CA3223191A1

CA3223191A1 - Compositions and methods for diagnosing, detecting and treating eosinophil-related diseases

Info

Publication number: CA3223191A1
Application number: CA3223191A
Authority: CA
Inventors: Gerald J. Gleich; Kristin M. Leiferman; Kathryn A. Peterson
Original assignee: University of Utah Research Foundation UURF
Current assignee: University of Utah Research Foundation UURF
Priority date: 2021-09-03
Filing date: 2022-09-02
Publication date: 2023-03-09
Also published as: WO2023034993A1

Abstract

Disclosed are compositions and methods for diagnosing, detecting and treating eosinophil-related disease or eosinophil-related inflammation in a subject. Also disclosed are compositions comprising the pro-piece of pro-eosinophil granule major basic protein- 1 or fragment thereof and methods of using the same.

Description

COMPOSITIONS AND METHODS FOR DIAGNOSING, DETECTING AND
TREATING EOSINOPHIL-RELATED DISEASES
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No.
63/240,470, filed September 3, 2021. The content of this earlier filed application is hereby incorporated by reference herein in its entirety.
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
This invention was made with government support under grant number A109728 and 1R43A1162331 awarded by National Institutes of Health. The government has certain rights in the invention.
REFERENCE TO A SEQUENCE LISTING
The Sequence Listing submitted herein as a text file named "21101 0394P1 SL.txt,"
created on September 2, 2022, and having a size of 24,576 bytes is hereby incorporated by reference pursuant to 37 C.F.R. 1.52(e)(5).
BACKGROUND
Diseases demonstrating inflammation with eosinophils have been identified by showing that peripheral blood eosinophils are increased (that is, by performing a complete blood count and differential white blood cell count). These white blood cells were named for the affinity of their cytoplasmic granules for the red dye, eosin, which makes them easily identifiable compared to other blood cells. Notably, the current technology for assessing eosinophil counts is based on the same staining methods that have been employed since about 1895. However, it is now recognized that the eosinophil is an important mediator of injury to tissues and organs in various diseases. Likely, its role in disease is underestimated because the cell loses its morphologic integrity (and is no longer identifiable) while depositing its toxic granule proteins in tissues, and the relationship of this pathophysiology with respect to peripheral blood eosinophil counts is variable.
An important element for diagnosing eosinophilic esophagitis (EoE) in a biopsy specimen is the presence of eosinophils. Normal esophageal tissue does not contain eosinophils (Kato et at, 1998). Normally, eosinophils reside in the blood stream, stomach, small and large intestine, and lymphatic system (Kato et al., 1998) but infiltrate pathologically into the esophagus in EoE. In biopsy specimens, an eosinophil can be identified as a cell 12-17 i.tm in diameter with a hi-lobed nucleus and cytoplasmic granules staining red with acidic dyes, for example, eosin. A tissue count of eosinophils in excess of 15 per field of view at high microscope power (greater than or equal to 15 per high-powered field (hp) indicates EoE.
Some clinical evidence suggests that inflammation, with accompanying disease manifestations, increases with eosinophil concentration. Strong evidence in eosinophil-related disorders involving many tissues and organs links the extracellular deposition of eosinophil granule proteins to disease manifestations, even in the absence of identifiable intact cells.
Currently, to identify an eosinophil-related disease, methods include associating an increase in eosinophil numbers in blood counts and/or demonstrating increased (morphologically identifiable) intact eosinophils in affected tissues. The identification of eosinophil granule proteins in tissues, outside of cells, is limited and problematic because of the properties of the granule proteins themselves and the relative paucity of detecting agents and methods.
In preclinical evaluations of both rat and primate models, major basic protein-1 (eMBP1) was shown to be directly responsible for an increase in airway responsiveness and bronchocon-striction as seen in clinical presentation of asthmatic patients. In the antigen-challenged guinea pig model, pretreatment with antibody to eMBP1 prevents airway hyperresponsiveness, demonstrating that blockade of eMBP1 has anti-inflammatory effects on targeted tissues. Further studies in the antigen-challenged guinea pig model shows treatment with heparin reverses vagally in-ducedbronchoconstriction.
The current treatments for eosinophil-related diseases depend on medications to suppress eosinophil production and/or the inflammation associated with eosinophil infiltration.
Glucocorticoids have long been the mainstay treatment of eosinophil-related diseases, and they both reduce inflammation and eosinophil production. Recently, new biologicals to suppress eosinophil-related inflammation by neutralization of TL-5 or by actual destruction of eosinophils using an antibody to the 1L-5 receptor a have been approved. Earlier, the role of the eosinophils in disease was debated, but now the results of clinical studies across a number of inflammatory conditions have shown that reducing eosinophils conveys clinical benefit.
Further, treatment with benralizumab (which specifically abolishes eosinophils from the body and has no other effects) is a useful medication in bronchial asthma with preliminary data for chronic rhinosinusitis, eosinophilic granulomatosis with polyangiitis as well as the hypereosinophilic syndrome.
The therapeutic agents available are used in large part to manage symptoms and reduce inflammation and are not without side effects and are associated with limited success. The main approach is to prevent allergy-related eosinophil-related diseases and disorders by controlling the body's allergic reaction. Alternatively, eosinophil-related diseases may be a sign of an

- 2 -

3 underlying condition that may not be preventable. Other treatment strategies include diet and dilation of the esophagus. Thus, alternative compositions and methods are needed to better diagnose and treat eosinophil-related diseases.
SUMMARY
Disclosed herein are methods of producing a medical image of a tissue, organ, or body part, the methods comprising: a) administering to a subject a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to label, wherein the pro-piece of pro-eMBP1 or fragment thereof binds to one or more eosinophil granule proteins in the tissue, organ, or body part of the subject, wherein said binding generates a pro-piece-eMBP1-labeled protein complex; and b) detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part, whereby detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part produces a medical image of the eosinophil granule protein localization in the tissue, organ, or body part.
Disclosed herein are methods of diagnosing eosinophil-related inflammation in a subject, the methods comprising: a) administering to a subject a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to label, wherein the pro-piece of pro-eMBP1 or fragment thereof binds to one or more eosinophil granule proteins in a tissue, organ, or body part of the subject, wherein said binding generates a pro-piece-eMBP1-labeled protein complex; and b) detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part, whereby detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part indicates eosinophil-related inflammation in the subject.
Disclosed herein are methods of detecting eosinophil degranulation in a subject, the methods comprising: a) administering to a subject a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to label, wherein the pro-piece of pro-eMBP1 or fragment thereof binds to one or more eosinophil granule proteins in a tissue, organ, or body part of the subject, wherein said binding generates a pro-piece-eMBP1-labeled protein complex; and b) detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part, whereby detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part in the subject indicates eosinophil degranulation.
Disclosed herein are methods of detecting eosinophil granule protein localization including from eosinophil degranulation in a subject, the methods comprising:
a) administering to a subject a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to label, wherein the pro-piece of pro-eMBP1 or fragment thereof binds to one or more eosinophil granule proteins in a tissue, organ, or body part of the subject, wherein said binding generates a pro-piece-eMBP1-labeled protein complex; and b) detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part, whereby detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part in the subject indicates eosinophil granule protein localization and/or eosinophil degranulation.
Disclosed herein are methods of detecting eosinophil granule proteins including from eosinophil degranulation in a subject, the methods comprising: a) utilizing a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to a supporting element, wherein the pro-piece of pro-eMBP1 or fragment thereof binds to one or more eosinophil granule proteins in a body fluid or tissue, organ, or body part extract from the subject, wherein said binding generates a pro-piece-eMBP1-support complex; and b) detecting the pro-piece-eMBP1 complex in the body fluid or tissue, organ, or body part extract, whereby detecting eMBP1-support element complex in the body fluid, tissue, organ, or body part extract from the subject indicates eosinophil granule protein localization and/or eosinophil degranulation.
Disclosed herein are methods of delivering a therapeutic agent to a diseased tissue, organ, or body part, the methods comprising, administering a therapeutically effective amount of a composition comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to a therapeutic agent to the subject.
Disclosed herein are methods of treating eosinophil-related inflammation in a subject, the methods comprising, administering a therapeutically effective amount of a composition comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof as a therapeutic agent to the subject, thereby treating eosinophil-related inflammation in the subject.
Disclosed herein are methods of neutralizing eMBP1 in a subject, the methods comprising, administering a therapeutically effective amount of a composition comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof as a therapeutic agent to the subject, thereby neutralizing eMBP1 in the subject.
Disclosed herein are methods of reducing inflammation in a subject with eosinophil-related inflammation, the methods comprising, administering a therapeutically effective amount

- 4 -of a composition comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof as a therapeutic agent to the subject, thereby reducing inflammation in the subject.
Disclosed herein are methods of treating eosinophil-related inflammation in a subject, the methods comprising, administering a therapeutically effective amount of a composition comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to a therapeutic agent to the subject, thereby treating eosinophil-related inflammation in the subject.
Disclosed herein are compositions comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to a therapeutic agent or a label.
Disclosed herein are compositions comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-C show the surface plasmon resonance (SPR) sensorgrams for pro-piece binding to eMBP-1. FIG. lA shows that triplicate 300 nM to 3.70 nM pro-piece injections over eMBP-1 surfaces do not overlay well. FIG. 1B shows 300 nM to 3.70 nM pro-piece injected over eMBP-1 surfaces. FIG. 1C shows that duplicat 300 nM pro-piece injections over EMBP-1 surfaces do not overlay well.
FIG. 2 shows the percent inhibition of eMBP1 by a pro-piece of pro-eosinophil granule major basic protein-1.
DETAILED DESCRIPTION
The present invention may be understood more readily by reference to the following detailed description of various aspects of the invention and the Examples included therein and to the Figures and their previous and following description.
Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that this invention is not limited to specific synthetic methods or specific radiolabeled contrast agents, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.
As used in the specification and the appended claims, the singular forms "a,"
"an," and -the" include plural referents unless the context clearly dictates otherwise.

- 5 -The word "or" as used herein means any one member of a particular list and also includes any combination of members of that list.
Ranges may be expressed herein as from "about" one particular value and/or to "about"
another particular value. When such a range is expressed, another aspect includesfrom the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent -about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant, both in relation to the other endpoint and independently of the other endpoint.
As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may or may not occur and that the description includes instances where said event or circumstance occurs and instances where it does not.
As used herein, the term "sample" is meant a tissue or organ from a subject; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; or a solution containing one or more molecules derived from a cell or cellular material (e.g., a polypeptide or nucleic acid), which is assayed as described herein. A sample may also be any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile) that contains cells or cell components.
As used herein the terms -amino acid" and -amino acid identity" refers to one of the 20 naturally occurring amino acids or any non-natural analogues that may be in any of the antibodies, variants, or fragments disclosed. Thus "amino acid" as used herein means both naturally occurring and synthetic amino acids. For example, homophenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention.
"Amino acid" also includes amino acid residues such as proline and hydroxyproline. The side chain may be in either the (R) or the (S) configuration. In an aspect, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradation.
"Treatment" and "treating" refer to administration or application of a therapeutic agent (e.g., a pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof described herein) to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition. For example, a treatment may include administration of a pharmaceutically effective amount of a therapeutic agent.

- 6 -As used herein, the term -treating" refers to partially or completely alleviating, ameliorating, relieving, delaying onset of, inhibiting or slowing progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment can be administered to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. For example, the disease, disorder, and/or condition can be an eosinophoilic-related inflammation or disease.
The term -fragment" can refer to a portion (e.g., at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, etc. amino acids) of a peptide that is substantially identical to a reference peptide and retains the biological activity of the reference. In some aspects, the fragment or portion retains at least 50%, 75%, 80%, 85%, 90%, 95%
or 99% of the biological activity of the reference peptide described herein. Further, a fragment of a referenced peptide can be a continuous or contiguous portion of the referenced polypeptide (e.g., a fragment of a peptide that is ten amino acids long can be any 2-9 contiguous residues within that peptide).
In some aspects, a fragment of the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) binds to and is capable of neutralizing eMBP1 or any eosinophil granule proteins.
The terms "eosinophilic- can be used to mean relating to eosinophilia. In some aspects, the terms -eosinophilic-related" and -eosinophil-related" can be used interchangeably herein.
Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a radiolabeled contrast agent is disclosed and discussed and a number of modifications that can be made to a number of molecules including the radiolabel and contrast agent are discussed, each and evey combination and permutation of the radiolabel and contrast agent and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C is disclosed as well as a class of molecules D, E, and F and an example of a combination molecule A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F is specifically contemplated and should be considered disclosed from disclosure of

- 7 -A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-groups of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in the methods of making and using the disclosed compostions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific aspect or combination of aspects of the disclosed methods and that each such combination is specifically contemplated and should be considered disclosed.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific aspects of the methods and compositions described herein. Such equivalents are intended to be encompassed by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed methods and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed methods and compositions, the particularly useful methods, devices, and materials are as described.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinence of the cited documents.
It is understood that the disclosed methods and compositions are not limited to the particular methodology, protocols, and reagents described, as these may vary.
It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings. The word -comprise" and

