Methods and compositions for preventing or treating severe asthma

Abstract

Described herein are methods for preventing or treating severe asthma by administrating peptides related to Chaperonin 60.1.

Description

METHODS AND COMPOSITIONS FOR PREVENTING OR TREATING SEVERE ASTHMAFIELD OF THE INVENTION

[0001] The embodiments of the present invention relate to compositions and methods for the prevention or treatment of severe asthma with peptides related to Chaperonin 60.1 (Cpn60.1).BACKGROUND OF THE INVENTION

[0002] Asthma is a chronic and heterogeneous respiratory condition characterized by airflow obstruction, airway inflammation and AHR (airway hyperresponsiveness) with symptoms of wheeze, cough, chest tightness and shortness of breath.1It affects approximately 300 million people worldwide.2The majority of asthma patients with asthma can be treated effectively with the currently available medications. However, a significant proportion of patients, labelled as “severe refractory asthma” or “severe asthma,” remain a challenge for the treating clinician. Severe asthma encompasses a variety of subphenotypes of asthma that do not respond to current standard therapy, i.e., high doses of corticosteroid (ICS) in combination with long-acting P2-agonists.3’4Approximately 10% of asthma patients do not respond to available steroid treatments.5This condition is referred to as “severe asthma,” “severe refractory asthma,” “corticosteroid-refractory asthma” or “severe corticosteroid-refractory asthma.”

[0003] Although eosinophil accumulation and Th2 cell activation is fundamental to mild- moderate asthma, patients with severe asthma manifest a more diverse inflammatory pattern with several prominent subphenotypes.6’7Severe asthma is poorly understood and difficult to treat, and these patients pose a major healthcare problem as they have greater morbidity and mortality accounting for most asthma-related healthcare expenditure.89Management of severe asthma is associated with a high and disproportional consumption of healthcare resources.10,11Although patients with severe asthma only make up a small proportion of asthma patients, this subgroup accounts for more than 50% of direct and indirect healthcare costs associated with asthma.12Over the last decade, it has become increasingly clear that there is an urgent need for the development of new targeted therapies.

[0004] Accordingly, there is a need for effective compositions and methods for the prevention or treatment of severe asthma.BRIEF SUMMARY OF THE INVENTION

[0005] The embodiments of the present invention provide a method for treating or preventing severe asthma in a subject that is resistant to one or more corticosteroid drug, the method comprising administering to the subject, a therapeutically-effective amount of a peptide related to Chaperonin 60.1 (Cpn60.1). In some embodiments, the Cpn60.1 -related peptide isselected from: DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 1) (PIN201104); DGSVVVNKVSELPAGH (SEQ ID NO: 2); GLNVNTLSYGDLAAD (SEQ ID NO: 3);SELPAGHGLNVNLTS (SEQ ID NO: 4); DGSVVVNKVS (SEQ ID NO: 5); ELPAGHGLNV (SEQ ID NO: 6); NTLSYGDLAAD (SEQ ID NO: 7); or a functionally equivalent fragment or variant thereof. In one embodiment, the Cpn60.1 -related peptide is SEQ ID NO:1.

[0006] In some embodiments, the method further comprises the step of diagnosing the subject as having, or likely to develop, severe asthma prior to administration, or receiving the results of an assay that diagnoses the subject as having, or likely to develop, severe asthma.

[0007] In some embodiments, the Cpn60.1 -related peptide is administered prior to the first symptom of severe asthma. In alternative embodiments, the Cpn60.1 -related peptide is administered upon clinical signs of severe asthma.

[0008] In another aspect, the embodiments of the present invention provide a method for treating severe asthma in a subject resistant to one or more corticosteroid drug, the method comprising the steps of: (i) administering a peptide related to Cpn60.1)to the subject upon clinical signs of severe asthma, and (ii) stopping the administration of the peptide related to Cpn60.1 upon remission of the clinical signs of severe asthma. wherein the severe asthma in the subject is resistant to one or more corticosteroid drug. In some embodiments, the Cpn60.1 -related peptide is selected from: DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 1) (PIN201104);DGSVVVNKVSELPAGH (SEQ ID NO: 2); GLNVNTLSYGDLAAD (SEQ ID NO: 3);SELPAGHGLNVNLTS (SEQ ID NO: 4); DGSVVVNKVS (SEQ ID NO: 5); ELPAGHGLNV (SEQ ID NO: 6); NTLSYGDLAAD (SEQ ID NO: 7); or a functionally equivalent fragment or variant thereof. In one embodiment, the Cpn60.1 -related peptide is SEQ ID NO:1.

[0009] In some embodiments, the method further comprises the step of diagnosing the subject as having, or likely to develop, severe asthma prior to administration, or receiving the results of an assay that diagnoses the subject as having, or likely to develop, severe asthma.

[0010] In some embodiments, the Cpn60.1 -related peptide is administered prior to the first symptom of severe asthma. In alternative embodiments, the Cpn60.1 -related peptide is administered upon clinical signs of severe asthma.

[0011] Other implementations are also described and recited herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] For the purpose of illustration, certain embodiments of the present invention are shown in the 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:

[0013] FIG. 1A-B shows bar graphs representing the total cell count in the bronchoalveolar lavage fluid following treatment with vehicle (5 ml_ / kg, IV), IRL201104 (20 or 80 pg / kg, IV) or dexamethasone (1 mg / kg, IP) 1 hour before HDM challenge on day 14 in HDM(100 pg, s.c., day 0) sensitized mice and challenged (days 14) with PBS or HDM (25 pg, IN). See FIG. 1A. In FIG. 1 B, mice were also challenged with polylC on day 13, 24 hours before HDM challenge. Each column represents the mean, and each bar represents SEM of n=8. Vehicle + HDM or Vehicle + polylC + HDM was compared to all other groups using ANOVA followed by Dunnett’s test. **P<0.01, ***P<0.001.

[0014] FIG. 2A-B shows bar graphs representing the number of eosinophils in the bronchoalveolar lavage fluid following treatment with vehicle (5 mL / kg, IV), IRL201104 (20 or 80 pg / kg, IV) or dexamethasone (1 mg / kg, IP) 1 hour before HDM challenge on day 14 in HDM (100 pg, s.c., day 0) sensitized mice and challenged (days 14) with PBS or HDM (25 pg, IN). See FIG. 2A. In FIG. 2B, mice were also challenged with polylC on day 13, 24 hours before HDM challenge. Each column represents the mean, and each bar represents SEM of n=8. Vehicle + HDM or Vehicle + polylC + HDM was compared to all other groups using ANOVA followed by Dunnett’s test. **P<0.01, ***P<0.001.

[0015] FIG. 3A-B shows bar graphs representing the number of neutrophils in the bronchoalveolar lavage fluid following treatment with vehicle (5 mL / kg, IV), IRL201104 (20 or 80 pg / kg, IV) or dexamethasone (1 mg / kg, IP) 1 hour before HDM challenge on day 14 in HDM (100 pg, s.c., day 0) sensitized mice and challenged (days 14) with PBS or HDM (25 pg, IN). See FIG. 3A. In FIG. 3B, mice were also challenged with polylC on day 13, 24 hours before HDM challenge. Each column represents the mean, and each bar represents SEM of n=8. Vehicle + HDM or Vehicle + polylC + HDM was compared to all other groups using ANOVA followed by Dunnett’s test. **P<0.01, ***P<0.001.

[0016] FIG. 4A-B shows bar graphs representing the number of macrophages in the bronchoalveolar lavage fluid following treatment with vehicle (5 mL / kg, IV), IRL201104 (20 or 80 pg / kg, IV) or dexamethasone (1 mg / kg, IP) 1 hour before HDM challenge on day 14 in HDM (100 pg, s.c., day 0) sensitized mice and challenged (days 14) with PBS or HDM (25 pg, IN). See FIG. 4A. In FIG. 4B, mice were also challenged with polylC on day 13, 24 hours before HDM challenge. Each column represents the mean, and each bar represents SEM of n=8. Vehicle + HDM or Vehicle + polylC + HDM was compared to all other groups using ANOVA followed by Dunnett’s test. **P<0.01, ***P<0.001.

[0017] FIG. 5A-B shows bar graphs representing the number of lymphocytes in the bronchoalveolar lavage fluid following treatment with vehicle (5 mL / kg, IV), IRL201104 (20 or 80 pg / kg, IV) or dexamethasone (1 mg / kg, IP) 1 hour before HDM challenge on day 14 in HDM (100 pg, s.c., day 0) sensitized mice and challenged (days 14) with PBS or HDM (25 pg, IN). See FIG. 5A. In FIG. 5B, mice were also challenged with polylC on day 13, 24 hours before HDM challenge. Each column represents the mean, and each bar represents SEM of n=8. Vehicle + HDM or Vehicle + polylC + HDM was compared to all other groups using ANOVA followed by Dunnett’s test. **P<0.01, ***P<0.001.

