherein the food antigen is derived from milk and its products; eggs and its products; meat and its products; fish, molluscs and crustaceans and their products; Oils, fats and their products; cereals and their products; Beans, seeds, kernels, nuts and their products; vegetables and their products; fruits and their products; Mushrooms and their products; Sugar, sugar products, chocolate products and confectionery; and spices and herbs.

8. The method of any one of claims 1 to 7, wherein administration of the Cpn60.1-related peptide is acute or short-term.

9. The method of any one of claims 1 to 7, wherein administration of the Cpn60.1-related peptide is chronic.

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

11. A therapeutically effective amount of a Cpn60.1-related peptide for use as claimed in claim 10, 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 functionally equivalent fragments or variants thereof.

12. The therapeutically effective amount of a Cpn60.1-related peptide for use as claimed in claim 11, wherein the Cpn60.1-related peptide is SEQ ID NO:

1.

13. A therapeutically effective amount of a Cpn60.1-related peptide for use as described in any one of claims 10 to 12, further comprising, prior to use, diagnosing the subject as having or likely to develop food allergy or food intolerance, or receiving a test result diagnosing the subject as having or likely to develop food allergy or food intolerance.

14. A therapeutically effective amount of a Cpn60.1-related peptide for use as claimed in any one of claims 10 to 13, wherein the Cpn60.1-related peptide is used prior to first exposure to a potentially pathogenic food allergen, or upon the onset of clinical signs of atopic symptoms.

15. A therapeutically effective amount of a Cpn60.1 related peptide for use as claimed in any one of claims 10 to 14, wherein the subject has been diagnosed with at least one food allergy or food intolerance.

16. The therapeutically effective amount of Cpn60.1-related peptide for use according to any one of claims 10 to 15, wherein the food antigen is derived from milk and its products; eggs and its products; meat and its products; fish, molluscs and crustaceans and their products; Oils, fats and their products; cereals and their products; Beans, seeds, kernels, nuts and their products; vegetables and their products; fruits and their products; Mushrooms and their products; Sugar, sugar products, chocolate products and confectionery; and spices and herbs.

17. A therapeutically effective amount of a Cpn60.1 related peptide for use as claimed in any one of claims 10 to 16, wherein the use of the Cpn60.1 related peptide is acute or short-term.

18. A therapeutically effective amount of a Cpn60.1-related peptide for use as claimed in any one of claims 10 to 16, wherein the use of the Cpn60.1-related peptide is chronic.

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