- 8 -variations of the word, such as -comprising" and -comprises," means -including, but not limited to" and is not intended to exclude, for example, other additives, components, integers or steps.
"Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. As used herein, by "subject" is meant an individual. A
subject can be a mammal such as a primate, for example, a human. The term "subject" also includes domesticated animals such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, possums, etc.).
As used herein, the terms "subject" and "patient" are interchangeable.
Disclosed herein are methods and compositions that can be used to identify eosinophil-related diseases by imaging the deposition of eosinophil granule proteins in affected tissues.
Using the same approach, medications can be delivered to these diseased tissues and organs.
Disclosed are compositions and methods for diagnosing eosinophil-related inflammation, detecting eosinophil degranulation, and detecting a change in an eosinophil-related disease in a subject before, during, and after treatment of an eosinophil-related disease.
Thus, also provided herein is a method of producing a medical image of a tissue, organ or body in a subject using the compositions disclosed herein. The methods disclosed herein can comprise administering to a subject a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to label. The pro-piece of pro-eMBP1 or fragment thereof can bind to one or more eosinophil granule proteins in the tissue, organ, or body part of the subject. The binding to one or more eosinophil granule proteins can generate a pro-piece-eMBP1-labeled protein complex. The method can also comprise detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part, whereby detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part can produce a medical image of the eosinophil granule protein localization in the tissue, organ, or body part. In some aspects, the localization of eosinophil granule protein in a tissue, organ, or body part can be microscopically detected.
Disclosed are compositions and methods for treating eosinophil-related inflammation.
The methods disclosed herein can comprise administering to a subject a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof In some aspects, the pro-piece of pro-eMBP1 or fragment thereof can bind to one or more eosinophil granule proteins in the tissue, organ, or body part of the subject.
The binding to one or more eosinophil granule proteins can generate a pro-piece-eMBP1-labeled protein complex,

- 9 -thereby neutralizing the one or more eosinophil granule proteins. In some aspects, the methods can reduce inflammation in a tissue, organ, or body part.
As used herein, a "mucosal tissue" is a tissue lining various cavities within the body.
Examples of a mucosal tissue include, but are not limited to, mucosal tissue lining the nose, sinuses, bronchi, lungs, conjunctiva, oral cavity, tongue, esophagus, stomach, pylorus, duodenum, jejunum, ileum, ascending colon, caecum, appendix, transverse colon, descending colon, rectum, anus, urethra, and urinary bladder. A mucosal tissue comprises an epithelial surface, glandular epithelium which secretes mucus, basement membrane, and submucosa with connective tissue. Thus, a labeled pro-piece-eMBP1-labeled protein complex can be detected on the epithelial surface, in the glandular epithelial tissue, on or in the basement membrane, and in the submucosal connective tissue of a mucosal tissue in a subject.
The term -mucous membrane" can refer to a membrane that can line any body passage that communicates with air, such as the respiratory and alimentary tracts, and having cells associated with glands that secreate mucous. Mucous membrane can also be referred to as mucosa.
As used herein, mucosa-associated lymphoid tissue (MALT) (also referred to as mucosa-associated lymphatic tissue) is a diffuse system of small concentrations of lymphoid tissue found in various submucosal membrane sites of the body, including, for example, the gastrointestinal tract, oral passage, nasopharyngeal tract, thyroid, breast, lung, salivary glands, eye, and skin.
MALT is populated by lymphocytes such as T cells and B cells, as well as plasma cells and macrophages, each of which is well situated to encounter antigens passing through the mucosal epithelium. In the case of intestinal MALT, M cells are also present, which sample antigen from the lumen and deliver it to the lymphoid tissue.
As used herein, -eosinophil degranulation" can include active piecemeal degranulation, cytolytic degranulation, and downstream granule protein deposition.
As used herein, an "eosinophil granule protein- is a protein that is contained in granules in eosinophils. When an eosinophil is activated, granule proteins are released from the cell into the surrounding tissue. The released granule proteins can cause pathologic inflammatory responses in the surrounding tissue, for example, in a tissue (e.g, mucosal tissue), organ or body part. Examples of eosinophil granule proteins include, but are not limited to, major basic protein (eMBP), major basic protein-1 (eMBP1), major basic protein-2 (eMBP2), eosinophil derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO). Other examples of eosinophil granule proteins are provided in Kita et al., Biology of Eosinophils,

- 10 -Chapter 19 of Immunology, which is hereby incorporated by reference for its teaching of examples of eosinophil granule proteins. In one aspect, an eosinophil granule protein can be eMBP-1.
As used herein, a "radiolabel- is an isotopic composition that can be attached to a substance, for example heparin, to track the substance as it passes through a system, a tissue, an organ or a body part. A non-limiting example of a radiolabeled substance is radiolabeled heparin. In one aspect, a radiolabedled heparin can be 991r1Tc-heparin.
Examples of other radiolabels include, but are not limited to, "In, 14C, 3H, 13N, 18F, 51cr, 1251,133, xe, 81mKr, and 1311. Other radiolabels that can be attached to a substance, for example, heparin, can be found in Table 1. A radiolabel, for example, 99mTc, can be attached to a substance, for example heparin, using commercially available reagents well known to persons of ordinary skill in the art.
Table 1. Examples of Commonly utilized radiolabels Nuclide Physical half-life 3H 12.3 years 20.4 minutes 13N 10 minutes 14C 5730 years 150 2 minutes 18F 110 minutes 32P 14.3 days 51cr 27.7 days 'Fe 8.3 hours 57Co 271 days 58Co 71 days "Fe 45 days 60co 5.2 years 62zn 9.3 hours 62cu 9.7 minutes 64cu 12.7 hours 'Cu 2.6 days 67Ga 78.2 hours 68Ga 68 minutes 76Br 16 hours 81mKr 8216 75 seconds 'Sr 25.5 days 86y 14.74 hours 89Zr 3.27 days 89sr 50.6 days 90Sr 28.5 years 90y 2.7 days 99Mo 66 hours

-11 -99mrrc 6.0 hours "In 2.8 days 1131n 100 minutes 1231 13.2 hours 1241 4.2 days 1251 60 days 1311 8.0 days 133Xe 5.3 days 137Cs 30 years 153 sm 1.9 days 186Re 3.8 days 2oin 73 hours The eosinophil is a peripheral blood leukocyte containing an abundance of cytoplasmic granules, rich in cationic protein toxins. Among these, the most abundant on a molar basis is the major basic protein-1, eMBP1. eMBP1 kills helminths, bacteria, and numerous cells, such as respiratory epithelium, but also activates cells, including basophils and mast cells. Studies of human diseases show that eMBP1 is present in secretions from patients with eosinophil-mediated diseases, including asthma, chronic rhinosinusitis, chronic sinus inflammation, allergic inflammation, and gastrointestinal diseases, and it is deposited on damaged targets. These studies show that the eosinophil mediates its damage to parasites and tissues by discharging its toxin rich granules onto microbial targets and tissues. eMBP1 is synthesized as a precursor, pro-eMBP1, composed of eMBP1 and a remarkably acidic pro-piece sequence. Developing eosinophils synthesize pro-eMBP1, and the pro-piece is removed during granule maturation.
Analyses of propiece in different models show that it can neutralize the toxic eMBP1 effect and also the toxicity of the eosinophil cationic protein (ECP). Disclosed herein are compositions and methods for the neutralization of eMBP1 that can be used as a treatment to mitigate tissue damage in eosinophil-related diseases. Described herein are compositions comprising the pro-piece of pro-eMBP1 and fragments thereof for treatment of eosinophil-mediated diseases by neutralization of eMBP1 and other granule toxins. Currently, no therapies for these diseases address the neutralization of granule proteins.
Eosinophil granule proteins, including eMBP1 are toxic and highly basic, and implicated in the pathogenesis of various target tissues including but not limited to the lung, GI tract, si-nuses, skin, and the eye. eMBP1 is released by activated eosinophils, eMBP1 drives inflamma-tion and clinical symptomatology in subjects. It also remains in target tissue much longer than eosinophils and it can be considered a "footprint" of eosinophils. Despite new therapies currently

- 12 -on the market for treating eosinophil-related disease, significant unmet need remains for detection and treatment of eosinophil-related diseases including but not limited to demonstrating strong binding to eMBP1.
Described herein are compositions and methods that can be used to neutralize eosinophil granule proteins, and thus, disrupt their cytotoxic effects on tissues. In some aspects, the compo-sitions and methods disclosed herein can be used to treat asthma, and reduce bronchocon-striction.
Further described herein are compositions that can be used as therapeutic agents to neutralize the effects of eMBP1 and many of the other eosinophil granule proteins. As a series of diseases are now identified in which the eosinophil plays a detrimental role, the disclosed compositions and methods can also be useful for topical use (e.g., sprays and eye drops) in diseases including but not limited to eosinophil-related bronchial asthma, eosinophil associated chronic rhinosinusitis and ocular diseases, including atopic kerato-conjunctivitis, vernal conjunctivitis and giant papillary conjunctivitis. Further, the disclosed composition can also be utilized as an orally administered medication to treat the eosinophil-mediated inflammation in eosinophilic esophagitis. Presently, no therapies address the neutralization of the granule proteins (which arguably are the mediators of eosinophil tissue damage).
COMPOSITIONS
Disclosed herein are compositions comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof Eosino_phil granule major basic protein. Eosinophil granule major basic protein (eMBP) comprises the crystalloid core of the eosinophil granule and is a 13.8 kD
single polypeptide.
eMBP plays an important role in eosinophil function; and is likely involved in endothelial injury in hypereosinophilic states. eMBP is also referred to as PRG2, BMPG, MBP, MBP1, proteoglycan 2, eosinophil granule major basic protein, proMBP, and pro eosinophil major basic protein). eMBP1 is a 117 amino acid protein (SEQ ID NO: 1) that predominates in eosinophil granules. The eMBP sequence is preceded by a putative signal peptide (e.g., SEQ ID NO: 2), and a middle sequence of amino acids (e.g. SEQ ID NO: 3); neither of which are present in the mature protein. An example of a full-length mRNA sequence for Homo sapiens proteoglycan 2, pro eosinophil major basic protein (PRG2), transcript variant 3 can be found at NCBI Reference Sequence: NM 001302926.2

- 13 -Table 2. Examples of Sequences.
SEQ ID Sequence Name NO.
1 TCRYLLVRSLQTFSQAWFTCRRCYRGNLVSIHNFNI eMBP1 GSRWNFAYWAAHQPWSRGGHCVALCTRGGHWR
RAHCLRRLPFICSY
2 MKLLLLLALLLGAVSA Putative Signal Peptide 3 LHLRSETSTFETPLGAKTLPEDEETPEQEMEETPCRE Middle Sequence;
LEEEEEWGSGSEDASKKDGAVESISVPDMVDKNLT
CPEEEDTVKVVGIPGCQ Pro-piece of Pro-eMBP 1 4 LHLRSETSTFETPLGAKTLPEDEETPEQEMEETPCRE Pro-eMBP1 LEEEEEWGSGSEDASKKDGAVESISVPDMVDKNLT
CPEEEDTVKVVGIPGCQTCRYLLVRSLQTFSQAWF
TCRRCYRGNLVSIHNFNINYRIQCSVSALNQGQVWI
GGRITGSGRCRRFQWVDGSRWNFAYWAAHQPWS
RGGHCVALCTRGGHWRRAHCLRRLPFICSY
atgaaactccccctacttctggctcttctatttggggcagtttctgct Nucleic acid sequence encoding the putative signal peptide 6 cttcatctaagglctgagacttccaccifigagacccattgggtgctaagacgctg Nucleic acid cctgaggatgaggagacaccagagcaggagatggaggagaccccttgcaggc sequence encoding agctggaggaagaggaggagtggggctctggaagtgaagatgcctccaagaa agatggggctgttgagtctatctcagtgccagatatggtgcacaaaaaccttacgt the Middle gtcctgaggaagaggacacagtaaaagtggtgggcatccctgggtgccag Sequence; Pro-piece of Pro-eMBP 1 7 acctgccgctacctcctggtgagaagtatcagacgtttagtcaagcttggtttactt Nucleic acid gccggaggtgctacaggggcaacctggittccatccacaccttcaatattatttatc sequence encoding gaatccagtgttctgtcagcgcgctcaaccagggtcaagtctggattggaggca ggatcacaggctcgggicgctgcagacgctitcagtgggttgacggcagccgct eMBP1 ggaactttgcgt, actgggctgctcaccagccctggt, cccgcggtggt, cactgcgt, ggccctg,tg,tacccgagcaggctactggcg,tcgagcccactgcctcagaagact tccificatctgttcctac S cttcatctaaggtctgagacttccacattgagacccctttgggtgctaagacgctg Nucleic acid cctgaggatgaggagacaccagagcaggagatggaggagaccccttgcaggc sequence encoding agctggaggaagaggaggagtggggctctggaagtgaagatgcctccaagaa agatggggctgttgagtctatctcagtgccagatatggtgcacaaaaaccttacgt Pro-eMBP1 gtcctgaggaagaggacacagtaaaagtgglgggcatccctgggtgccagacc