[0018] FIG. 6A-B provides line graphs showing the change in total resistance (Rl) following 20 sec methacholine (MCh, 1-30 pg / mL) aerosol following treatment with vehicle (5 mL / kg, IV), IRL201104 (20 or 80 pg / Kg, IV) or dexamethasone (1 mg / Kg, IP) 1 hour before HDM challenge on day 14 in HDM (100 pg, s.c., day 0) sensitized mice and challenged (days 14) with PBS or HDM (25 pg, IN). See FIG. 6A. In FIG. 6B, mice were also challenged with polylC on day 13, 24 hours before HDM challenge. Total resistance changes to MCh were conducted 24 h after the day 14 HDM challenge. Each symbol represents the mean, and each bar represents SEM of n=8. Vehicle + HDM or Vehicle + polylC + HDM was compared to all other groups using ANOVA followed by Dunnett’s test. Vehicle + polylC + HDM was compared to Vehicle + HDM (#) using unpaired student f-test. **P<0.01, ###P<0.001.

[0019] FIG. 7A-B provides line graphs showing the change in dynamic compliance (Cdyn) following 20 sec methacholine (MCh, 1-30 pg / mL) aerosol following treatment with vehicle (5 mL / kg, IV), IRL201104 (20 or 80 pg / Kg, IV) or dexamethasone (1 mg / Kg, IP) 1 hour before HDM challenge on day 14 in HDM (100 pg, s.c., day 0) sensitized mice and challenged (days 14) with PBS or HDM (25 pg, IN). See FIG. 7A. In FIG. 7B, mice were also challenged with polylC on day 13, 24 hours before HDM challenge. Dynamic compliance changes to MCh were conducted 24 h after the day 14 HDM challenge. Each symbol represents the mean, and each bar represents SEM of n=8. Vehicle + HDM or Vehicle + polylC + HDM was compared to all other groups using ANOVA followed by Dunnett’s test. Vehicle + polylC + HDM was compared to Vehicle + HDM (#) using unpaired student f-test. **P<0.01, ###P<0.001.

[0020] FIG. 8A-D provides bar graphs showing the effects of treatment on HDM- induced cellular infiltration on eosinophils (FIG. 8A), neutrophils (FIG. 8B), macrophages (FIG. 8C), and lymphocytes (FIG. 8D) lung infiltrate. Data are expressed as cells per mL of BALF, mean ± SEM. Groups were compared to HDM / vehicle group using a one-way ANOVA, followed by a Dunnett’s test, ***P<0.001 ; n=8.

[0021] FIG. 9A-D provides bar graphs showing the effects of treatment on HDM- induced cellular infiltration exacerbated by Poly l:C exposure on eosinophils (FIG. 9A), neutrophils (FIG. 9B), macrophages (FIG. 9C), and lymphocytes (FIG. 9D) lung infiltrate. Data are expressed as cells per mL of BALF, mean ± SEM. Groups were compared to HDM / Poly I :C / vehicle group using a one-way ANOVA, followed by a Dunnett’s test;*P<0.05,***P<0.001. A Student’s t-test was used when comparing HDM / saline / vehicle vs HDM / Poly l:C / vehicle group; #P<0.05; n=8.

[0022] FIG. 10A-B provides bar graphs showing the effects of treatment of Dexamethasone and ‘1104 on serum HDM specific IgE in the absence (FIG. 10A) or presence (FIG. 10B) of Poly l:C exacerbation. Data are expressed as pg / mL, mean ± SEM. Groups were compared to either to HDM / vehicle (FIG. 10A) or HDM / Poly l:C / vehicle (FIG. 10B) using aone-way ANOVA, followed by a Dunnett’s test, ;**P<0.01 ,***P<0.001. A Student’s t-test was used when comparing HDM / saline / vehicle vs HDM / Poly l:C / vehicle group; #P<0.05; n=8.

[0023] FIG. 11 A-D provides line graphs showing the effects of treatment on AHR to methacoline (MCh). Effect of treatment on total lung resistance (Rl) and dynamic compliance (Cdyn) in the absence (FIG. 11A+C) or presence (FIG. 11 B+D) of Poly l:C exacerbation. Data are expressed as area under the curve (AUC), mean ±SEM. Groups were compared to either to HDM / vehicle (FIG. 11A+C) or HDM / Poly l:C / vehicle (FIG. 11B+D) groups using a one-way ANOVA, followed by a Dunnett’s test; **P<0.01, ***P<0.001. A Student’s t-test was used when comparing HDM / saline / vehicle vs. HDM / Poly l:C / vehicle group; ###P<0.001; n=8.DETAILED DESCRIPTION OF THE INVENTION

[0024] The subject innovation is now described with reference to the drawings, wherein like reference numerals are used to refer to like elements throughout. In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It may be evident, however, that the present invention may be practiced without these specific details. In other instances, well- known structures and devices are shown in block diagram form in order to facilitate describing the present invention. It is to be appreciated that certain aspects, modes, embodiments, variations and features of the invention are described below in various levels of detail in order to provide a substantial understanding of the present invention.DEFINITIONS

[0025] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0026] 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, and the like.

[0027] As used herein, the term "approximately" or "about" in reference to a value or parameter are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% orexceed 100% of a possible value). As used herein, reference to "approximately" or "about" a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to "about X" includes description of "X".

[0028] As used herein, the term “or” means “and / or.” The term "and / or" as used in a phrase such as "A and / or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0029] As used herein, the term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation.

[0030] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0031] 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 or functional characteristic(s) of that embodiment of the invention.

[0032] The term "statistically significant" or "significantly" refers to statistical significance and generally means a two-standard deviation (2SD) or greater difference.

[0033] As used herein, the term "subject" refers to a mammal, including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate. Subjects can be house pets (e.g., dogs, cats), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), but are not so limited. Subjects include human subjects. The human subject may be a pediatric, adult, or a geriatric subject. The human subject may be of either sex.

[0034] As used herein, the terms "effective amount" and “therapeutically-effective amount” include an amount sufficient to prevent or ameliorate a manifestation of severe asthma. It will be appreciated that there will be many ways known in the art to determine the effective amount for a given application. For example, the pharmacological methods for dosage determination may be used in the therapeutic context. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds.

[0035] As used herein, the terms "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed disease or infection and (2) prophylactic or preventative measures that prevent or slow the development of a disease or infection.

[0036] As used herein, the terms “treat," “treatment,” “treating," or “amelioration” when used in reference to a disease, disorder or medical condition, refer to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a condition is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized ( / .e., not worsening) state of an immune disorder, delay or slowing of an immune disorder, and an increased lifespan as compared to that expected in the absence of treatment. Signs and symptoms of an asthma attack include, but are not limited to, severe shortness of breath, chest tightness or pain, coughing or wheezing, and low peak expiratory flow (PEF) readings, using a peak flow meter. Severe asthma is present when adequate control of asthma cannot be achieved by high-dose treatment with inhaled corticosteroids and additional controllers (long-acting inhaled beta 2 agonists, montelukast, and / or theophylline) or by oral corticosteroid treatment (for at least six months per year) or is lost when the treatment is reduced.

[0037] The term “acute treatment” is used to mean that the peptide is administered at the onset of or during a period of relapse of the condition, but that it is not necessary for the peptide to be continuously administered. In particular, it may not be necessary for the peptide to be administered during periods of remission of the condition. Thus, the “acute treatment” according to the present invention can be distinguished from known methods for the treatment of relapsing-remitting conditions which provide chronic therapy requiring continuous, long-term administration of the pharmaceutical without any breaks in treatment. The provision of an acute treatment provides significant advantages to the patient. Since the peptides of the invention only need to be administered over a short period of time, side effects, for example injection site reactions are reduced. In addition, during periods of remission patients enjoy an improved lifestyle, without the need to remember dosage regimens. Without being bound by theory, it is understood that the peptides of the invention do not simply affect the symptoms of the condition or disease, but rather they modify the underlying condition or disease itself. Thus, administration of the peptide of the invention has a long-term effect.

[0038] In some embodiments, a single dose of the peptide molecule is administered to the subject. In alternative embodiments, two or more doses (e.g., 3, 4, 5, or more doses) are administered over a short period of time, for example over a period of one day, three days, 28 days, 56 days or 112 days. The time between dose administration to the subject may be three hours, one day, 14 days, 28 days or 56 days after the previous dose.

[0039] Remission usually comprises the reduction, alleviation or elimination of one or more symptoms of the condition. Typically, remission or clinical remission comprises a period with no symptoms associated with the relapsing-remitting disease or a period during which the symptoms associated with the disease have decreased in severity and / or in number. A symptom associated with a condition, disease or disorder includes any clinical or laboratory manifestation associated with the disease or disorder. Clinical remission may therefore be measured according to the relevant scale or remission indicators, and well known in the medical field by for example, clinicians and researchers. Conversely, relapse of the condition may be defined as the increase or appearance of one or more symptoms of the condition. For example, symptoms of asthma may be shortness of breath, difficulty breathing, chest tightness, coughing, reduced lung capacity, trouble sleeping caused by shortness of breath, coughing or wheezing, a whistling or wheezing sound when inhaling, coughing or wheezing attacks that are worsened by a respiratory virus such as cold or flu. Additionally, symptoms may include hospitalization or loss of work / school attendance.

[0040] A reduction or elimination of one or more symptoms is typically a significant reduction or elimination of one or more symptoms as identified by a physician. Symptoms of the relapsing-remitting conditions can be measured and quantified using well-known diagnostic tests. For example, lung function tests such as spirometry and methacholine challenge tests can be used to quantify the symptoms of asthma, using ACQ scores. The ACQ is a simple questionnaire to measure the adequacy of asthma control and change in asthma control which occurs either spontaneously or as a result of treatment. ACQ has a multidimensional construct assessing symptoms (5 items-self-administered) and rescue bronchodilator use (1 item-self- administered) and forced expiratory volume in 1 minute (FEV1) (1 item) completed by clinic staff.13

[0041] As well as providing a clinical definition of remission, it is also possible to define a biological or mechanistic definition of remission. In some embodiments, the condition is associated with eosinophilia and / or neutrophilia. In this case, remission comprises a significant reduction in the number of neutrophils and / or the number of eosinophils trafficking to a site of inflammation in the human or animal subject relative to a control subject who has not been administered the peptide molecule. If the condition is a pulmonary condition, remission comprises a significant reduction in the number of neutrophils and / or the number of eosinophils recruited to the lungs or found within the circulatory system.

[0042] Remission may also be associated with a significant reduction in the number of lymphocytes or a significant increase in the number of macrophages in the human subject relative to a control subject. Remission may further be associated with a significant change in the amount of one or more inflammatory markers such as cytokines, for example IL-4, IL-5, IL-10, or IL-13 in the human subject relative to a control subject. Remission may comprise a significant increase in the amount of IL-10 in the human subject relative to a control subject. Remission may comprise a significant reduction in the amount of IL-4, IL-5 or IL-13 in the human subject relative to a control subject.

[0043] A relapsing-remitting condition is any condition which has one or more periods of relapse, wherein each relapse is followed by a period of remission. During these symptom free periods or periods of remission, patients do not require quantifiable circulating levels of the therapeutic peptide. In a preferred embodiment remission is maintained when the plasma peptide concentration is below the lower limit of quantification. This limit of quantification may vary depending on the detection method employed. Typically, the plasma peptide concentration is undetectable at circulating levels of less than 40 ng / mL, for example less than 30 ng / mL or 20 ng / mL. A typical method for determining the plasma peptide concentration is high resolution accurate mass (HRAM) LC-MS / MS. In a preferred embodiment remission of the condition is for a period of at least 7 days, for example 14 days, at least 28 days, more preferably at least 6 months after the concentration of the peptide molecule in the plasma of the subject is undetectable. In another embodiment remission of the condition is for a period of at least 7 days, optionally at least 14 days, optionally at least 28 days, optionally at least 6 months after administration of the final dose of the peptide.

[0044] As used herein, the term "long-term" administration means that the therapeutic agent or drug is administered for a period of at least 12 weeks. This includes that the therapeutic agent or drug is administered such that it is effective over, or for, a period of at least 12 weeks and does not necessarily imply that the administration itself takes place for 12 weeks, e.g., if sustained release compositions or long-acting therapeutic agent or drug is used. Thus, the subject is treated for a period of at least 12 weeks. In many cases, long-term administration is for at least 4, 5, 6, 7, 8, 9 months or more, or for at least 1 , 2, 3, 5, 7 or 10 years, or more.

[0045] The administration of the compositions contemplated herein may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. In a preferred embodiment, compositions are administered parenterally. The phrases “parenteral administration” and “administered parenterally” as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrastemal 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.

[0046] The terms “decrease,” “reduced,” “reduction,” or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment or agent) and can include, for example, a decrease by 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, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0047] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example 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 a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.Biopharmaceutical definitions:

[0048] As used herein, the terms “protein" and “polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus,exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0049] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant" where the alteration 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 disclosure.

[0050] In some embodiments, the polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a fragment or segment of a peptide which retains at least 50% of the wildtype reference polypeptide’s activity according to the assays described below herein. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

[0051] In some embodiments, the polypeptide described herein can be a variant of a sequence described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Variant polypeptide-encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains activity. A wide variety of PCR-based site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan.

[0052] As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, thenucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA or cDNA. Suitable RNA can include, e.g., mRNA.

[0053] In some embodiments of any of the aspects, a polypeptide, nucleic acid, or cell as described herein can be engineered. As used herein, “engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered" when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature. As is common practice and is understood by those in the art, progeny of an engineered cell are typically still referred to as “engineered" even though the actual manipulation was performed on a prior entity.

[0054] In some embodiments, a nucleic acid encoding a polypeptide as described herein (e.g., an antibody or antibody reagent) is comprised by a vector. In some of the 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 can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.

[0055] As used herein, the term "expression vector" refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification. The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g., 5’ untranslated (5’UTR) or "leader" sequences and 3’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0056] The term “isolated” or “partially purified” as used herein refers, in the case of a nucleic acid or polypeptide, to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide as found in its natural source and / or that would be present with the nucleic acid or polypeptide when expressed by a cell, or secreted in the case of secreted polypeptides. A chemicallysynthesized nucleic acid or polypeptide or one synthesized using in vitro transcription / translation is considered “isolated.” The terms “purified” or “substantially purified” refer to an isolated nucleic acid or polypeptide that is at least 95% by weight 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 antibody, antigen-binding portion thereof, or chimeric antigen receptor (CAR) described herein is isolated. In some embodiments, the antibody, antibody reagent, antigen-binding portion thereof, or CAR described herein is purified.

[0057] As used herein, “engineered” refers to the aspect of having been manipulated by the hand of man. For example, an antibody, antibody reagent, antigen-binding portion thereof, CAR or bispecific antibody is considered to be “engineered” when the sequence of the antibody, antibody reagent, antigen-binding portion thereof, CAR or bispecific antibody is manipulated by the hand of man to differ from the sequence of an antibody as it exists in nature. As is common practice and is understood by those in the art, progeny and copies of an engineered polynucleotide and / or polypeptide are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.PHARMACEUTICAL COMPOSITIONS

[0058] The compositions and methods of the present invention may be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration ( / .e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0059] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention. Such physiologically acceptable agentsinclude, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-micro emulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[0060] The phrase "pharmaceutically acceptable" is employed herein to refer 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 problem or complication, commensurate with a reasonable benefit / risk ratio.

[0061] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc;(8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;(12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water;(17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0062] A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The compound may also be formulated forinhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same 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, as well as in patents cited therein.

[0063] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

[0064] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the 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 liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0065] Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.

[0066] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), 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 starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0067] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropyl methyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surfaceactive or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0068] The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropyl methyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

[0069] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, micro-emulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0070] 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.

[0071] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0072] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

[0073] The ointments, pastes, creams and gels may contain, in addition to an active compound, 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.

[0074] Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0075] Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

[0076] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraocular (such as intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may bereconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0077] Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0078] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0079] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0080] Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

[0081] For use in the methods of this invention, active compounds can be given perse or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0082] Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow-release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.

[0083] 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.

[0084] 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 ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound(s) 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.

[0085] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art. See, e.g., Isselbacher et al. (1996).14

[0086] In general, a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest doseeffective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

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

[0088] The patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines bovine, porcine, sheep, feline, and canine; poultry; and pets in general.

[0089] In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent.

[0090] The present disclosure includes the use of pharmaceutically acceptable salts of compounds of the invention in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1 H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2- hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the invention include, but are 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-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1 ,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic 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, I- malic acid, malonic acid, mandelic acid, methanesulfonic acid , naphthalene-1 ,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid salts.

[0091] The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

[0092] 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.

[0093] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0094] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. 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 defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy;15The Encyclopedia of Molecular Cell Biology and Molecular Medicine;16Molecular Biology and Biotechnology: a Comprehensive Desk Reference;17Immunology;18Janeway's Immunobiology;19Lewin's Genes XI;20Molecular Cloning: A Laboratory Manual.;21Basic Methods in Molecular Biology;22Laboratory Methods in Enzymology;23Current Protocols in Molecular Biology (CPMB);24Current Protocols in Protein Science (CPPS);25and Current Protocols in Immunology (CPI).26

[0095] In some embodiments of any of the aspects, the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.