- 14 -tgccgclaccicclgglgagaagtclicagacgttlaglcaagctlgglItacttgcc ggaggtgctacaggggcaacctgg,iftccatccacaccttcaatattatttatcgaa tccagtgttctgtcagcgcgctcaaccagggtcaagtctggattggaggcaggat cacaggctcgggtcgctgcagacgattcagtgggttgacggcagccgctgga actagcgtactgggctgctcaccagccctggtcccgcggtggtcactgcgtggc cctgtgtacccgagcaggctactggcgtcgagcccactgcctcagaagacttcct ticatetglicctac 9 MKLLLLLALLLGAVS Putative Signal Peptide ALHLRSETSTFETPLGAKTLPEDEETPEQEMEETPC A variant of Pro-RELEEEEEWGSGSEDASKKDGAVESISVPDMVDKN .
LTCPEEEDTVKVVGIPGCQ piece of Pro-eMBP 1 11 HLRSETSTFETPLGAKTLPEDEETPEQEMEETPCREL A variant of EEEEEWGSGSEDASKKDGAVESISVPDMVDKNLTC
PEEEDTVKVVGIPGCQ
Pro-piece of Pro-eMBP 1 ; bold indicates N-linked gly can 12 MKLLLLLALLLGAVSAL Putative Signal Peptide 13 ALHLRSETSTFETPLGAKTLPEDEETPEQEMEETPC Pro-eMBP1 with a RELEEEEEWGSGSEDASKKDGAVESISVPDMVDKN
LTCPEEEDTVKVVGIPGCQTCRYLLVRSLQTFSQA variant of pro-WFTCRRCYRGNLVSIHNFNINYRIQCSVSALNQGQ piece of pro-VWIGGRITGS GRCRRFQWVDGSRWNFAYWAAHQP
eMBP1 WSRGGHCVALCTRGGHWRRAHCLRRLPFIC SY
14 HLRSETSTFETPLGAKTLPEDEETPEQEMEETPCREL Pro-eMBP1 with a EEEEEWGSGSEDASKKDGAVESISVPDMVDKNLTC
PEEED'TVKVVGIPGCQTCRYLLVRSLQTF S QAWFT variant of pro-CRRCYRGNLVSIHNFNINYRIQCSVSALNQGQVWIG piece of pro-GRITGSGRCRRFQWVDGSRWNFAYWAAHQPWSR
eMBP1 GGHCVALCTRGGHWRRAHCLRRLPFICSY
MKLLLLLALLLGAV SAL Putative Signal HLRSETSTFETPLGAKTLPEDEETPEQEMEETPCREL
EEEEEWGSGSEDASKKDGAVESISVPDMVDKNLTC Peptide and Pro-PEEEDTVKVVGIPGCQ Piece 16 ETSTFETPLGAKTLPEDEETPEQEMEETPCRELEEEE Fragment of Pro-EWGSGSEDASKKDGAVESISVPDMVDKNLTCPEEE
DTVKVVGIPGCQ
piece of Pro-eMBP1

- 15 -17 HLRSETSTFETPLGAKTLPEDEETPEQEMEETPCREL Fragment of Pro-EEEEEWGSGSEDASKKDGAVESISVPDMVDKNLTC
PEEEDTVKVVGIPGCQ piece of Pro-eMBP1 18 SETSTFETPLGAKTLPEDEETPEQEMEETPCRELEEE Fragment of Pro-EEWGSGSEDASKKDGAVESISVPDMVDKNLTCPEE
EDTVKVVGIPGCQ piece of Pro-eMBP1 19 SETSTFETPLGAKTIPEDEETPEQEMEETPCRELEEE Fragment of Pro-EEWGSGSEDASKKDGAV
piece of Pro-eMBP1 20 EEEEEWGSGSEDASKKDGAV Fragment of Pro-piece of Pro-eMBP1 In some aspects, the Putative Signal Peptide sequence can be one amino acid shorter than SEQ ID NO: 2. For example, in some aspects the Putative Signal Peptide sequence can be MKLLLLLALLLGAVS (SEQ ID NO: 9). In some aspects, the Putative Signal Peptide sequence can be one amino acid shorter than SEQ ID NO: 2. For example, in some aspects, the Putative Signal Peptide sequence can be MKLLLLLALLLGAVSAL (SEQ ID NO: 12).
In some aspects, the Middle Sequence (Pro-piece of pro-eMBP1sequence) can be one amino acid longer than SEQ ID NO: 3. For example, in some aspects the Middle Sequence (Pro-piece of pro-eMBP1sequence) sequence can be ALHLRSETSTFETPLGAKTLPEDEETPEQEMEETPCRELEEEEEWGSGSEDASKKDGAVE
SISVPDMVDKNLTCPEEEDTVKVVGIPGCQ (SEQ ID NO: 10). In some aspects, the the Middle Sequence (Pro-piece of pro-eMBP1sequence) can be one amino acid shorter than SEQ
ID NO: 3. For example, in some aspects, the Middle Sequence (Pro-piece of pro-eMBP1sequence) can be HLRSETSTFETPLGAKTLPEDEETPEQEMEETPCRELEEEEEWGSGSEDASKKDGAVESIS
VPDMVDKNLTCPEEEDTVKVVGIPGCQ (SEQ ID NO: 11).
In some aspects, the Pro-eMBP1 sequence can be one amino acid longer than SEQ
ID
NO: 4. For example, the Pro-eMBP1 sequence can be ALHLRSETSTFETPLGAKTLPEDEETPEQEMEETPCRELEEEEEWGSGSEDASKKDGAVE
SISVPDMVDKNLTCPEEEDTVKVVGIPGCQTCRYLLVRSLQTFSQAWFTCRRCYRGNLV
SIHNFNINYRIQCSVSALNQGQVWIGGRITGSGRCRRFQWVDGSRWNFAYWAAHQPWS

- 16 -RGGHCVALCTRGGHWRRAHCLRRLPFICSY (SEQ ID NO: 13). In some aspects, the Pro-eMBP1 sequence can be one amino acid shorter than SEQ ID NO: 4. For example, the Pro-eMBP1 sequence can be HLRSETSTFETPLGAKTLPEDEETPEQEMEETPCRELEEEEEWGSGSEDASKKDGAVESIS
VPDMVDKNLTCPEEEDTVKVVGIPGCQTCRYLLVRSLQTFSQAWFTCRRCYRGNLVSIH
NFNINYRIQCSVSALNQGQVWIGGRITGSGRCRRFQWVDGSRWNFAYWAAHQPWSRG
GHCVALCTRGGHWRRAHCLRRLPFICSY (SEQ ID NO: 14).
In some aspects, the fragments of pro-piece of pro-eMBP1 can comprise a sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% identity to SEQ ID
NO: 3. In some aspects, the fragment or portion of pro-eMBP1 retains at least 50%, 75%, 80%, 85%, 90%, 95% or 99% of the biological activity of the reference protein described herein.
In some aspects, the variants of pro-piece of pro-eMBP1 can comprise a sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% identity to SEQ
ID NOs:
10 or 11. In some aspects, the variants of pro-eMBP1 retains at least 50%, 75%, 80%, 85%, 90%, 95% or 99% of the biological activity of the reference protein described herein.
In some aspects, the fragments of pro-eMBP1 can comprise a sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% identity to SEQ ID NO:
4. In some aspects, the fragment or portion of pro-eMBP1 retains at least 50%, 75%, 80%, 85%, 90%, 95% or 99% of the biological activity of the reference protein described herein.
In some aspects, the fragment of the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) can comprise 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 amino acids of SEQ ID NO: 3 or another number in between. In some aspects, the fragment of the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) can comprise SEQ
ID NOs: 16, 17, 18, 19 or 20. In some aspects, the fragment or portion of pro-eMBP1 retains at least 50%, 75%, 80%, 85%, 90%, 95% or 99% of the biological activity of the protein comprising the sequence of SEQ ID NO: 3 described herein.
In some aspects, the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment are chemical analogs or peptidomimetics.
An -analog" of a molecule such as a peptide refers to a molecule similar in function to either the entire molecule or to a fragment thereof The term "analog" is also intended to include induced variants. Analogs typically differ from naturally occurring peptides at one or a few positions, often by virtue of conservative substitutions. Analogs typically exhibit at least 80 or 90% sequence identity with natural peptides. Some analogs also include unnatural amino acids or

- 17 -modifications of N or C terminal amino acids. Examples of unnatural amino acids are, for example but not limited to; di substituted amino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline, y-carboxyglutamate, c-N,N,N- trimethyllysine, c-N-acetyllysine, 0-phosphoserine, N-acetylserine, N- formylmethionine, 3-methylhistidine, 5-hydroxylysine, G-N-methyl arginine.
In some aspects, the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof disclosed herein can further comprise a peptide or polypeptide having one or more amino acid residues with a modified side chain. In some aspects, one or more amino acids of any of the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof disclosed here can have a modified side chain.
Examples of side chain modifications include but are not limited to modifications of amino acid groups, such as reductive alkylation; amidination with methylacetimidate; acylation with acetic anhydride;
carbamolyation of amino groups with cynate; trinitrobenzylation of amino acid with 2,4,6-trinitrobenzene sulfonic acid (TNBS); alkylation of amino groups with succinic anhydride; and pyridoxylation with pridoxa1-5-phosphate followed by reduction with NaBH4.
The pro-piece of pro-eosinophil granule major basic protein-1 contains carbohydrates. In some aspects, one or more of the carbohydrates present in the pro-piece of pro-eosinophil granule major basic protein-I can be used to label for example, with Technetium99m. See, for example, Popken-Harris et al., The Journal of Immunology, 1995, 155:1472-1480, which is incorporated herein by reference. While eosinophil major basic protein is highly basic, the pro piece of pro-eosinophil granule major basic protein-1 comprises acidic amino acids as well as heparan-like glycosaminoglycan. In some aspects, the pro-piece of pro-eosinophil granule major basic protein-1 comprises a serine at position 46 of SEQ ID NO: 3 that is derivatized with a carbohydrate. In some aspects, the carbohydrate can be a glycosaminoglycan. In some aspects, the glycosaminoglycan at position 46 of SEQ ID NO: 3 can be associated with a change in the charge. For example, in some aspects, the negative charge can be due to sulfation of the glycosaminoglycan moiety. In some aspects, the carbohydrate is heparan. Thus, the pro-piece of pro-eosinophil granule major basic protein-1 is capable of binding to eosinophil major basic protein by the same mechanism as heparin. In some aspects, the pro-piece of pro-eosinophil granule major basic protein-I (pro-eMBP1) or fragment thereof disclosed herein can combine binding to eMBP1 and a heparin/heparan binding.
In some aspects, one or more site(s) of carbohydrate attachment can be at one or more amino acid residues of SEQ ID NO: 3. In some aspects, one or more site(s) of carbohydrate

- 18 -attachment can be at one or more of the amino acid residues 7T, 8S, 9T, I 8T, 46S, or 70N of SEQ ID NO: 3. In some aspects, amino acid residue 70N can be the site of an N-linked glycan attachment. In some aspects, amino acid residues 7T, 8S, 9T, and 18T can be the site of an 0-linked glycan attachment. In some aspects, amino acid residue 46S can be the site of an 0-linked glycosaminoglycan attachment.
In some aspects, the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof can be modified. hi some aspects, pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof can be be amidated, acetylated, or both. Such modifications can be at the amino or carboxy terminus of the polypeptide. The pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof can also include peptidomimetics.
In some aspects, the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof described herein can be further modified to improve stability. In some aspects, any of the amino acid residues of the the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof described herein can be modified to improve stability. In some aspects, the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof can have at least one amino acid residue that has an acetyl group, a fluorenylmethoxy carbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, or polyethylene glycol. In some aspects, an acetyl protective group can be bound to the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof described herein.
In aspects, the the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof can have at least one amino acid substitution (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 substitutions), insertion, or deletion. The pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof can contain, for example, 1 to 12, 1 to 10, 1 to 5, or 1 to 3 amino acid substitutions, or 1 to 10 (e.g., to 9, 8, 7, 6, 5, 4, 3, 2) amino acid substitutions. The amino acid substitution(s) may be conservative or non-conservative.
As used herein, the term -stability" refers to storage stability (e.g., room-temperature stability) as well as in vivo stability. The foregoing protective group can protect the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof described herein from the attack of protein cleavage enzymes in vivo. In some aspects, the construction of the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof can include incorporating one or more D-amino acids into the polypeptide. In some aspects, the

- 19 -construction of the pro-piece of pro-eosinophil granule major basic protein- I
(pro-eMBP1) or fragment thereof can include incorporating one or more L-amino acids into the polypeptide.
The construction of peptides incorporating D-amino acids can be particularly useful when greater in vivo or intracellular stability is desired or required. More specifically, D- peptides are resistant to endogenous peptidases and proteases, thereby providing better delivery, improved bioavailability, and prolonged intravascular and interstitial lifetimes when such properties are desirable.
In some aspects, the pro-eMBP1 peptide has a calculated mass of 23.5 kDa. In some aspects, the the pro-eMBP1 peptide has a measured mass of 50-90kDa (CH0=26-76kDa). In some aspects, the pro-piece of pro-eMBP1 has a calculated mass of 10kDa. In some aspects, the pro-piece of pro-eMBP1 has a measured mass of approximately 25kDa (CH0-15kDa).
In some aspects, functional equivalents of the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof are included with the scope of the invention described herein. As used herein, the term "functional equivalents" can refer to amino acid sequence variants having an amino acid substitution, addition, or deletion in some of the amino acid sequence of the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof while simultaneously having similar or improved biological activity, compared with the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof as described herein. In some aspects, the amino acid substitution can be a conservative substitution. Examples of the naturally occurring amino acid conservative substitution include, for example, aliphatic amino acids (Gly, Ala, and Pro), hydrophobic amino acids (Ile, Leu, and Val), aromatic amino acids (Phe. Tyr, and Trp), acidic amino acids (Asp and Glu), basic amino acids (His, Lys, Arg, Gin, and Asn), and sulfur-containing amino acids (Cys and Met). In some aspects, the amino acid deletion can be located in a region that is not directly involved in the activity of the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof disclosed herein.
In some aspects, the amino acid sequence of the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof described herein can include a peptide sequence that has substantial identity to any of sequence of the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof disclosed herein. As used herein, the term "substantial identity" means that two amino acid sequences, when optimally aligned and then analyzed by an algorithm normally used in the art, such as BLAST, GAP, or BESTFIT, or

- 20 -by visual inspection, share at least about 60%, 70%, 80%, 85%, 90%, or 95%
sequence identity.
Methods of alignment for sequence comparison are known in the art.
In some aspects, the amino acid sequence of the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof described herein can include a peptide sequence that has some degree of identity or homology to any of sequences of the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof disclosed herein.
The degree of identity can vary and be determined by methods known to one of ordinary skill in the art. The terms "homology- and -identity" each refer to sequence similarity between two polypeptide sequences. Homology and identity can each be determined by comparing a position in each sequence which can be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same amino acid residue, then the polypeptides can be referred to as identical at that position; when the equivalent site is occupied by the same amino acid (e.g., identical) or a similar amino acid (e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous at that position. A percentage of homology or identity between sequences is a function of the number of matching or homologous positions shared by the sequences. The pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof described herein can have at least or about 25%, 50%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity or homology to the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof, wherein the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof is SEQ
ID NO: 3.
In some aspcts, the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof can be produced by recombinant technology or chemical synthesis.
Therapeutic agents and labels. In some aspects, the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof can be conjugated to a therapeutic agent or a label. In some aspects, the therapeutic agent can be benralizumab. In some aspects, the therapeutic agent can be an IL-6 inhibitor, an IL-17 antibody, an IL-12/23 antibody, an IL-5 antibody or IL-5 inhibitor, and IL-4/13 antibody, an integrin inhibitor, a JAK
kinase inhibitor, sphingosine phosphate inhibitor, a calcineurin inhibitor (e.g., cyclosporine or tacrolimus), an anti-IgE antibody, anti-inflammatory agent (e.g., anti-eotaxin, anti-siglet 8, anti-TSLP, anti-IL-31, anti-TNF, anti-TFG, interferon) or a combination thereof. In some aspects, the therapeutic agent can be one that disrupts inflammation. For example, the therapeutic agent can be one that disrupts protein synthesis or reduces cell proliferation. In some aspects, the therapeutic agent

- 21 -can be ruxolitnib. In some asepcts, the pro-piece of pro-eosinophil granule major basic protein- I
(pro-eMBP1) or fragment thereof can be conjugated to a polymer of aspartic acid or glutamic acid. See, for example, Barker RL, et al., J Clin Invest 88:798-805, 1991, which is incorporated herein by reference.
In some aspects, one or more of the cysteine residues present in the Pro-piece of pro-eMBP1 or fragment thereof can be a site for conjugation. In some aspects, one or more of the cvsteine residues at positions 35, 73, or 88 as identified in SEQ ID NO: 3 in the Pro-piece of pro-eMBP1 or fragment thereof can serve as a reactive sulfhydryl group. In some aspects, the reactive sulfhydryl group can be used to label for example, with Technetium99m. In some aspects, one or more of the lysine residues present in the Pro-piece of pro-eMBP1 or fragment thereof can be a site for conjugation.
In some aspects, the label can be fluorochrome, an enzyme, a substrate, a glycoprotein, a solid-phase immobilizing agent, an ultrasound signal-detection agent, a laser-induced ultrasound optoacoustic signal-dection agent, a radioisotope, or a radiolabel. In some aspects, the label can be a radioisotope. In some aspects, the label can be Technetium99m, barium or gadolinium. In some aspects, the radioisotope can be Technetium99m (991u1Tc). Examples of radiolabels can be found in Table 1.
PHARMACEUTICAL COMPOSITIONS
As disclosed herein, are pharmaceutical compositions, comprising pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof described herein.
Also disclosed herein, are pharmaceutical compositions, comprising pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof described herein and a pharmaceutical acceptable carrier. Further disclosed herein are pharmaceutical compositions for producing a medical image of a tissue, organ, or body part; diagnosing eosinophil-related inflammation in a subject; detecting a change in an eosinophil-related disease in a subject diagnosed with an eosinophil-related disease; and detecting eosinophil degranulation in a subject.
Also disclosed herein are pharmaceutical compositions for delivering a therapeutic agent to a diseased tissue, organ, or body part; and treating eosinophilic-related inflammation in a subject.
In some aspects, the pharmaceutical compositions can comprise: a therapeutically effective amount of a composition comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof disclosed herein; and a pharmaceutically acceptable carrier.

- 22 -The pharmaceutical compositions described above can be formulated to include a therapeutically effective amount of a pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof Therapeutic administration encompasses prophylactic applications. Based on genetic testing and other prognostic methods, a physician in consultation with their patient can choose a prophylactic administration where the patient has a clinically determined predisposition or increased susceptibility (in some cases, a greatly increased susceptibility) to a type of eosinophil-related inflammation or eosinophil-related disease.
The pharmaceutical compositions described herein can be administered to the subject (e.g., a human patient) in an amount sufficient to delay, reduce, or preferably prevent the onset of clinical disease. Accordingly, in some aspects, the patient can be a human patient. In therapeutic applications, compositions can be administered to a subject (e.g., a human patient) in an amount sufficient to at least partially improve a sign or symptom or to inhibit the progression of (and preferably arrest) the symptoms of the condition, its complications, and consequences (e.g., eosinophil-related inflammation or eosinophil-related disease). An amount adequate to accomplish this is defined as a "therapeutically effective amount." A
therapeutically effective amount of a pharmaceutical composition can be an amount that achieves a cure, but that outcome is only one among several that can be achieved. As noted, a therapeutically effective amount includes amounts that provide a treatment in which the onset or progression of eosinophil-related inflammation or eosinophil-related disease or a symptom of eosinophil-related inflammation or eosinophil-related disease is ameliorated. One or more of the symptoms can be less severe.
Recovery can be accelerated in an individual who has been treated.
In some aspects, the pharmaceutical composition can be formulated for intravenous administration. In some aspects, the pharmaceutical composition can be formulated for subcutaneous, intranasal or oral administration. In some aspects, the compositions of the present disclosure also contain a therapeutically effective amount of the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof alone or conjugated to a therapeutic agent. In some aspects, the therapeutic agent can be glucocorticoid, a tumor necrosis factor (TNF) inhibitor, a phosphodiesterase (PDE) inhibitor, a calcineurin inhibitor, a thiopurine, a integrin inhibitor, eotaxin inhibitor, a JAK kinase inhibitor, IL13 inhibitor, a IL4/13 inhibitor, or a transforming growth factor (TGF) modulator. The compositions can be formulated for administration by any of a variety of routes of administration, and can include one or more physiologically acceptable excipients, which can vary depending on the route of administration.
As used herein, the term -excipient" means any compound or substance, including those that can

- 23 -also be referred to as -carriers" or -diluents." In some aspects, the compositions can include a pharmaceutically acceptable adjuvant, carrier or other diluent. Examples of adjuvants, include, but are not limited to aluminum-(alum-)free adjuvants, which are formulated in the absence of any such aluminum salts. Alum-free adjuvants include oil and water emulsions, such as water-in-oil, and oil-in-water (and variants therof, including double emulsions and reversible emulsions), liposaccharides, lipopolysaccharides, liposomes, and various combinations of such components.
Preparing pharmaceutical and physiologically acceptable compositions is considered routine in the art, and thus, one of ordinary skill in the art can consult numerous authorities for guidance if needed.
Pharmaceutically acceptable carriers and excipients can be incorporated (e.g., water, saline, aqueous dextrose, and glycols, oils (including those of petroleum, animal, vegetable or synthetic origin), starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monosterate, sodium chloride, dried skim milk, glycerol, propylene glycol, ethanol, and the like). The compositions may be subjected to conventional pharmaceutical expedients such as sterilization and may contain conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting or emulsifying agents, salts for adjusting osmotic pressure, buffers, and the like. Suitable pharmaceutical carriers and their formulations are described in -Remington's Pharmaceutical Sciences" by E.W. Martin, which is herein incorporated by reference. Such compositions will, in any event, contain an effective amount of the compositions together with a suitable amount of carrier so as to prepare the proper dosage form for proper administration to the patient.
The pharmaceutical compositions as disclosed herein can be prepared for oral or parenteral administration. Pharmaceutical compositions prepared for parenteral administration include those prepared for intravenous (or intra-arterial), intramuscular, subcutaneous, intraperitoneal, transmucosal (e.g., intranasal, intravaginal, or rectal), or transdermal (e.g., topical) administration. Aerosol inhalation can also be used to deliver the decoy peptide. Thus, compositions can be prepared for parenteral administration that includes the the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment or a therapeutic agent dissolved or suspended in an acceptable carrier, including but not limited to an aqueous carrier, such as water, buffered water, saline, buffered saline (e.g., phosphate buffered saline (PBS)), and the like. One or more of the excipients included can help approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents, and the like. Where the compositions include a solid component (as they may for oral

- 24 -administration), one or more of the excipients can act as a binder or filler (e.g., for the formulation of a tablet, a capsule, and the like). Where the compositions are formulated for application to the skin or to a mucosal surface, one or more of the excipients can be a solvent or emulsifier for the formulation of a cream, an ointment, and the like. In some aspects, the compositions described herein can be formulated as a spray. In some aspects, the compositions described herein can be formulated as eye drops.
The pharmaceutical compositions can be sterile and sterilized by conventional sterilization techniques or sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation, which is encompassed by the present disclosure, can be combined with a sterile aqueous carrier prior to administration. The pH of the pharmaceutical compositions typically will be between 3 and 11 (e.g., between about 5 and 9) or between 6 and 8 (e.g., between about 7 and 8). The resulting compositions in solid form can be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules. The composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.
In some aspects, the compositions described herein can be pharmaceutical compositions.
In some aspects, the compositions described herein can be administered to a subject orally, intravenously, topically (e.g., skin or mucuous membranes, including oral, genital and conjunctival), intra-orificially (e.g., through a body orifice, including oral, intra-nasal, intra-auricle, inner or outer ear, intra-vesicular, per rectum), intra-articularly, or ocularly. In some aspects, the compositions described herein can be administered to a subject via a medical device or tube. In some aspects, the medical device or tube can be a nasal-gastric tube or a radiology-placed cannula. Oral dosing, for example, can entail ingestion similar to routine barium studies of the esophagus. Any of the compositions described herein (e.g., pro-piece of pro-eosinophil granule major basic protein-I (pro-eMBP1) or fragment thereof conjugated to label) can be suspended in a thickened mixture (i.e., sucralose). Examples of thickening agents include, but are not limited to, dietary starches, such as agar-agar, alginate, carrageenan, cassia gum, cellulose gum, gellan gum, guar gum, hydroxypropylcellulose, konjac gum, locust bean gum, methylcellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, pectin, and xanthan gum. Other viscosifiers include honey, agave nectar, date nectar, Kuzu or Kudzu root, arrow root, corn syrup, thick juices, maple syrup, coconut oil, and palm oil.
METHODS OF DIAGNOSIS AND DETECTION

- 25 -Disclosed herein are methods of producing a medical image of a tissue, organ, or body part or a combination thereof Disclosed herein are methods of producing a medical image of one or more of a tissue, organ, or body part or a combination thereof Also disclosed herein are methods of diagnosing eosinophil-related inflammation in a subject. Further disclosed herein are methods of detecting eosinophil degranulation in a subject. Further disclosed herein are methods of detecting an eosinophil granule protein (e.g., major basic protein 1) in a sample. Further disclosed herein are methods of detecting eosinophil degranulation in a sample.
In some aspects, the methods can comprise administering to a subject a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to label. In some aspects, the pro-piece of pro-eMBP1 or fragment thereof can bind to one or more eosinophil granule proteins in the tissue, organ, or body part of the subject. In some aspects, said binding can generate a pro-piece-eMBP1-labeled protein complex. In some aspects, the methods can include detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part, whereby detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part can produce a medical image of the eosinophil granule protein localization in the tissue, organ, or body part. In some aspects, the localization of eosinophil granule protein in a tissue, organ, or body part can be microscopically detected. In some aspects, the methods can comprise detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part, whereby detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part can indicate eosinophil-related inflammation in the subject. In some aspects, the methods can comprise detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part, whereby detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part in the subject can indicate eosinophil degranulation.
Disclosed herein are methods of detecting eosinophil granule protein localization including from eosinophil degranulation in a subject. The methods can comprise: administering to a subject a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to label. In some aspects, the pro-piece of pro-eMBP1 or fragment thereof can bind to one or more eosinophil granule proteins in a tissue, organ, or body part of the subject. In some aspects, the binding can generate a pro-piece-eMBP1-labeled protein complex. In some aspects, the methods can further comprise detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part, whereby