[0096] Other terms are defined herein within the description of the various aspects of the invention.SEVERE ASTHMA

[0097] Asthma is a complex, respiratory disease characterized by airway inflammation and bronchoconstriction, which make it difficult to breathe. Asthma affects approximately 300 million people worldwide.27There are heterogeneous clinical symptoms with varying degrees of response to therapy in asthma. Inhaled corticosteroid (ICS) is a common therapy for asthma, to which controllers such as a long-acting [32 agonist are added if required, and if these fail oral corticosteroids are also added.28,29Approximately 10% of asthma patients do not respond to available steroid treatments.30This condition is referred to as “severe asthma,’’ “severe refractory asthma,’’ “corticosteroid refractory asthma” or “severe corticosteroid refractory asthma.” In 2014, a task force of ERS / ATS defined severe asthma as “asthma which requires treatment with high dose inhaled corticosteroids (ICS) plus a second controller (and / or systemic corticosteroids) to prevent it from becoming ‘uncontrolled,’ or which remains ‘uncontrolled’ despite this therapy.”31

[0098] Although patients with severe asthma make up a small proportion of asthma patients, this subgroup accounts for more than 50% of direct and indirect healthcare costs associated with asthma.32Research in the last three decades has shown that there are multiple phenotypes or subgroups in severe asthma, with differences in clinical symptoms and molecular profiles. There is also a prominent sex-related disparity, as severe asthma disproportionally affects adult females compared to males. Some studies demonstrate that almost 2 / 3 of severe asthmatics are females.33Resistance to corticosteroids has been attributed to a variety of components, from genetic variability to various molecular factors such as defective glucocorticoid receptor (GR) function with increased expression of the non- responsive isoform of GR[3, different transcription factor and signaling pathways, as well as specific cytokine-mediated downstream responses.34,35,36Diagnosis:

[0099] Asthma is among the most common chronic diseases in the developed and developing world, but its diagnosis can be difficult. Although symptoms including wheeze, chest tightness and shortness of breath are often considered essential features of asthma in humans, the adage ‘all that is asthma does not wheeze and all that wheezes is not asthma’ holds true. Epidemiological studies that rely on ‘doctor diagnosis’ of asthma overestimate the true disease prevalence owing to misclassification.37A diagnosis of asthma usually begins when a child or adult presents with a range of spontaneous respiratory symptoms including recurrent cough and nocturnal awakening, along with symptoms triggered by external stimuli, such as allergens, viral infections, exercise and cold air. In adults, a history of asthma or recurrent ‘bronchitic episodes’ in childhood holds an important clue to diagnosis. However, a diagnosis of asthma should not be made on clinical characteristics alone. The definition of asthma requires a combination of appropriate clinical symptoms in association withdocumented reversible airflow limitation and / or airway hyper-responsiveness38Although reversibility of airway obstruction and hyper-responsiveness are considered hallmarks of asthma, the sensitivity and specificity of the diagnostic criteria to identify these symptoms are poorly defined. Guidelines have added the important extra dimension of airway inflammation to the diagnosis of asthma, which is measured by eosinophil counts in sputum or blood and / or increased fractional exhaled nitric oxide (FeNO).39,40

[0100] Physiologically determined abnormalities, such as reduced spirometry, are also of value in establishing an asthma diagnosis early in the course of the condition. Patients should be tested on a spirometer that is equipped with population normal values and, ideally, one that generates a flow-volume loop that can be evaluated for both inspiratory and expiratory effort. FEV1, forced vital capacity (FVC) and the FEV1 / FVC ratio should be reported alongside reversibility of lung function with an inhaled SABA. Symptomatic asthma is often associated with a predicted FEV1 of <80% and an age- adjusted FEV1 / FVC of <75%. Testing should be repeated after inhalation of a SABA to establish reversibility of airway obstruction, a hallmark of asthma, although as noted, both bronchodilator reversibility and peak expiratory flow (PEF) variability have poor sensitivity and specificity for the diagnosis of asthma. By convention, a diagnosis of asthma requires at least a 12% improvement in FEV1 over baseline and a total improvement of at least 200 mL. As asthma is frequently highly variable, normal spirometry results do not exclude the disease. Additional diagnostic aids include repeat testing over time and diurnal PEF monitoring using a portable PEF meter. If spirometry results remain normal, bronchial provocation testing with inhaled methacholine or mannitol should be considered to establish if airway hyper-responsiveness exists as another characteristic feature of asthma, although some variability in responses can be seen. To perform this test, the patient inhales increasing concentrations of the challenge substance until there is a >20% fall in the FEV1 from the saline control value. Each challenge agent has a threshold concentration for the fall that identifies asthma. Exercise testing or, as an alternative, eucapnic hyperventilation, which mimics the volume of air exchanged during exercise, is another method for uncovering hyperresponsiveness and is especially useful in diagnosing asthma in children.

[0101] The documentation of asthma-related airway inflammation is an important recent development in asthma diagnosis and is especially useful for ruling out asthma, as many diseases can produce asthma-like symptoms and provide positive results in the tests described above. Recent UK National Institute for Health and Care Excellence (NICE) guidelines for asthma diagnosis highlighted an urgent need to mainstream the use of inflammatory biomarkers, such as FeNO and sputum eosinophil counts, for reliable diagnosis of T2-type asthma162 (FIG. 10).41If diagnosis is still questionable, then a practical approach is to treat the patient with medications that are appropriate to their level of severity defined by national or international asthma guidelines. If the patient’s symptoms become markedly better in responseto this treatment, then asthma is the likely cause. Possible other causes of wheezing and asthma-like symptoms should always be considered in the face of a poor response to a trial of treatment, and can include COPD, upper airway obstruction and laryngospasm in adults and viral-associated infection in children.Biomarkers:

[0102] Many different ‘subphenotypes’ of asthma with differing characteristics are becoming increasingly recognized.42In terms of disease classification, perhaps the most important distinction to make is whether the patient has evidence of an eosinophilic T2-type inflammatory process. This is important for disease management, as evidence is emerging that responses to ICSs and biologic therapies that target IgE, the I L-4— I L-13 pathway and IL-5 are all greater in patients with evidence of T2-type inflammation than in patients without evidence of T2-type inflammation.43'44'45A characteristic feature of this immune activation profile is the appearance of eosinophils in the blood and induced sputum as well as increased FeNO. ICS treatment monitored by sputum eosinophil testing is highly effective,4647and both sputum and blood eosinophil counts are being used to effectively identify patients who might be responsive to biologic therapies that target IL-4, IL-13 and IL-5.48’49Measurement of sputum and blood eosinophilia is, unfortunately, not widely implemented.50

[0103] Nitric oxide produced by inducible nitric oxide synthase in the bronchial epithelium increases in response to IL-4 and IL-13, is a marker for T2-type inflammation and is highly corticosteroid sensitive.51 52Although measurement of the FeNO requires a specific analyzer, the test itself is easy to perform, is reproducible and can be measured as a point-of- care biomarker with instant results. Elevated FeNO increases the likelihood of an asthma diagnosis involving T2-type inflammation and can be used as a predictor of and to follow therapeutic responses to biologies that are targeted at IgE, the IL-4 receptor, IL-13 and I [__Cj 53,54,55

[0104] Expression of periostin, an extracellular matrix protein, is induced by IL-4 and IL-13 in airway epithelial cells and lung fibroblasts,56and periostin is secreted as a soluble peptide from the basolateral surface from which it gains access to the circulation.57Periostin functions as a ligand for aV[33 and aV[35 integrins to promote adhesion and migration of epithelial cells and aids in the crosslink-age of submucosal collagen.58As a T2-type immunity biomarker, reduced serum levels of periostin predict the clinical efficacy of biologies targeting the I L-4— I L-13 pathway and discriminate patients with high numbers of eosinophils in their airways.5960As a high proportion of patients expressing T2-type airway inflammation are atopic, assessment of allergen-specific IgE in the serum provides information on patientspecific allergic triggers.61

[0105] Perhaps the most crucial use of these biomarkers will be to identify various molecular phenotypes of asthma, in particular severe asthma, where biologic agents are likelyto be targeted. To date, the biomarkers identified are all linked to T2-type inflammatory phenotypes, which might either predict or be responsive to these T2-type-targeted therapies. Blood eosinophil counts of ~150 per .L seem to both predict responses to IL-5-targeted therapies and fall in response to these treatments.62At present, it is unclear which biomarker will best predict response to I L-4— I L-13 pathway-targeted therapies, as all have shown some predictive ability. However, for I L-4— I L-13 pathway-targeted therapies, whereas blood eosinophil counts predict response, these counts do not decline in response to therapy.6364

[0106] Most recently, a cross-sectional study of patients with asthma of varying severity and endobronchial tissue gene expression analysis has revealed three major patient clusters: TH2-high, TH17-high, and TH2 / TH17-low.65In individual patient samples, TH2-high and TH17- high patterns were mutually exclusive, and their gene signatures were inversely correlated and differentially regulated by IL-13 and IL-17A. In a mouse model of allergen-induced lung inflammation, IL-4— IL- 3 blockade caused an increase in TH17 cells and neutrophilic inflammation, whereas neutralization of IL-13 and IL-17 protected mice from eosinophilia, mucus metaplasia and airway hyper-responsiveness as well as causing an attenuation of neutrophilic inflammation. It was concluded that combination therapy targeting both pathways may maximize therapeutic efficacy across a patient population comprising both TH2 and TH17 endotypes.CHAPERONIN 60.1 -RELATED PEPTIDES

[0107] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0108] Compositions useful in the methods of the present invention include, but are not limited to:DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 1) (PIN201104); DGSVVVNKVSELPAGH (SEQ ID NO: 2); GLNVNTLSYGDLAAD (SEQ ID NO: 3); SELPAGHGLNVNLTS (SEQ ID NO: 4); DGSVVVNKVS (SEQ ID NO: 5); ELPAGHGLNV (SEQ ID NO: 6); NTLSYGDLAAD (SEQ ID NO: 7); or a functionally equivalent fragment or variant thereof.