- 26 -detecting the pro-piece-eMBP I -labeled protein complex in the tissue, organ, or body part in the subject can indicate eosinophil granule protein localization and/or eosinophil degranulation.
Disclosed herein are methods of detecting one or more eosinophil granule proteins in a subject, the methods comprising: utilizing a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to a supporting element, wherein the pro-piece of pro-eMBP1 or fragment thereof binds to one or more eosinophil granule proteins in a body fluid or tissue, organ, or body part extract from the subject, wherein said binding generates a pro-piece-eMBP1-support complex; and b) detecting the pro-piece-eMBP1-support complex in the body fluid or tissue, organ, or body part extract, whereby detecting the pro-piece-eMBP1-support complex in the body fluid, tissue, organ, or body part extract from the subject indicates eosinophil granule protein localization. In some asepcts, detecting the pro-piece-eMBP1 complex in the body fluid, tissue, organ, or body part extract from the subject indicates eosinophil degranulation. In some aspects, the pro-piece-eMBP1-support complex can be 99mTc-pro-eMBP1/MBP-1, 99mTc- pro-eMBP1/MBP, 99mTc-pro-eMBP1/MBP-2, 99mTc- pro-eMBP1/EDN, 99mTc- pro-eMBP1/ECP, and 99mTc- pro-eMBP1/EPX. In some aspects, a support element can be 99" "In-heparin, In-heparin, or 14C-heparin. Thus, a labeled pro-piece-eMBP1-support complex can be detected on the epithelial surface, in the glandular epithelial tissue, on or in the basement membrane, and in the submucosal connective tissue of a mucosal tissue in a subject.
Disclosed herein are methods of detecting one or more eosinophil granule proteins in a sample, the methods comprising: contacting a sample with a pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to a supporting element, wherein the pro-piece of pro-eMBP1 or fragment thereof binds to one or more eosinophil granule proteins in the sample, wherein said binding generates a pro-piece-eMBP1-support complex; and detecting the pro-piece-eMBP1-support complex in the sample, whereby detecting the pro-piece-eMBP1-support complex in the sample thereby detecting a eosinophil granule protein in the sample.
Disclosed herein are methods of detecting one or more eosinophil granule proteins in a sample, the methods comprising: contacting a sample with a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to a supporting element under conditions to allow the pro-piece of pro-eMBP1 or fragment to bind to one or more eosinophil granule proteins in a sample, wherein said binding

- 27 -generates a pro-pi ece-eMBP 1-support complex; and detecting the pro-pi ece-eMBP I-support complex in the sample, thereby detecting a eosinophil granule protein in the sample.
Disclosed herein are methods of detecting one or more eosinophil granule proteins in a sample, the methods comprising: utilizing a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to a supporting element, wherein the pro-piece of pro-eMBP1 or fragment thereof binds to one or more eosinophil granule proteins in a sample. wherein said binding generates a pro-piece-eMBP1-support complex; and detecting the pro-piece-eMBP1-support complex in the sample, whereby detecting the pro-piece-eMBP1-support complex in the sample indicates eosinophil granule protein localization.
In some aspects, the sample can be a body fluid or tissue, organ, or body part extract. In some aspects, the sample can be obtained from a subject. In some aspects, the sample can be a biopsy. In some aspects, the biopsy can be an esophageal biopsy. In some aspects, the esophageal biopsy can be from the distal esophagus, proximal esophagus or a combination thereof In some aspects, the esophageal biopsy can be taken from a furrow, plaque, or edema area. In some asepcts, detecting the pro-piece-eMBP1 complex in the sample indicates eosinophil degranulation. In some aspects, the pro-piece-eMBP1-support complex can be 99"Tc-pro-eMBP1/MBP-1, 99mTc- pro-eMBP1/MBP, 99mTc- pro-eMBP1/MBP-2, 99mTc- pro-eMBP1/EDN, 99mTc- pro-eMBP1/ECP, and 99mTc- pro-eMBP1/EPX. In some aspects, a support element can be 99"Tc-heparin, "In-heparin, or 14C-heparin. In some aspects, in any of the methods disclosed herein, the pro-piece-eMBP1 can be used as a reagent. As disclosed herein, a labeled pro-piece-eMBP1-support complex can be detected on the epithelial surface, in the glandular epithelial tissue, on or in the basement membrane, and in the submucosal connective tissue of a mucosal tissue inform or in a subject.
Disclosed herein are methods detecting a change in an eosinophil-related disease in a subject. In some aspects, the subject can be diagnosed with an eosinophil-related disease. In some aspects, the method can comprise a) producing a first medical image of a tissue, organ, or body part in a subject diagnosed with an eosinophil-related disease. In some aspects, the method can comprise administering to a subject a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to label.
In some aspects, the pro-piece of pro-eMBP1 or fragment thereof can bind to one or more eosinophil granule proteins in the tissue, organ, or body part of the subject.
In some aspects, said binding can generate a pro-piece-eMBP1-labeled protein complex. In some aspects, the method

- 28 -can comprise b) producing a second medical image of the tissue, organ, or body part in the subject of step (a) by repeating the step of administering to the subject subject a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to label. In some aspects, the pro-piece of pro-eMBP1 or fragment thereof can bind to one or more eosinophil granule proteins in the tissue, organ, or body part of the subject. In some aspects, said binding can generate a pro-piece-eMBP1-labeled protein complex. In some aspects, the method can comprise c) comparing the medical image of step (b) with the medical image of step (a), whereby detecting a change in the medical image of step (b) compared to the medical image of step (a) can detect a change in the eosinophil-related disease in the subject.
In some aspects, the eosinophil granule protein can be major basic protein 1 (eMBP1). In some apects, the eosinophil granule protein can be eosinophil cationic protein (ECP). In some apects, the eosinophil granule protein can be eosinophil peroxidase (EPX).
In some aspects, the label can be a fluorochrome, an enzyme, a substrate, a glycoprotein, a solid-phase immobilizing agent, an ultrasound signal-detection agent, a laser-induced ultrasound optoacoustic signal-dection agent, a radioisotope, or a radiolabel.
Examples of fluorochromes include fluorescent dyes. Examples of enzymes and substrates include peroxidases and/or peroxidase substrates). Examples of glycoproteins include avidin and biotin.
In some aspects, the label can be a radioisotope. In some aspects, the radioisotope can be Technetium99m (99"iTc).
A person of ordinary skill in the art can administer to a subject a composition comprising pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof alone or conjugated to label to enhance the detection of eosinophil granule proteins in a tissue, organ or body part in a subject. For example, such a composition can be conjugated to 99mTc-heparin, "In-heparin, or 14C-heparin, or any combination thereof For example, such a composition can be conjugated to a radioisotope or label as described herein without using heparin. In some aspects, a label (or radioisotope) can be Technetium99m (99mTc). Examples of a pro-piece-eMBP1-labeled protein complex include, but are not limited to, 99mTc-pro-eMBP 1/MBP-1, 99'"Tc- pro-eMBP 1/MBP, 99"11"c- pro-eMBP 1 /MBP-2, 99mTc- pro-eMBP 1 /EDN, 99mTc- pro-eMBP1/ECP, and 99mTc- pro-eMBP1/EPX.
The dwell time, for example, in the esophagus can be controlled by varying the viscosity of the compositions and by increasing the time interval between swallows, thereby providing more time for the composition to contact and bind to an eosinophil granule protein. Further,

- 29 -having a subject lie down with head below feet, so that there is some reflux within the esophagus, can prolong contact between the composition and the mucosal tissue of the esophagus in a subject.
Any of the compositions described herein can be administerd to a subject in a volume from about 0.1 mL to about 1,000 mL, including all volumes in between 0.1 mL
and 1,000 mL.
A person of ordinary skill can determine by methods well known in the art the volume of the compositions described herein to be administered to a subject based on the age, sex, weight, and overall condition of a subject. For example, in some aspects, the volume of the composition administered to a subject can be from about 0.1 mL to about 5 mL. Thus, the volume of the composition that can be administered to a subject can be, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, or 5 mL, and all volumes in between. In some apects, the volume of the composition administered to a subject can be about 0.1 mL to about 1 mL. Thus, the volume of the composition that can be administered to a subject can be, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mL, and all volumes in between.
In some apects, the volume of the composition administered to a subject can be about 1 mL to about 5 mL. Thus, the volume of the composition that can be administered to a subject can be, for example, about 1, 2, 3, 4, or 5 mL, and all volumes in between. In some aspects, the volume of the composition administered to a subject can be from about 5 mL to about 250 mL. In some aspects, the volume of the composition administered to a subject can be from about 10 mL to about 125 mL. In some aspects, the volume of the composition administered to a subject can be from about 15 mL to about 100 mL. Thus, the volume of the composition that can be administered to a subject can be, for example, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,

30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mL, and all volumes in between.
After administering to a subject a composition comprising pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof alone or conjugated to label, for example 99mTc, a person of skill can use one or more technologies and processes to detect pro-piece-eMBP1-labeled protein complexes in a tissue, organ, or body part in a subject. For example, pro-piece-eMBP1-labeled protein complexes can be detected where eosinophils have degranulated and caused one or more patches of inflammation, to create a medical image to map the distribution of inflmmation and deposition of eosinophil granule proteins to study the anatomy and/or pathophysiology of eosinophil-related inflammation, and changes in eosinophil-related inflammation or disease. Examples of technologies that can be used to create a medical image include, but are not limited to, single photon emission computed tomography (SPECT), positron emission (PET) scans, X-ray, conventional or computed tomography (CT), a combination of SPECT and CT, or magnetic resonance imaging (MRI). In one aspect, for example, SPECT can optionally be used in combination with MRI and/or CT scans to produce a medical image of a tissue, organ or body part having patches of eosinophilic-related inflammation or disease. Fiduciary markers on the skin of a subject can also be used to position a subject so that the subject can be imaged from day to day. For example, lasers can be used to position a subject reproducibly. This permits use of multiple scans to be precisely compared. In some aspects, a medical image can be three-dimensional. In some aspects, a medical image can be two-dimensional. In some aspects, the medical image can be a microscopic image. In some aspects, the medical image can be used to assess pathology specimens.
In some apects, any of the methods disclosed herein can comprise one or more of the following steps. In some aspects, the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof can be conjugated to reduced Technetium-99m (Tc99m) using a cysteine residue as the amino acid for attachment. In some aspects, 3 mg pro-piece of pro-eMBP1 can be labeled with 3 mCi Tc99m. In some aspects, the pro-piece of pro-eMBP1-Tc99m (3 mCi and 3 mg pro-piece of pro-eMBP1) can be present in 15 mL of water. In some aspects, the methods can comprise a subject swallowing the pro-piece of pro-eMBP1-Tc99m in 1 mL sips over a 15 minute period (start at zero time). In some aspects, at t=30 minute, SPECT/CT can be performed. In some aspects, at 60 minutes, a subject can swallow 100 mL of water (as a wash) in 15 swallows of about 6.5 mL over a 15 minute period. In some aspects, at 75 minutes the SPECT/CT can be repeated.
The radiolabeled pro-piece of pro-eMBP1 or fragment thereof disclosed herein can be prepared at various doses. For example, the radiolabeled pro-piece of pro-eMBP1 or fragment thereof can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 mCi. In some aspects, the dose of radiolabeled pro-piece of pro-eMBP1 or fragment thereof can be 0.3 mCi. In some aspects, the dose of radiolabeled pro-piece of pro-eMBP1 or fragment thereof can be 1.0 mCi. In some aspects, the dose of radiolabeled pro-piece of pro-eMBP1 or fragment thereof can be 10 mCi.
In some aspect, the radiolabeled pro-piece of pro-eMBP1 or fragment thereof can be Tc-99m-pro-piece of pro-eMBP1. In some aspects, the doses of Tc-99m-pro-piece of pro-eMBP1 can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0,

- 31 -9.5, or I0.() mCi. In some aspects, the dose of Tc-99m-pro-piece of pro-eMBP I
can be 0.3 mCi.
In some aspects, the dose of Tc-99m-pro-piece of pro-eMBP1 can be 1.0 mCi. In some aspects, the dose of Tc-99m-pro-piece of pro-eMBP1 can be 10 mCi. In some aspects, 1-100 mg of pro-piece of pro-eMBP1 can be labeled with 0.1 to 30 mCi of a radiolabel (e.g.
technetium-99M). In some aspects, the ratio of pro-piece of pro-eMBP1 to Tc-99m can be 1:1.
In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be tissue- or organ-specific. In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be specific for the gastrointestinal tract, lung, nose, eye, skin, one or more joints, one or more muscles, one or more nerves, heart, kidney, bladder, uterus, prostate, breast, lymph or blood.
In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be an eosinophilic gastrointestinal disorders. Examples of an eosinophilic gastrointestinal disorders include but are not limited to eosinophilic esophagitis, eosinophilic gastritis, eosinophilic enteritis, eosinophilic cholecystitis, and eosinophilic colitis. In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be an eosinophilic pancreatitis. In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be an eosinophilic hepatitis. In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be an eosinophilic ascites. In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be a pulmonary eosinophoilic syndrome. Examples of a pulmonary eosinophilic syndrome include but are not limited to eosinophilic asthma, eosinophilic bronchitis, eosinophilic pneumonia, and eosinophil pleuritic. In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be an eosinophilic myocarditis. In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be eosinophilic coronary periarteritis. In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be eosinophilic rhinosinusitis. In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be eosinophilic nasal polyposis. In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be an eosinophilic ocular disorder. Examples of eosinophilic ocular disorder include but are not limited to allergic conjunctivitis (e.g., seasonal and perennial), giant papillary conjunctivitis, and keratoconjunctivitis (atopic and vernal)). In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be an eosinophilic nephritis. In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be an eosinophilic cystitis. In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be an eosinophilic