[0109] Other compositions useful in the methods of the present invention include, but are not limited to, Cpn60.1 -related peptides disclosed in United States Published Patent Application No. 200401321636Sand United States Patent Nos. 11 ,098,090;679,320,791 ;689, 085, 632.69

[0110] In certain aspects, a Cpn60.1 -related peptide is composed of amino acid residues. As used herein, the term “amino acid residue” is used interchangeably with the terms “amino acid” or “aa” to refer to an amino acid which is part of a peptide or protein. In some such aspects, an agonist or ligand of the present invention is composed of amino acids with the standard structure NH2 — C(H)(R) — COOH, where R represents an individual amino acid side chain. In certain aspects, an agonist or ligand is composed of amino acid residues which are naturally occurring amino acids. In certain aspects, a naturally occurring amino acid includes one of the twenty standard amino acids found in naturally occurring peptides and proteins. In some such aspects, an agonist or ligand is composed of at least one naturally occurring amino acid residue which is alanine (“A”), arginine (“R”), asparagine (“N”), aspartic acid (“D”), cysteine (“C”), glutamine (“Q”), glutamic acid (“E”), glycine (“G”), histidine (“H”), isoleucine (“I”), leucine (“L”), lysine (“K”), methionine (“M”), phenylalanine (“F”), proline (“P”), serine (“S”), threonine (“T”), tryptophan (“W’), tyrosine (“Y”), or valine (“V”).

[0111] In other aspects a Cpn60.1 -related peptide used in the methods of the present invention is composed of at least one amino acid residue which is an unnatural or synthetic amino acid. In some such aspects, an unnatural or synthetic amino is a chemically modified amino acid including but not limited to amino acids which have been modified by methylation, amidation, acetylation, protecting groups, and / or substitution with other chemical groups that can change the physiochemical properties of a peptide. In further aspects, an unnatural or synthetic amino is a chemically modified amino acid which has been 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, and the like).

[0112] In further aspects, a Cpn60.1 -related peptide used in the methods of the present invention is composed of at least one amino acid which has an L-configuration (the chirality of an L-amino acid). In additional aspects, a Cpn60.1 -related peptide used in the methods of the present invention is composed of at least one amino acid which has a D-configuration (the chirality of a D-amino acid).

[0113] In certain aspects, an agonist or ligand of the present invention is about 50 aa in length; 49 aa in length; 48 aa in length; 47 aa in length; 46 aa in length; 45 aa in length; 44 aa in length; 43 aa in length; 42 aa in length; 41 aa in length; 40 aa in length; 39 aa in length; 39 aa in length; 38 aa in length; 37 aa in length; 36 aa in length; 35 aa in length; 34 aa in length;33 aa in length; 32 aa in length; 31 aa in length; 30 aa in length; 29 aa in length; 28 aa in length; 27 aa in length; 26 aa in length; 25 aa in length; 24 aa in length; 23 aa in length; 22 aa in length; 21 aa in length; or 20 aa in length; 19 aa in length; 18 aa in length; 17 aa in length; 16 aa in length; 15 aa in length; 14 aa in length; 13 aa in length; 12 aa in length; 11 aa in length;10 aa in length; 9 aa in length; 8 aa in length; 7 aa in length; 6 aa in length; or 5 aa in length.

[0114] In certain aspects, compositions of the present invention are administered to a patient by any appropriate route known and / or employed by those skilled in the art. In some aspects, compositions of the present invention are administered by oral (PO), intravenous (IV), intramuscular (IM), intra-arterial, intramedullary, intrathecal, subcutaneous (SQ), intraventricular, transdermal, interdermal, intradermal, rectal (PR), vaginal, intraperitoneal (IP), intragastric (IG), topical (e.g., by powders, ointments, creams, gels, lotions, and / or drops), mucosal, intranasal, buccal, enteral, intravitreal, sublingual, by intratracheal instillation, bronchial instillation, and / or inhalation, as an oral spray, nasal spray, aerosol, and / or through a portal vein catheter.

[0115] In certain aspects, a Cpn60.1-related peptide used in the methods of the present invention and / or pharmaceutical compositions thereof may be administered intravenously, for example, by intravenous infusion. In further aspects, a Cpn60.1 -related peptide used in the methods of the present invention and / or pharmaceutical compositions thereof may be administered by intramuscular injection. In more aspects, a Cpn60.1 -related peptide used in the methods of the present invention and / or pharmaceutical compositions thereof may be administered by intratumoral injection. In certain aspects, a Cpn60.1 -related peptide used in the methods of the present invention and / or pharmaceutical compositions thereof may be administered by subcutaneous injection. In further aspects, a Cpn60.1 -related peptide used in the methods of the present invention and / or pharmaceutical compositions thereof may be administered via portal vein catheter. In more aspects, the invention encompasses the delivery of a Cpn60.1 -related peptide used in the methods of the present invention and / or pharmaceutical compositions thereof by any appropriate route taking into consideration likely advances in the art of drug delivery.

[0116] In some aspects, a Cpn60.1 -related peptide used in the methods of the present invention and / or pharmaceutical compositions thereof may be administered at dosage levels sufficient to deliver from about 0.001 mg / kg to 100 mg / kg, from about 0.01 mg / kg to 50 mg / kg, from about 0.1 mg / kg to 40 mg / kg, from about 0.5 mg / kg to 30 mg / kg, from about 0.01 mg / kg to 10 mg / kg, from about 0.1 mg / kg to 10 mg / kg, or from about 1 mg / kg to 25 mg / kg of patient body weight per day to obtain the desired therapeutic effect. In certain aspects, the desired dosage may be delivered more than three times per day, three times per day, two times per day, once per day, once every other day, once every third day, once every 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 certain aspects, the desired dosage may be delivered using multiple administrations (e g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). In certain aspects, the desired dosage may be delivered using one or more administrations during an initial period of time, followed by a period of time in which no dosage is administered.

[0117] In further aspects, a Cpn60.1 -related peptide used in the methods of the present invention may be utilized for prophylactic applications. In more aspects, prophylactic applications involve systems and methods for preventing, inhibiting progression of, and / or delaying the onset of severe asthma, in individuals susceptible to and / or displaying symptoms of severe asthma.

[0118] In certain aspects, a Cpn60.1-related peptide used in the methods of the present invention is administered to a target cell in vivo. In other aspects, a Cpn60.1 -related peptide used in the methods of the present invention is administered to a target cell ex vivo. In additional aspects, a Cpn60.1 -related peptide used in the methods of the present invention is administered to a target cell ex vivo, then the target cell is re-introduced into an organism. In some such aspects, the target cell is cultured into multiple progeny cells ex vivo before being re-introduced in an organism. In more aspects, the organism is a human. In further aspects, the organism is a human patient. In certain aspects, the target cell was originally derived from the organism to which it is re-introduced. In other aspects, the target cell was originally derived from a different organism to which it is re-introduced.

[0119] In certain aspects, a Cpn60.1-related peptide used in the methods of the present invention and / or pharmaceutical compositions thereof are employed in combination therapies for treating or reducing the risk of severe asthma. In such aspects, administration can be in combination with one or more additional therapeutic agents. As used herein, the phrases “combination therapy,” “combined with,” “in combination,” and the like, refer to the use of more than one medication or treatment simultaneously to increase the response. In certain aspects, a Cpn60.1 -related peptide used in the methods of the present invention and / or pharmaceutical compositions thereof are administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In certain aspects, a Cpn60.1 -related peptide used in the methods of the present invention and / or pharmaceutical compositions thereof are administered in combination together in a single composition or administered separately in different compositions.

[0120] In certain aspects, the particular combination of therapies to employ in a combination regimen will generally consider the compatibility of the desired therapeutics and / or procedures, and the desired therapeutic effect to be achieved. In further aspects, the therapies employed may achieve a desired effect for the same purpose (e.g., a Cpn60.1 -related peptide used in the methods of the present invention which is useful for treating, preventing, and / or delaying the onset of severe asthma may be administered concurrently with another therapeutic agent which is also useful for treating, preventing, and / or delaying the onset of severe asthma symptoms), or they may achieve different effects. In further aspects, the combination of therapies employed may achieve the same or a substantially similar desired effect for the same disease, condition or disorder; may achieve the same or a substantially similar desired effect for one ormore different diseases, conditions or disorders; may achieve different desired effects for the same disease, condition or disorder; or may achieve different desired effects for one or more different diseases, conditions or disorders.