- 32 -prostatitis. In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be an eosinophilic endometritis. In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be an eosinophilic myometritis (uterus). In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be an eosinophilic mastitis. In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be an eosinophil-related neuropathy. In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be an eosinophilic synovitis. In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be an eosinophilic myositis. In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be an eosinophilic panniculitis. In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be an eosinophilic fasciitis (Shulman syndrome). In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be chronic rhinosinusitis.
In some aspects, the eosinophil-related disease can be eosinophilic cystitis, eosinophilic fasciitis, eosinophilic colitis, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic granulomatosis with polyangiitis, eosinophilic pneumonia, hypereosinophilic syndrome, vernal conjunctivitis, giant papillary conjunctivitis, atopic dermatitis, chronic rhinosinusitis or transplant rejection In some aspects, the eosinophil-related inflammation can be caused by a parasitic disease;
an allergic reaction; asthma; an autoimmune disease; a drug reaction; an environmental exposure; a topical contact; a genetic disease; a transplant rejection, a hematologic or lymphocytic disease, or an inflammatory or immunological reaction with expression of eosinophil differentiating, chemoattracting, activating factors or a combination thereof.
Examples of a parasitic disease can include but is not limited to helminthic infections and ectoparasites. Examples of drug reactions include but are not limited to drug hypersensitivity reactions (e.g., drug reactions with eosinophilia and systemic symptoms (DRESS) with potential for prolonged sequelae). In some aspects, the eosinophil-related inflammation can be caused by a solid tumor (e.g., a malignancy), a lymphoma or a leukemia. In some aspects, the activating factor can be a marker for a cancer. In some aspects, eosinophils can indicate a gastrointestinal cancer.
In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be eosinophil-related syndrome.
In some aspects, eosinophil-related syndromes can include eosinophilia myalgia syndrome (EMS) and toxic oil syndrome (TOS). Eosinophilia myalgia syndrome and toxic oil

-33 -syndrome include but are not limited to severe myalgi a plus hypereosinophilia (peripheral blood and/or tissue) or eosinophilia, often accompanied by neurologic symptoms and skin changes.
Epidemic cases of EMS have been attributed to contaminated L-tryptophan exposure. Epidemic cases of TOS have been attributed to rapeseed oil denatured with aniline.
In some aspects, eosinophil-related syndromes can include eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome). Symptoms of eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome) include but are not limited necrotizing vasculitis with hypereosinophilia; antineutrophil cytoplasmic antibodies (e.g., ANCA1 and subvariants); 4 of 6 criteria including asthma, eosinophilia, history of allergy, nonfixed pulmonary infiltrates, paranasal sinus abnormalities, and extravascular eosinophils.
In some aspects, eosinophil-related syndromes can include episodic angioedema with eosinophilia (Gleich syndrome). Episodic angioedema with eosinophilia (Gleich syndrome) can include but is not limited to cyclic recurrent angioedema, hypereosinophilia, and increased IgM
levels, often with clonal T cells, one of several possible clinical presentations of secondary/reactive hypereosinophilic syndromes). Hypereosinophilic syndromes can include peripheral blood hypereosinophilia, hypereosinophilia-related organ damage.
In some aspects, eosinophil-related syndromes can include hyper-IgE syndromes.
Hyper-IgE syndromes can include but are not limited to hereditary immunodeficiency syndromes with hypereosinophilia and increased IgE levels, often with eczema and facial anomalies; and known gene mutations: autosomal dominant hyper-IgE syndrome, signal transducer and activator of transcription 3 (STAT3) mutations and autosomal recessive hyper-IgE syndrome, dedicator of cytokinesis 8 (DOCK8) mutations.
In some aspects, eosinophil-related syndromes can include IgG4-related diseases. IgG4-related diseases include but are not limited to a spectrum of disorders with fibrosis as a major finding, tumor-like swelling of tissues and organs, tissue eosinophilia, and increased IgG4.
In some aspects, eosinophil-related syndromes can include Omenn syndrome.
Omenn syndrome includes but is not limited to severe combined immunodeficiency with hypereosinophilia, often with erythroderma, hepatosplenomegaly, and lymphadenopathy and autosomal recessive genetic disease (recurrent mutations in recombination-activating gene (e.g., RAG1 or RAG2).
In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be an eosinophil-related dermatoses. Disclosed herein are different and overlapping compartments associated with a varity of diseases, including but not limited to: epidermis 4 eosinophilic

- 34 -spongiosis; dermis, connective tissue eosinophilic cellulitis; dermis, blood vessels eosinophilic vasculitis; hair follicles 4 eosinophilic folliculitis;
subcutaneous fat 4 eosinophilic panniculitis; fascia 4 eosinophilic fasciitis; muscle 4 eosinophilic myositis;
and nerve 4 eosinophilic neuritis.
In some aspects, the eosinophil-related inflammation or eosinophil-related disease can be allergic contact dermatitis; angiolymphoid hyperplasia with eosinophilia;
annular erythema of infancy; atopic dermatitis; bullous pemphigoid and pemphigoid variants;
coccidiomycosis; drug eruptions; eosinophilic fasciitis; eosinophilic, polymorphic, and pruritic eruption associated with radiotherapy; eosinophilic pustular folliculitis: all variants; erythema toxicum neonatorum;
eosinophilic ulcer of the oral mucosa; eosinophilic vasculitis; granuloma faciale; infestations (parasites/ectoparasites, including scabies, bed bugs, and cutaneous larva migrans); incontinentia pigmenti; kimura disease; langerhans cell histiocytosis; mycosis fungoides and Sezary syndrome/cutaneous lymphoma; pachydermatous eosinophilic dermatitis;
pemphigoid variants, including bullous pemphigoid and pemphigoid gestationis; pemphigus variants;
pregnancy-related dermatoses; pseudolymphoma; urticaria/angioedema; vasculitis; or Wells syndrome (various diseases with eosinophilic cellulitis). See Starr J, et al, Mayo Clin Proc 75:755-759, 2000; incorporated herein by reference.
In some aspects, the eosinophil-related disease can be eosinophilic cystitis, eosinophilic fasciitis, eosinophilic colitis, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic granulomatosis with polyangiitis, eosinophilic pneumonia, hypereosinophilic syndrome, vernal conjunctivitis, giant papillary conjunctivitis, atopic dermatitis, or transplant rejection.
Additional examples of eosinophil -related diseases, eosinophil-related etiopathogenesis, and patterns of eosinophil involvement that can be treated or diagnosed using the compsotions disclosed herein can include one or more of the disease or disorders provided in the Tables disclosed herein.
Additional diseases or disorders that can be diagnosed or treated using the disclosed compostions include, but are not limited to the diseases or disorders described in Table E2 in Valent P. et al. Contemporary consensus proposal on criteria and classification of eosinophilic disorders and related syndromes. J Allergy Clin Immunol. 2012;130(3):607-12;
and Table 1 in Mejia R, Nutman TB. Evaluation and differential diagnosis of marked, persistent eosinophilia.
Semin Hematol 2012;49(2):149-59; Leiferman KM, Peters MS. Eosinophils in Cutaneous Diseases, Chap 36, Table 36-11 in Fitzpatrick's Dermatology in General Medicine, 8th Edition,

-35 -Goldsmith LA, Katz SI, Gilchrest BA, Paller AS, Leffell DJ, Wolff K, eds.
McGraw Hill Medical, San Francisco, pp 386-400, 2012; Leiferman KM, Peters MS. Eosinophils in Cutaneous Diseases, Chap 36, Table 36-5 in Fitzpatrick's Dermatology in General Medicine, 8th Edition, Goldsmith LA, Katz SI, Gilchrest BA, Paller AS, Leffell DJ, Wolff K, eds.
McGraw Hill Medical, San Francisco. pp 386-400, 2012; Leiferman KM, Gleich GJ, Peters MS.
Dermatologic manifestations of the hypereosinophilic syndromes, Box 1, Immunol Allergy Clin North Am.
2007;27(3):415-41, Epub 2007/09/18; Table 1 in Valent P. et al., Contemporary consensus proposal on criteria and classification of eosinophilic disorders and related syndromes. J Allergy Clin Immunol. 2012;130(3):607-12 e9, Epub 2012/03/31; Leiferman KM. Eosinophil-Associated Diseases with Dermatologic Manifestations. Table 9-7 In: Callen JP, Jorizzo JL, editors.
Dermatological Signs of Systemic Disease: Elsevier; 2017. p. 69-76; Leiferman KM, Peters MS.
Eosinophil-Associated Dermatoses, Table 25-1 In: Bolognia JL, Schaffer JV, Cerroni L, editors.
Dermatology: Elsevier 2017. p. 440-52; and McKenna JK, Leiferman KM.
Dermatologic drug reactions In Immunol Allergy Clin North Am 2004 (24, pp. 399-423, vi) which are all hereby incorporated by reference.
Table 3. Diseases/disorders with eosinophil-related etiopathogenesis Strong/direct Weaker/indirect Allergic reactions Adrenal insufficiency (Addison disease) Atheroembolic disorders/cholesterol embolization Atopic diseases Autoimmune lymphoproliferative syndrome Drug reactions Chronic graft-versus-host disease Ectoparasitic infestations:
Chronic inflammatory disorders, including inflammatory bowel scabies, pediculosis, disease pthiriasis, cimicosis Helminth infections Fibrotic reactions Fungal infections: allergic bronchopulmonary aspergillosis, Hypereosinophilic coccidioidomycosis, paracoccidioidomycosis, syndromes basidiobolomycosis, histoplasmosis, cryptococcosis Immunobullous diseases: Human Immunodeficiency Virus (HIV) and Human T-Cell particularly, pemphigoid Lymphotropic Virus (HTLV) I and II
Myiasis IgG4-related diseases Immunodeficiency syndromes: hyperimmunoglobulin E
syndromes, Omenn syndrome, IPEX, ZAP-70 deficiency Irradiation

- 36 -Langerhans cell histiocytosis Leukemias: acute myelogenous, B-cell, acute lymphoblastic/lymphocytic Lymphomas: Hodgkin's, T-cell, B-cell Mastocytosis Sarcoidosis Solid tumors/malignancy (development or progression) Table 4. Cutaneous manifestations demonstrating extracellular eosinophil granule protein deposition and patterns of eosinophil involvement Clinical presentations = Eczematous dermatoses o Atopic dermatitis o Onchocercal dermatitis o Prurigo nodularis o Pachydermatous eosinophilic dermatitis = Edema o Chronic urticaria o Solar urticaria o Delayed pressure urticaria o IgE-mediated late phase reaction, many possible allergens o Episodic angioedema with eosinophilia (Gleich syndrome) o Facial edema with eosinophilia o Nodules, eosinophilia, rheumatism, dermatitis, swelling syndrome (NERDS) o Granulocyte macrophage-colony stimulating factor (GM-CSF) reaction o Interleukin (IL)-2 capillary leak syndrome o Eosinophilic cellulitis (Wells syndrome) = Blistering o Pemphigoid (herpes) gestationis o Bullous pemphigoid o Bullous morphea o Eosinophilic granulomatosis with polyangiitis blisters

- 37 -o Incontinentia pigmenti = Sclerosis or Fibrosis o Eosinophilic fasciitis o Eosinophili a myalgi a syndrome o Toxic oil syndrome = Vasculitis o Recurrent cutaneous eosinophilic vasculitis o Eosinophilic vasculitis in connective tissue disease o Eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome) Patterns of eosinophil involvement based on eosinophil granule protein tissue staining = Eosinophil infiltration of few to many intact cells = Intact eosinophils with extracellular eosinophil granule protein deposition proportionate to the numbers of infiltrating eosinophils = Intact eosinophils with extensive extracellular eosinophil granule protein deposition disproportionately greater than the numbers of infiltrating eosinophils = Extensive extracellular eosinophil granule protein deposition with few or no infiltrating eosinophils (in this pattern, eosinophil involvement may not be recognized on histopathological examination) Each pattern may be found either with or without peripheral blood eosinophilia/hypereosinophilia Table 5. Eosinophils in histopathological diagnoses Eosinophils are an important feature in diagnosis = Annular erythema of infancy = Eosinophilic, polymorphic, and pruritic eruption associated with radiotherapy = Eosinophilic pustular folliculitis o Classical (Ofugi disease) o Infantile/neonatal o Human immunodeficiency virus-associated = Eosinophilic annular erythema = Eosinophilic cellulitis (Wells syndrome) = Eosinophilic dermatosis of hematologic malignancy = Erythema toxicum neonatorum = Eosinophilic ulcer of oral mucosa = Hypereosinophilic dermatitis of Nir-Westfried

-38 -= Hypereosinophilic syndromes = IgG4-related cutaneous diseases o Angiolymphoid hyperplasia with eosinophilia o Granuloma faciale o Kimura disease = Juvenile temporal arteritis = Pachydermatous eosinophilic dermatitis Diseases commonly associated with tissue eosinophils = Arthropod bite and sting reactions = Autoimmune blistering diseases o Pemphigoid o Pemphigus o Epidermolysis bullosa acquisita = Dermatoses of pregnancy o Pemphigoid gestationis o Polymorphic eruption of pregnancy, pruritic urticarial papules and plaques of pregnancy = Drug reactions o DRESS (drug reaction with eosinophilia and systemic symptoms) also known as drug-induced hypersensitivity syndrome o Interstitial granulomatous drug reaction o Cutaneous involvement other, many drugs and many reaction patterns = Eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome) = Hereditary syndromes o Incontinentia pigmenti o Hyper IgE syndromes and other immunodeficiency syndromes (Table 2) o Autoimmune lymphoproliferative syndrome = Histiocytic diseases o Langerhans cell histiocytosis o Juvenile xanthogranuloma = Itchy, red bump disease (papular dermatitis) = Oid-oid disease (exudative discoid and lichenoid chronic dermatosis of Sulzberger and Garbe) = Papuloerythroderma of Ofugi = Parasitic diseases/infestations o Cysticercosis, dirofilariasis, fascioliasis, gnathostomiasis, larva migrans, loiasis, myiasis, onchocerciasis, paragonimiasis, schistosomiasis, stronffloidiasis, tungiasis o Scabies, pediculosis, pthiriasis, cimicosis o Swimmer's itch (cercarial dermatitis) and seabather's itch = Pruritic papular eruption of human immunodeficiency virus disease = Sclerosis or fibrosis o Eosinophilic fasciitis o Eosinophilia myalgia and toxic oil syndromes o Drugs including statins o Iron infusion o Lymphoma and leukemia

-39 -o Graft versus host disease following stem cell or bone marrow transplantation = Urticaria and angioedema = Vasculitis Eosinophils of limited or unreliable value in histopathological diagnosis = Granuloma annulare = Interstitial granulomatous dermatitis = Lymphoproliferative disorders (except HES variants) o Mycosis fungoides o Anaplastic large cell lymphoma (except neutrophil-rich and eosinophil-rich variant) o Lymphomatoid papulosis = Mastocytosis = Neoplasms o Keratoacanthoma-type squamous cell carcinoma o Invasive squamous cell carcinoma Table 6. Flame figures in skin biopsy specimens Flame figures in skin biopsy specimens = Angiolymphoid hyperplasia with eosinophilia = Arthropod bite and sting reaction = Dermographism = Drug reaction = Eczema = Eosinophilic cellulitis (Wells syndrome) = Eosinophilic fasciitis = Eosinophilic granulomatosis with polyangiitis (Churg Strauss syndrome) = Eosinophilic pustular folliculitis = Fungal disease including dermatophyte infection (Splendore Hoeppli phenomenon may be same/similar) = Herpes simplex infection = Human immunodeficiency virus infection = Hypereosinophilic syndromes = Immunobullous diseases (pemphigoid, pemphigoid gestationis, pemphigus vegetans) = lncontinentia pigmenti = Kimura disease = Malignancies, solid organ (bronchogenic carcinoma, colonic adenocarcinoma) = Mastocytoma = Molluscum contagiosum = Myeloproliferative disorders involving skin = Parasite infestation (ascariasis, gnathostomiasis, onchocerciasis, toxocariasis) = Urticaria = Vaccination site reactions = Varicella

- 40 -Table 7. Mucocutaneous manifestations in hypereosinophilic syndromes Angioedema Bulla(e) Dermographism Digital gangrene Eczema Eosinophilic cellulitis (flame figures of Wells syndrome) Erosions Erythema Erythema annulare centrifugum Erythroderma Excoriation(s) Livedo reticularis Lymphomatoid papulosis Macules Mucosal ulcer(s) (oral and genital) Nail fold infarction(s) Necrosis Nodules Papules Patches Pruritus Purpura Raynaud's phenomenon Splinter hemorrhage(s) Ulcer(s) Urticaria Vasculitis

- 41 -Table 8. Eosinophil-related cutaneous disease; Biopsy specifications for testing and site selection.
Test performed on sections from Additional Biopsy site by lesion type skin/mucosal specimen*
considerations New pink-erythematous macular or papular lesion Histopathology More than one biopsy OR may be indicated if Brightfield microscopic Border of erythematous or edematous clinical morphology is examination of formalin-fixed, lesion varied paraffin-embedded, hematoxylin and eosin stained tissue sections OR
Blister edge, or entire blister if small (less than 4-mm punch) In a patient with Border of a new erythematous or generalized pruritus but edematous skin lesion no primary skin lesions, Direct immunofluorescence (DIF)** DIF may be obtained from normal-appearing OR
Testing for immunoglobulins, Perilesional tissue immediately flexural skin of upper complement, and fibrinogen adjacent to a blister arm or thigh or from the performed on sections from tissue trunk placed in Michel or Zeus transport Skin or mucosal biopsy For mucosa, medium or from snap-frozen tissue from a blister or erosion, non-lesional tissue from region of must be mostly "intact"
active disease (if biopsy too close to Note: DIF cannot be performed on (epithelium attached to lesion, the epithelium is likely to formalin-fixed tissue underlying detach) dermis/stroma) Eosinophil-specific immunostain New pink-slightly erythematous lesion/affected area Complete blood count, Cellular and extracellular with differential white eosinophil granule protein blood cell count, to localization on sections from tissue placed in Michel or Zeus transport OR
assess peripheral blood eosinophil level medium, formalin-fixed or snap- Perilesional tissue immediately frozen tissue adjacent to a blister * Biopsy size = A 4-mm or 5-mm punch biopsy is preferred if the specimen is submitted for a single test = A 6-mm or larger diameter punch biopsy is preferred when a single specimen will be split into 2 parts:
1/2 for histopathology and 1/2 for direct immunofluorescence = Note that the preferred site for the two tests may differ = If using a single specimen for 2 tests, carefully bisect the tissue to minimize epithelial separation ** Serum testing by indirect immunofluorescence and/or ELISA for antibodies associated with immunobullous diseases may provide diagnostic information

- 42 -Table 9. Definition and gradations of peripheral blood eosinophilia and tissue hypereosinophilia in eosinophil-related diseases.
(1) Peripheral Blood Counts x 1094_, blood Eosinophils >1.5 recorded on at least 2 determinations with a minimum time interval of 4 weeks (unless immediate designation is Hypereosinophilia juired to initiate therapy because of eosinophil-related organ dysfunction) Eosinophilia 0.5-1.5 0.05-0.5 Normal (1-6% of white blood cell count range) (2) Tissue hypereosinophilia The percentage of eosinophils exceeds 20% of all nucleated bone marrow cells, and / or Pathologist is of the opinion that tissue eosinophil infiltration is extensive (massive) [pared with the normal physiological range, compared with other inflammatory cells or both, and / or A specific eosinophil granule protein stain (e.g. eosinophil granule major basic protein 1, 3P1, or eosinophil-derived neurotoxin, EDN) demonstrates extensive extracellular deposition cative of local eosinophil activation and degranulation even in the absence of local eosinophil tration No eosinophils or extracellular eosinophil granule protein 3sition except as known in specific organs and tissues (bone Normal row, spleen, thymus, lymph nodes, gastrointestinal tract il to esophagus); occasional intravascular eosinophils may be rved by histopathology, particularly with increased pheral blood eosinophils Table 10. Histopathological patterns with eosinophil-related inflammation in different cutaneous compartments.
Histopathological patterns with eosinophil-related inflammation in different cutaneous compartments Epidermis Eosinophilic spongiosis Connective tissue in dermis Eosinophilic cellulitis Blood vessels in dermis Eosinophilic vasculitis Hair follicles in dermis Eosinophilic folliculitis Subcutaneous fat Eosinophilic panniculitis Fascia Eosinophilic fasciitis

- 43 -Muscle Eosinophilic myositis = Each pattern may be found in various diseases = Overlapping patterns in one disease are common = Patterns may change as a disease develops/persists Table 11. Cutaneous presentations with different diagnoses of eosinophil-related diseases Cutaneous signs I symptom Eosinophil-related disease diagnostic differential Angioedema / dermographism / Cellulitis edema / urticaria Drug reaction Eosinophilia myalgia and toxic oil syndromes Eosinophilic cellulitis (Wells syndrome) Erysipelas Hereditary angioedema Hypereosinophilic syndromes Immunodeficiency syndromes Insect bite or sting Mast cell disease Parasitic infection Urticaria Urticarial vasculitis Blisters and / or ulceration Aphthous stomatitis Behcet syndrome Drug reaction Erythema multiforme Herpes simplex infection Hypereosinophilic syndromes Immunobullous disease Incontinentia pigmenti Insect bite or sting Lichen planus Lesions in diseases in the box above (angioedema /
dermographism / edema / urticaria) may blister Dermatitis / eczema / pruritus Atopic dermatitis Contact dermatitis Drug reaction Ectoparasite infestation Fungal infection Hypereosinophilic syndromes Immunodeficiency syndromes Parasitic infection Erythema / pruritus Drug reaction Ectoparasite infestation Hypereosinophilic syndromes Sezary syndrome Seborrheic dermatitis Fibrosis / sclerosis IgG4-related diseases

- 44 -Eosinophilia myalgia and toxic oil syndromes Eosinophilic fasciitis Morphea Hyperpigmentation Adrenal insufficiency (Addison disease) Drug reaction Hemochromatosis Hyperthyroidism Nodules / papules / plaques / Acne, including acne neonatorum pustules Acropustulosis Epith el i oi d angi sarcoma Epithelioid hernangioendothelioma Erythema toxicum neonatorurn Folliculitis Follicular mucinosis Fungal infection Hypereosinophilic syndromes IgG4-related diseases Angiolymphoid hyperplasia with eosinophilia Granuloma faciale Kimura disease Kaposi sarcoma Lichen planus Lupus erythematosus Mast cell disease Morphea Mycosis fungoides Nodules, eosinophilia, rheumatism, dermatitis and swelling (NERDS) Palmar¨plantar pustular psoriasis Pityriasis lichenoides chronica Pyogenic granuloma T-cell lymphoma Vasculitis Various, miscellaneous disease- Asthma associated Crohn disease Erythema multiforme Fungal infection Immunodeficiency syndromes including hyper IgE
syndromes Reactive arthritis (formerly Reiter syndrome) Sarcoidosis Syphilis Ulcerative colitis Table 12. Eosinopohil-related cutaneous diseases; clinical clues.
Diagnosis Clinical clues Allergic contact dermatitis History of exposure; distribution pattern; positive patch test Angiolymphoid hyperplasia with Single or multiple nodules of face, scalp and ears; vascular

- 45 -eosinophilia proliferation with prominent endothelial cells, and mixed mainly lymphocytic inflammation, with eosinophils Annular erythema of infancy Annular or serpiginous non-pruritic plaques in infants Arthropod bite and sting reactions Primarily involves exposed skin; spiders, ticks, fleas, mites and mosquitoes; exaggerated reactions in patients with chronic lymphocytic leukemia Atopic dermatitis History of atopy; pruritus; flexural and extremity accentuation Autoimmune bullous dennatoses Pemphigoid (particularly bullous pemphigoid, pemphigoid gestationis), pemphigus, linear IgA bullous dermatosis, epidermolysis bullosa acquisita and less often dermatitis herpetiformis; initial phase of bullous pemphigoid and pemphigoid gestationis may be urticarial Eosinophilic spongiosis, eosinophil-rich subepidermal bulla, mixed superficial dermal inflammation including eosinophils; eosinophilic spongiosis may be a marker for immunobullous disease, particularly pemphigus and pemphigoid, but more often correlates with a clinical diagnosis of dermatitis Drug eruptions Drug history, especially new drugs within previous 2 weeks Eosinophils observed in many types of drug eruptions but absent in about 50% of cases and present in many non-drug related dermatoses - therefore, an unreliable marker of drug etiology Eosinophilic dermatosis of Often in setting of chronic lymphocytic leukemia but hematologic malignancy reported in patients with other hematologic malignancies (eosinophilic dermatosis of Considered an insect bite-like reaction but without history of myeloproliferative disease) bites, presenting as pruritic, papules, nodules, and sometimes vesicles Superficial and deep dermal lymphohistiocytic inflammation with eosinophils Eosinophilic fasciitis (Shulman Sudden onset of symmetrical induration of skin and syndrome) subcutaneous tissues of the limbs with -peau d'orange"
texture, peripheral blood eosinophilia Eosinophilic granulomatosis with Palpable purpura and tender papulonodules;
asthma and polyangiitis (Churg Strauss peripheral blood eosinophilia;
peripheral neuropathy, syndrome) cardiac involvement and glomerulonephritis may be present; cutaneous small-vessel vasculitis; prominent IgG4 and IgE responses Eosinophilic (pustular) folliculitis HIV variant: HIV+ patient with severe pruritus and papules on the face and upper trunk Ofuji 's disease: Japanese patient with chronic, recurrent follicular pustules in a seborrheic distribution with tendency to form circinate plaques Pediatric: follicular pustules on the scalp of an infant Eosinophilic, polymorphic and Local and generalized pruritus, erythematous papules, pruritic eruption associated sometimes with vesicles with radiotherapy Eosinophilic vasculitis Pruritic urticarial and purpuric papules; angioedema, juvenile