[0121] In additional aspects, the delivery of a Cpn60.1 -related peptide used in the methods of the present invention as a pharmaceutical composition is in combination with one or more additional components that may improve the bioavailability of the Cpn60.1 -related peptide used in the methods of the present invention, reduce and / or modify its metabolism, inhibit its excretion, and / or modify its distribution in the body.

[0122] In certain aspects, combination therapy may involve administrations of a plurality of Cpn60.1 -related peptides in accordance with the present invention. In further aspects, combination therapy may involve administrations of a plurality of a Cpn60.1 -related peptides that treat, prevent, improve, achieve remission of, and / or reduce the risk of severe asthma. In more aspects, combination therapy can be a plurality of Cpn60.1 -related peptides that treat, prevent, improve, achieve remission of, and / or reduce the risk of severe asthma.

[0123] In certain aspects, a Cpn60.1-related peptide used in the methods of the present invention is combined with at least one pharmaceutically acceptable excipient, in the form of a pharmaceutical composition. As used herein, “pharmaceutical composition” refers to a formulation containing an active ingredient, and optionally a pharmaceutically acceptable carrier, diluent or excipient. The term “active ingredient” can interchangeably refer to an “effective ingredient,” and is meant to refer to any agent that is capable of inducing a sought- after effect upon administration. Examples of active ingredient include, but are not limited to, chemical compound, drug, therapeutic agent, small molecule, and the like.

[0124] In certain aspects of the present invention, the active ingredient is a Cpn60.1 -related peptide as disclosed herein. In particular aspects, the active ingredient is PIN201104, or a derivative thereof. In certain aspects, the active ingredient is PIN201360, PIN201361 , PIN201362, PIN201116, PI N201105 or a derivative thereof. In further aspects, the active ingredient is a peptide described in W02009 / 106819, or a derivative thereof.

[0125] In certain aspects, the pharmaceutical compositions are useful in medicine or the manufacture of medicaments. In further aspects, the pharmaceutical compositions are useful in one or more of the therapeutic applications disclosed herein, for example, in an individual suffering from an autoimmune disorder. In additional aspects, the pharmaceutical compositions are formulated for administration to a human patient.

[0126] In certain aspects, the pharmaceutical composition is in a sterile injectable form (e.g., a form that is suitable for subcutaneous injection or intravenous infusion). In more aspects, the pharmaceutical composition is in a liquid dosage form that is suitable for injection. In further aspects, the pharmaceutical composition is in a powder (e.g., lyophilized and / or sterilized), optionally under vacuum, which is reconstituted with an aqueous diluent (e.g., water;buffer; salt solution, and the like) prior to injection. In additional 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, and the like). In certain aspects, the pharmaceutical composition is in a form that can be refrigerated and / or frozen. In further aspects, the pharmaceutical composition is in a form that cannot be refrigerated and / or frozen. In certain aspects, the pharmaceutical composition is a reconstituted solution and / or liquid dosage form which can be stored for a certain period of time after reconstitution (e.g., 2 hours, 12 hours, 24 hours, 2 days, 5 days, 7 days, 10 days, 2 weeks, a month, two months, or longer).

[0127] In certain aspects, preparatory methods for pharmaceutical compositions include the step of bringing the active ingredient (e.g., a Cpn60.1 -related peptide used in the methods of the present invention) into association with one or more pharmaceutically acceptable excipients and then shaping and / or packaging the product into a desired single- or multi-dose unit. A pharmaceutical composition in accordance with the invention may be prepared, packaged in bulk, packaged as a single unit dose, and / or packaged as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition including a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to a dose that would be administered to a subject and / or a convenient fraction of such a dose such as, for example, one-half or one-third of such a dose. The relative amounts of active ingredient, pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition in accordance with the invention may vary, depending upon the identity, size, and / or condition of the subject treated and / or depending upon the route by which the composition is to be administered. In certain aspects, for example, the composition may include between about 0.1 % to 100% (w / w) of an active ingredient.

[0128] In another aspect, the present invention includes kits that are useful for carrying out the methods of the present invention. The components contained in the kit depend on a number of factors, including the particular application (e.g., the particular route of administration to be employed, or the particular disease, condition or disorder to be treated). In certain aspects, the present invention provides a kit for administering a Cpn60.1 -related peptide in accordance with the present invention to treat a disease, condition or disorder disclosed herein. In some such aspects, the kit further includes instructions for administration. In certain aspects, the kit is for administering a Cpn60.1 -related peptide to treat a patient with severe asthma. In certain aspects, the kits contain one or more a Cpn60.1 -related peptides. In certain aspects, the kit includes a number of unit doses of a pharmaceutical composition containing a Cpn60.1- related peptide. In additional aspects, kits for use in accordance with the present invention include instructions (e.g., for administration, for storage, and the like), buffers and / or otherreagents. In some such aspects, the kit includes (i) at least one Cpn60.1 -related peptide, (ii) a syringe, needle, applicator, or the like for administration of the at least one Cpn60.1 -related peptide to a patient, and (iii) instructions for use. In further aspects, the kit includes a treatment schedule designating when the unit dosages are to be administered. In more aspects, placebo dosages, either in a form similar to or distinct from the dosages of the pharmaceutical compositions, are included. In certain aspects, kits include one or more containers so that certain of the individual components or reagents may be separately housed. In certain aspects, kits may include a means for enclosing the individual containers in relatively close confinement for commercial sale, e.g., a plastic box, in which instructions, packaging materials such as Styrofoam, and the like, may be enclosed.

[0129] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while 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 concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0130] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0131] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.EXAMPLES

[0132] The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention.EXAMPLE 1 EFFECTIVENESS OF CPN60.1 -RELATED PEPTIDES IN AN ANIMAL MODEL OF SEVERE ASTHMA

[0133] Acute exacerbations are significant events in severe asthma and have enormous implications for patients, their caregivers and healthcare providers.70Asthma exacerbations are characterized by progressive shortness of breath, chest tightness, cough and wheezing, or a combination of these, and increased airflow obstruction that is manifested by reductions in lung function. Exacerbations accelerate disease progression, impair quality of life, cause significant morbidity and are a major cause of mortality. Preventing exacerbations is a major therapeutic goal not achieved with currently available treatments.

[0134] Viral infections are major triggers for asthma exacerbations in both adults and children.71Most viruses, including those that cause respiratory infections (rhinovirus, influenza, respiratory syncytial virus), produce dsRNA at some point during their replication.72Consequently, the TLR3 (Toll-like receptor 3) agonist, polyinosinic:polycytidylic acid (poly(l:C), poly IC or polylC), a synthetic analogue of dsRNA, has been used as a surrogate to mimic viral infections.73In this animal model, the use of poly IC was shown to elicit an exacerbated response in a model that resembles a severe asthma phenotype.74

[0135] Cpn60.1 -related peptides have already been shown to be effective for the treatment of asthma induced by house dust mite.75However, the effectiveness of Cpn60.1- related peptides have never been assessed in an animal model of severe asthma. Accordingly, present study assessed the anti-inflammatory and pulmonary functional effect of IRL201104 (20 and 80 pg / kg, i.v.) in a mouse model of asthma induced by house dust mite, as well as in a mouse model of severe asthma induced by house dust mite exacerbated by poly-IC.MATERIALS AND METHODS

[0136] The design protocol and endpoints are summarized in Table 1.Table 1 - Severe Refractory Asthma Model

[0137] Animals: Male BalbC mice (20-30 g on arrival, Charles Rivers, UK) were used in the present study.

[0138] Animal welfare: On arrival from the supplier, animals were acclimatized for period of 7 days before start of experimental procedures. Mice were housed in cages of four on arrival based on weight (equal distribution of animal weights amongst each of the cages by the animal technician) with a 12 hour light dark cycle. Room temperature and humidity were maintained between 17-24°C and 40-70%, respectively. Environmental enrichment was provided in all cages. Mice had access to standard chow ad libitum and Water was available from bottles ad libitum.

[0139] HDM sensitization and challenge: Mice were actively sensitized with house dust mite (HDM, 100 pg, s.c.) in FCA (Sigma Aldrich) on days 0. In order to elicit a local inflammatory response in the lungs, mice were intranasally challenged on day 14 with an HDM (25 pg) or saline under isoflurane (5% in 02) anesthesia. The HDM or saline was instilled into each nostril in a drop wise fashion alternating between the two until a volume of 50pL had been delivered.

[0140] Polyinosinic-polyctidylic acid challenge: High molecular mass poly l:C(30 pg / animal) or saline was administered intranasally under isoflurane (5% in O2) anesthesia 24 hours before the HDM challenge. The poly l:C or saline was instilled into each nostril in a drop wise fashion alternating between the two until a volume of 50 pL has been delivered.

[0141] Treatment of animals with dexamethasone, ‘1104 or vehicle: Non-fasted mice were weighed and placed into a hotbox for 5 min before being placed in a whole body restrainer to receive an intravenous administration (5 mL / kg) via the tail vein of eitherI RL201104 or vehicle (15 min before HDM challenge on day 14). Dexamethasone (1 mg / kg) treatment was administered orally (10 mL / kg) 1 hour before HDM challenge.

[0142] Assessment of airway hyperresponsiveness (AHR): Airway hyperresponsiveness (AHR) is the predisposition of a patient’s airways to narrow excessively in response to stimuli that would produce little or no effect in healthy subjects. 24 hours after HDM challenge, mice were anaesthetized with 100mg / kg ketamine, 10 mg / kg xylazine and 3mg / kg acepromazine. Mice were laid supine, and a tracheotomy was performed with a cannula (17 gauge) inserted into the trachea. After surgery, the mice were placed in a whole body plethysmograph and the tracheotomy tube connected to a mechanical ventilator set at a rate of 150 breaths per minute with a tidal volume of 0.15 - 0.2 mL.

[0143] The flow signal was recorded using the plethysmograph and the pressure signal was recorded from a sidearm of the tracheal catheter. The flow and pressure signals were processed together to determine lung resistance (RL) and dynamic compliance (Cdyn) using a software analyzer provided in the FinePointe resistance-compliance software (DSI Inc.). Dynamic compliance is the continuous measurement of pulmonary compliance calculated at each point representing schematic changes during rhythmic breathing.76It monitors both elastic and airway resistance. Airway resistance depends on the air viscosity, density, and length, and radius of airways.

[0144] After surgery the animals were allowed to stabilize for a period of 10 min, after which initial baseline readings were taken. Bronchoconstriction was then evoked with 10 pL aerosolized methacholine (MCh 3, 10 and 30 pg mL1for 20 s). Changes evoked by methacholine to Cdynand RLwere calculated from the difference between the baseline level (20 breath averaged before challenge) and maximum effect below or above baseline level within 5 min of challenge.

[0145] The following end point samples will be collected in a separate cohort of n=8 mice per group 48 hrs after the HDM challenge.

[0146] Blood sample collection: A terminal blood sample was collected via cardiac puncture and placed into a serum tube. Each serum sample were kept at room temperature for 45 minutes to allow coagulation, before being centrifuged (2000g, 15 min at 4°C) from which the resulting supernatant was extracted, aliquoted and stored at -80°C for analysis.

[0147] Bronchoalveolar Lavage (BAL) and Cell Counts: The trachea was cannulated to allow the lungs to be lavaged by aspirating 0.5 mL of phosphate buffered saline into the lungs. This procedure was repeated until the recovered volume was 1 ,6mL. The isolated BALF was then centrifuged at 1500 rpm for 10 mins at 4°C and the supernatant was aliquoted (400 pL) at -80°C for cytokine analysis. The cell pellets were then re-suspended in 1.6 mL of phosphate buffered saline and the BAL cells were then analyzed for total and differential numbers.

[0148] Total and differential cell counts of the BAL fluid samples were measured using a XT-2000iV analyzer (Sysmex). Results were expressed as cells / mL (total and differential). Cell types differentially classified were neutrophils, eosinophils, lymphocytes or macrophages.

[0149] Lung Tissue Collection: Following BALF collection the thoracic cavity was opened to expose the lungs, which were dissected free of the animal. The left lung lobe was placed into a sterile container and immersed in 10% neutral buffered formalin for 48 hours before being transferred to 70% ethanol for any future histopathology. Right lung lobes were placed into a sterile container and snap frozen before being stored at -80°C for future cytokine analysis.

[0150] Cytokine Analysis: Cytokine levels (see below for details of cytokines to be evaluated) of BALF supernatant (all groups) were measured using magnetic multiplex assays as per the manufacturer’s instructions. Levels were measured using a Magpix system (Luminex Corp.).

[0151] Cytokine levels of 13 mouse cytokine / chemokine using a magnetic multiplex panel (Biotechne): IFNg, IL-4, IL-5, IL-6, IL-10, IL-12(p40), IL-13, Eotaxin, IL-17, G-CSF, KC, RANTES, and MCP-1.

[0152] HDM specific IgE ELISA assay: Serum supernatant was evaluated for HDM specific IgE concentrations using ELISA kit (Condrex Inc.) as per the manufacturer’s instructions. Optical density was measured at 450 nM using a microplate reader (SpectraMax 340PC). Concentrations of IgE were determined using SoftMax Pro v. 6.4 (Molecular Devices). Data were reported as HDM specific IgE (pg / mL), mean ± S.E.M. (standard error of the mean).

[0153] Data Analysis: Inter-group deviations were statistically analyzed by a one-way analysis of variance (ANOVA). In the case of significant difference in the mean values among the different levels of treatment, comparisons versus the vehicle group will be carried out using the Dunnett’s test. In case the equal variance test fails, a Kruskal-Wallis one-way analysis of variance on ranks followed by a Dunn’s test will be proposed. p< 0.05 will be considered statistically significant.RESULTS

[0154] Bronchoalveolar Lavage (BAL) and Cell Counts: Total cell counts were significantly elevated in mice challenged with HDM (FIG. 1A) and in mice challenged with HDM and poly IC (FIG. 1 B). Cells that were elevated included eosinophils (FIG. 2A-B), neutrophils (FIG. 3A-B), macrophages (FIG. 4A-B), and lymphocytes (FIG. 5A-B). Treatment with IRL201104 was able to significantly reduce HDM-induced elevation in total cell counts (FIG. 1A), eosinophils (FIG. 2A-B), neutrophils (FIG. 3A-B), and lymphocytes (FIG. 5A-B). In mice challenged with both HDM and poly IC, IRL201104 (80 pg / kg) was able to significantly reduce HDM-induced elevation in total cell counts (FIG. 1 B), eosinophils (FIG. 2B), and neutrophils (FIG. 3B).

[0155] Assessment of airway hyperresponsiveness (AHR): Although the reductions in the elevated cell counts induced by HDM and HDM and poly IC are important, it was very important to confirm that the effects of IRL201104 also restored functional parameters.

[0156] As shown in FIGS. 6-7, lung resistance (R ) was significantly increased, and dynamic compliance (Cdyn) was significantly decreased in mice challenged with HDM (FIG. 6A and FIG. 7A, respectively) and in mice challenged with HDM and poly IC (FIG. 6B and FIG. 7B, respectively) compared to the vehicle + saline mice. Both doses of IRL201104 (20 and 80 pg / kg) significantly prevented the increase in lung resistance (RL) and decrease in dynamic compliance (Cdyn) induced by HDM (FIG. 6A and FIG. 7A, respectively) and HDM and poly IC (FIG. 6B and FIG. 7B, respectively).

[0157] Accordingly, the data of the present study indicate that I RL201104 is able to mitigate the consequences of exposure to HDM and to HDM and poly IC at both a cellular and a functional level in an animal model of severe asthma.EXAMPLE 2 FURTHER ASSESSMENT OF THE EFFECTIVENESS OF CPN60.1 -RELATED PEPTIDES IN AN ANIMAL MODEL OF SEVERE ASTHMA

[0158] The data in the previous Example support that Cpn60.1 -related peptides are effective in an animal model of severe asthma. The previous study demonstrated the antiinflammatory and pulmonary functional effect of ‘1104 (20 and 80 pg / kg, i.v.) in a mouse model of asthma induced by house dust mite, as well as in a mouse model of severe asthma induced by house dust mite exacerbated by poly-IC.

[0159] The present study provided further assessments using the higher dose of ’1104 (80 pg / kg, i.v.). Furthermore, we have also studied for the first time the effect of 1104 on airway hyperresponsiveness (AHR) measured by Resistance / Compliance.MATERIALS AND METHODS

[0160] Model: Mice were sensitized with HDM (100 pg, s.c., GREER) in FCA (Sigma) at day 1 and then intranasally challenged at day 14 with either HDM (25 pg) or saline. A second group of animals also received high molecular mass Poly l:C (30 pg / animal) or saline administered intranasally 24 hrs before HDM / saline challenge. All endpoints were taken 48h after HDM challenge, other than AHR which was measured 24 hrs after challenge.

[0161] 1104 (80 pg / kg, iv) or vehicle was administered 15 min before challenge.Dexamethasone (1 mg / kg, po) was administered 1 hour before challenge.

[0162] BALF collection, differential cell counts and cytokine / chemokine measurements: Animals were overdosed with pentobarbitone 48 hrs after HDM challenge, and bronchoalveolar lavage was carried out using phosphate buffered saline. The isolated bronchoalveolar lavage fluid (BALF) was then centrifuged at 1500 rpm for 10 min at 4°C andthe supernatant was aliquoted and stored at-80°C for cytokine analysis. Cell pellets were then re-suspended in 0.2% w / v NaCI to induce hemolysis of any erythrocytes. After isotonization with the same volume of 1 .6% w / v NaCI, the BALF cells were analyzed for total and differential cell numbers using a XT-2000iV analyzer (Sysmex).

[0163] A 14-Plex cytokine / chemokine panel (IFN-y, I L-4, IL-5, IL-6, KC, IL-10, I L-12(p40), IL-13, IL-17, Eotaxin, G-CSF, GM-CSF, RANTES and MCP-1) was run in BALF supernatant using a magnetic multiplex assay as per the manufacturer’s instructions (Biotechne Ltd). Levels were measured using a Magpix system (Luminex Corp).

[0164] HDM specific IgE ELISA assay: A terminal blood sample was collected via cardiac puncture 48 hrs after HDM challenge, and serum separated. HDM specific IgE concentration in serum was determined using an ELISA kit (Condrex Inc.) as per the manufacturer’s instructions. Optical density was measured at 450 nM using a microplate reader (SpectraMax 340PC). Concentrations of IgE were determined using SoftMax Pro v. 6.4 (Molecular Devices).

[0165] Assessment of airway hyper-responsiveness (AHR): Mice were anaesthetized and tracheotomized 24 hrs after HDM challenge. After surgery, they were placed in a whole-body plethysmograph and the tracheotomy tube was connected to a mechanical ventilator (rate 150 breaths / minute; tidal volume 0.15 - 0.2 mL). Flow signal was recorded using the plethysmograph and pressure signal was recorded from a sidearm of the tracheal catheter. Flow and pressure signals were processed together to determine lung resistance (RL) and dynamic compliance (Cdyn) using a software analyzer provided in the FinePointe resistance- compliance software (DSI Inc.).

[0166] After 10 min stabilization, initial baseline readings were taken.Bronchoconstriction was then evoked with 10 pL aerosolized methacholine (MCh 3, 10 and 30 pg / ml for 20 sec). Changes to Cdyn and RL were calculated from the difference between the baseline level (20 breath averaged before challenge) and maximum effect below or above baseline level within 5 min of challenge.

[0167] Statistical analysis: Data are shown as mean ± S.E.M. (standard error of the mean). Inter-group deviations were statistically analyzed by a one-way analysis of variance (ANOVA) followed by a Dunnett’s test or a Student’s t-test when comparing HDM / saline vs HDM / Poly l:C groups. P< 0.05 was considered statistically significant.RESULTS

[0168] Effect of treatment on HDM induced cell infiltration in the presence / absence of Poly l:C exacerbation: Exposure to intranasal HDM, 48 hrs postchallenge, triggered inflammatory infiltration in the lung as measured by BALF differential cell counts. Surprisingly, as this model is usually refractory to steroids, both dexamethasone and1104 significantly reduced HDM-induced lung infiltration of eosinophils, neutrophils, and lymphocytes but did not affect macrophages. See, FIG. 8A-D.

[0169] Exposure to Poly I :C, 24 hrs before HDM challenge, elicited exacerbated inflammatory infiltration which was significant in macrophages and lymphocytes. See, FIG. 9C-D. Dexamethasone caused a significant inhibition of eosinophils, while ‘1104 significantly reduced both eosinophilic and neutrophilic infiltration. See, FIG. 9A-B.

[0170] Effect of treatment on HDM specific IgE in serum: HDM challenge elicited an increase in HDM specific IgE in serum with was exacerbated by Poly l:C exposure. See, FIG. 10A-B. Dexamethasone treatment significantly reduced HDM specific IgE in serum in the non-exacerbated model (FIG. 10A) while 1104 was effective in both the exacerbated and nonexacerbated model (FIG. 10B).

[0171] Effect of treatment on cytokine / chemokine release in BALF: 48 hrs after allergen challenge, HDM alone or in combination with Poly l:C elicited the release of IFN-y, IL-4, IL-5, IL-6, KC, IL-10, IL-12(p40), IL-13, IL-17, Eotaxin, G- CSF, GM-CSF, RANTES and MCP-1 in BALF supernatant. Both dexamethasone and 1104 significantly suppressed these cytokines / chemokines except for IL-10, IL-17 and RANTES. See, Table 2.Table 2 - Effect of treatment on cytokines / chemokines in BALF after HDM challengeData are expressed as picograms per mL of BALF, mean ± SEM.Groups were compared to HDM / vehicle group using a one-way ANOVA, followed by a Dunnett’s test;*P<0.05,*P<0.01 ,***P<0.001 ; n=8.

[0172] Poly l:C exposure 24 hours before HDM challenge significantly exacerbated the release of IFN-y, IL-5, IL-6, KC, IL-13, G-CSF, RANTES and MCP-1. As expected, the exacerbated model was largely refractory to steroids, with dexamethasone only significantlyimpacting IL-5 and IL-6. In contrast 1104 retained a significant effect on IFN-y, IL-4, IL-5, IL-6, KC, IL-13, Eotaxin and MCP-1. See, Table 3.Table 3 - Effect of treatment on cytokines / chemokines in BALF after HDM challenge and Poly l:C exacerbationData are expressed as picograms per mL of BALF, mean ± SEM.Groups were compared to HDM / Poly l:C / vehicle using a one-way ANOVA, followed by a Dunnett’s test; *P<0.05, **P<0.01 ,***P<0.001 . A Student’s t-test was used when comparing HDM / saline / vehicle vs. HDM / Poly l:C / vehicle; ##P<0.01 , ###P<0.001 ; n=8.

[0173] Effect of treatment on Airway Hyperresponsiveness (AHR): HDM challenge caused a significant increase in total resistance (Rl) and decrease in dynamic compliance (Cdyn), which were exacerbated with Poly l:C administration. See, FIG. 11. Both ‘1104 and dexamethasone had a significant effect on Rl and Cdyn in the absence of exacerbation (FIG. 11A-C), but only 1104 remained efficacious after exacerbation (FIG. 11B-D).

[0174] These results indicate that ‘1104 showed efficacy on inflammatory markers and lung function in a HDM drivel model of allergic inflammation. Unlike steroid dexamethasone, 1104 maintained its efficacy even after Poly l:C exacerbation. The present work shows the potential of IRL201104 in asthma and other allergic inflammatory diseases even in phenotypes that do not respond to steroids.CONCLUSIONS

[0175] In this model of HDM driven allergic inflammation, with a mixed T cell background, ‘1104 showed a significant impact on inflammatory infiltration, HDM specific IgE,relevant cytokines / chemokines and lung function which was similar or better than positive control dexamethasone. As previously described, Poly l:C exacerbation rendered the model less sensitive to steroids. In the exacerbated model, unlike dexamethasone, 1104 largely maintained its efficacy on inflammatory infiltration, HDM specific IgE, relevant cytokines / chemokines and lung function. The results of present studies using a mouse model of severe asthma suggests that Cpn60.1 -related peptides are effective for the prevention and treatment of severe asthma, even in phenotypes that do not respond to steroids.REFERENCES:1Holgate, S.T. (2011). “Asthma: a simple concept but in reality a complex disease.” Eur. J. Clin. Invest.,41 : 1339-1352.2Nunes, C., et al. (2017). “Asthma costs and social impact.” Asthma Res. 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[0176] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present 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 as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0177] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the present aspects and embodiments. The present aspects and embodiments are not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect and other functionally equivalent embodiments are within the scope of the disclosure. Various modifications in addition to 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 objects described herein are not necessarily encompassed by each embodiment. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method for treating or preventing severe asthma in a subject in need thereof, the method comprising administering to the subject, a peptide related to Chaperonin 60.1 (Cpn60.1), wherein the severe asthma in the subject is resistant to one or more corticosteroid drug.

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

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

4. The method of any one of claims 1-3, further comprising, prior to administration, diagnosing the subject as having, or likely to develop, severe asthma, or receiving the results of an assay that diagnoses the subject as having, or likely to develop, severe asthma.

5. The method of any one of claims 1-4, wherein the Cpn60.1 -related peptide is administered prior to the first symptom of severe asthma, or upon clinical signs of severe asthma.

6. A method for treating severe asthma in a subject in need thereof, the method comprising the steps of:(i) administering a peptide related to Chaperonin 60.1 (Cpn60.1) to the subject upon clinical signs of severe asthma, and(ii) stopping the administration of the peptide related to Cpn60.1 upon remission of the clinical signs of severe asthma, and wherein the severe asthma in the subject is resistant to one or more corticosteroid drug.

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

8. The method of claim 7, wherein the Cpn60.1 -related peptide is SEQ ID NO:1.

9. The method of any one of claims 6-8, further comprising, prior to administration, diagnosing the subject as having severe asthma, or receiving the results of an assay that diagnoses the subject as having severe asthma.

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