- 46 -temporal arteritis; histopathological reaction pattern rather than specific dermatosis Eosinophilic ulcer of the oral Rapidly enlarging nodule that develops ulceration; tongue mucosa most common site; in addition to eosinophils, brisk lymphocytic infiltrate with component of CD30-positive cells and occasional clone suggests inclusion in spectrum of CD30+ lymphoproliferative disorders Epidermolysis bullosa acquisita Rare, acquired subepithelial immunobullous disease of skin (also see above, Autotimmune and mucous membranes Rullous Dermatoses) Erythema toxicum neonatorum Newborn with erythematous macules, papules and pustules, often with prominent erythematous flare; involving trunk.
Granuloma faciale Solitary to few, asymptomatic, red-brown to violaceous plaques on face, most common in middle age white men, IgG4-related disease; nasal mucosal variant termed eosinophilic angiocentric fibrosis Hypereosinophilic dermatitis of Pruritic, papular eruption with peripheral eosinophilia; often Nir-Westfried dapsone-responsive Hypereosinophilic syndromes Cutaneous lesions include pruritic erythematous papules or (HES) nodules as well as urticaria and angioedema in primary and secondary variants; mucosal ulcers are associated with an aggressive course.
Incontinentia pigmenti Vesicles and bullae along Blaschko lines in neonate (stage I);
blood eosinophilia; eosinophilic spongiosis and keratinocyte dyskeratosis Itchy red bump disease (papular Markedly pruritic pink to red papules;
may be associated dermatitis) with eczematous patches and/or dermographism Juvenile temporal arteritis Large-vessel eosinophilic vasculitis of children or young adults Juvenile xanthogranuloma Yellow to red¨brown papules or nodules on the head and neck, upper trunk or proximal extremities; early lesions may have numerous eosinophils Kimura disease Head/neck masses, peripheral eosinophilia, increased serum IgE; deep dermal and subcutaneous nodular lymphoid infiltrates with component of eosinophils Langerhans cell histiocytosis Disseminated pink and yellow¨brown papules (often with (especially eosinophilic crusts), nodulo-ulcerative periorificial lesions; infiltrate granuloma) of Langerhans histiocytes with variable, sometime prominent eosinophilic component Lymphoproliferative disorders, Patches, plaques or nodules; in mycosis fungoides, including mycosis fungoides, eosinophils often in tumor stage but uncommon in patch lymph om atoi d papul osi s, stage disease; a minor component of various cutaneous anaplastic large cell peripheral T-cell lymphomas and lymphomatoid lymphoma papulosis; prominent in eosinophil-rich neutrophil-rich variant of anaplastic large cell lymphoma Mastocytosis Pink¨tan to red¨brown macules, papules and plaques that urticate with stroking (Darier sign) Nummular dermatitis Discrete round eczematous plaques on the extremities;
pruritic

- 47 -Oid-oid disease (exudative Middle-aged, predominantly Jewish, men; pruritic lesions discoid and lichenoid chronic commonly involving genitals;
progressive stages: discoid dermatosis of Sulzberger and ¨> exudative ¨> lichenified;
peripheral blood eosinophilia Garbe)8 Pachydermatous eosinophilic South African black teenage girls with generalized pruritic dermatitis papules, hypertrophic genital lesions and peripheral blood eosinophilia; dapsone-responsive; possibly a variant of hypereosinophilic dermatitis of Nir-Westfried Papular eruption of blacks Young, black men with intensely pruritic papules on the trunk and upper arms Papuloerythroderma of Ofuji Widespread red-brown papules and/or confluent erythroderma sparing skin folds ("deck-chair sign") in elderly men, blood eosinophilia, lymphopenia, high IgE, possible underlying T-cell lymphoma, gastric carcinoma, or other malignancy.
Parasitic infestations Cysticercosis, dirofilariasis, larva migrans, myiasis, onchocerciasis, tungiasis, schistosomiasis and others Pemphigoid variants (also see Bullous pemphigoid: Elderly patients, pruritic tense blisters, above, Autotimmune Bullous urticarial plaques, and itching without primary lesions, Dermatoses) Pemphigoid gestationis: Pregnant patient; urticarial papules, plaques and bullae of abdomen, including periumbilical.
Other pemphigoid variants Pemphigus variants (also see Flaccid vesicles and crusted erosions;
distribution depends on above, Autotimmune Bullous subtype Dermatoses) Polymorphic eruption of Primigravida in third trimester;
urticarial papules and pregnancy (also referred to as plaques, especially in striae pruritic urticarial papules and plaques of pregnancy) Pruritic papular eruption of HIV Symmetrical pruritic non-follicular papules; HIV infection;
disease' may be an exaggerated reaction to arthropod antigens without recognized bites Pseudolymphoma Association with arthropod bite or drug (especially phenytoin) Scabies Marked nocturnal pruritus; webspace, umbilical and groin involvement Seabather's eruption (larvae of Pruritic papules in distribution of swimsuit covering; occurs Linuche unguiculata and after ocean swimming Edwardsiella lineakt and other larval cnidarians) Swimmer's itch or cercarial Pruritic papules on uncovered skin;
occurs after fresh-water dermatitis (larvae of avian and swimming mammalian schistosome species) Urticaria Pruritic, migratory, transient (lasting <24 hours) wheals Urticarial allergic eruption Annular or gyrate urticarial plaques that persist >24 hours Urticarial dermatitis Very pruritic, eczematous and/or urticarial, often excoriated papules and plaques of trunk and proximal extremities, particularly of the elderly ; persist for days to weeks;
often accompanied by excoriated papules; minimally

- 48 -responsive to topical glucocorticoids and oral antihistamines Urticarial vasculitis Lesions may burn rather than itch and persist >24 hours;
associated with autoimmune connective tissue disease and decrease in complement levels Table 13. Cutaneous eruptions/lesion types in drug reactions Acanthosis nigricans Acneiform lesions Acute generalized exanthematous pustulosis Alopecia Angioedema Aphthous stomatitis Black hairy tongue Bullous eruptions Erythema multiforme and Stevens-Johnson syndrome Erythema nodosum Exanthemas Exfoliative dermatitis Fixed drug eruption Gingival hyperplasia Lichen planus-like (lichenoid) eruption Lupus erythematosus Onycholysis Pemphigus vulgaris Photosensitivity Pigmentation Pityriasis rosea-like eruption Pruritus Psoriasis Purpura Raynaud's phenomenon Toxic epidermal necrolysis Urticaria Vasculitis Xerostomia In some aspects, the organ can be an ovary, a breast, a brain, a muscle, a heart, a lung, a stomach, a proximal large intestine, a distal large intestine, a small intestine, a pancreas, a thyroid, skin, an eye, a testicle, a thymus, a gallbladder, a uterus, a liver, a spleen, a kidney, an esophagus, a bladder, a stomach, or a blood vessel. In some aspects, an organ can include one or more glands. In some aspects, the gland can be thymus. In some aspects, an organ can include one or more organ systems. In some aspects, an organ can include the nervous system. The

- 49 -nervous system includes, for example, nerves, neurons, spinal cord, brain and ganglia. The nervous system can also include sensory organs. Examples of sensory organs include eyes, ears, tongue, skin and nose. In some aspects, an organ can include the respiratory system. The respiratory system includes mouth, nose, pharynx, larynx, trachea, bronchi, lungs and diaphragm.
In some aspects, an organ can include the biliaiy system. The biliaiy system is a series of channels and ducts. In biliary system includes bile ducts. "Organ" as used herein can also refer to the forming a functional grouping together of two or more (or multiple) tissues.
In some aspects, the tissue can be epithelial, connective, muscular or nervous. In some aspects, the tissue can include "sub-tissues" located within each of the primary tissues. "Tissue"
as used herein can also mean an ensemble of similar cells from the same origin that work toegether to carry out a specific function. In some aspects, the tissue can be subcutaneous fat, fascia, muscle, endomysium, fibrous tissue, mesentery, or diaphragm. In some aspects, tissue can include a sinus. A sinus is a sac or cavity in any organ or tissue.
Examples of a sinus include nasal and paranasal.
In some aspects, the body part can be an extremity, including for example, an arm, a leg, neck, ear or nose. For example, while ultrasound can be used to aid the diagnosis of eosinophilic fasciitis; it is not precise. In some aspects, the compositions and methods disclosed herein can be used to precisely detect the location of eosinophilic fasciitis inflammation, for instance, in a leg.
In some aspects, the subject can be a mammal. In some aspects, the mammal can be a human, cat, a dog, a pig, or a cow. In some aspects, the subject can be a human. In some aspects, the subject can be a dog.
In some aspects, in any of the methods described herein, the pro-piece-eMBP1-labeled protein complex resulting from the pro-piece of pro-eMBP1 or fragment thereof binding to one or more eosinophil granule proteins in the tissue, organ, or body part of the subject can be detected using one or more of single-photon emission computed tomography (SPECT), positron emission tomography (PET), X-ray, conventional or computed tomography (CT), a combination of SPECT and CT, magnetic resonance imaging (Mm), ultrasound, laser, and light. In some aspects, the light can be visible, ultraviolet, or infrared (with and without magnification).
METHODS OF TREATING
Disclosed herein are methods of delivering a therapeutic agent to a diseased tissue, organ, or body part. In some aspects, the methods can comprise administering a therapeutically effective amount of a composition comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to a therapeutic agent to a subject.

- 50 -Disclosed herein are methods of treating eosinophil-related inflammation in a subject. In some aspects, the methods can comprise administering a therapeutically effective amount of a composition comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof as a therapeutic agent to a subject.
Disclosed herein are methods of treating eosinophil-related inflammation in a subject. In some aspects, the methods can comprise administering a therapeutically effective amount of a composition comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to a therapeutic agent to a subject.
Disclosed herein are methods of reducing inflammation in a subject with eosinophil-related inflammation. In some aspects, the methods can comprise, administering a therapeutically effective amount of a composition comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof as a therapeutic agent to the subject, thereby reducing inflammation in the subject.
Disclosed herein are methods of reducing inflammation in a subject with eosinophil-related inflammation. In some apects, the methods can comprise administering a therapeutically effective amount of a composition comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to a therapeutic agent to a subject.
In some aspects, the diseased tissue, organ or body part can be any tissue, organ or body part disclosed herein. In some aspects, the diseased tissue or organ or body part can be subcutaneous fat, fascia, muscle, endomysium, fibrous tissue, mesentery, an ovary, a breast, a brain, a muscle, a heart, a lung, a stomach, a proximal large intestine, a distal large intestine, a small intestine, a pancreas, a thyroid, skin, mucous membrane, an eye, a testicle, a thymus, a gallbladder, a uterus, a liver, a spleen, a kidney, an esophagus, a bladder, a bile ducts, a blood vessel, a sinus, a larynx, a trachea, a thymus, a nerve, spinal cord, ganglia or diaphragm. In some aspects, the diseased tissue, orgran or body part can be nasal and/or sinus mucosa. In some aspects, the diseased tissue, organ or body part can be cancerous.
In some aspects, the therapeutic agent can be a glucocorticoid, a tumor necrosis factor (TNF) inhibitor, a phosphodiesterase (PDE) inhibitor, a calcineurin inhibitor, a thiopurine, a integrin inhibitor, eotaxin inhibitor, a JAK kinase inhibitor, IL13 inhibitor, a IL4/13 inhibitor, or a transforming growth factor (TGF) modulator. In some aspects, the calcineurin inhibitor can be sirolimus, thiopurine, cyclosporine or tacrolimus. In some aspects, the glucocorticoid can be mometasone, fluticasone, budesonide, prednisone or solumedrol. In some aspects, the therapeutic agent can be CellCept0. In some aspects, the therapeutic agent can be benralizumab.

-51-In some aspects, the dose of benralizumab can be lower than the dose currently administered.
Examples of other therapeutic agents include but are not limited to 1L-5 inhibitor sor monoclonal antibodies, IL-4/IL-13 inhibitors, anti-IgE agents, anti-inflammatory agents (e.g., anti-eotaxin, anti-siglet 8, anti-TSLP, anti-IL-31, anti-TNF, anti-TGF, and interferon).
In some aspects, in any of the methods disclosed herein, disclosed herein is a step comprising identifying the subject in need thereof In some aspects, the subject in need thereof can be identified by any of the methods disclosed herein.
EXAMPLES
Example 1: Pro-piece of pro-eMBP1 (binding to eMBP-1 by surface plasmon resonance) Described herein are studies directed to designing peptides capable of neutralizing and, thus, mitigating toxic effects of eosinophil granule proteins as a treatment for eosinophil-related diseases.
The eosinophil is a peripheral blood leukocyte containing an abundance of cytoplasmic granules, rich in cationic protein toxins, which stain strongly with acidic dyes. Electron microscopy shows that the granules have a distinctive substructure with an electron dense core and a relatively radiolucent matrix. Studies of granule proteins have revealed remarkably cationic molecules with isoelectric points approaching pI values of 11. Among these, the most abundant on a molar basis is eMBP1, which is exceedingly cationic with a calculated pI of 11.6, is rich in arginine and has a molecular weight of 14 kDa. The eMBP1 crystal structure reveals an atypical lectin that binds heparin. Analyses of genes expressed by developing eosinophils showed that eMBP1 is the most highly expressed gene at 8% of transcripts;
these analyses also revealed a homologue of eMBP1 with a reduced isoelectric point, referred to as eMBP2.
eMBP1 is toxic to helminths, bacteria and numerous cells, such as respiratory epithelium, and is stimulatory to other cells, including basophils and mast cells.
Analyses of the biological effects of eMBP1 have shown that it kills parasites including schistosomules of Schistosoma mcmsoni, bloodstream forms of Trypanosomci cruzi, newborn larvae of Trichinellct spiral's and microfilariae of Brugla pahangi and Brugla malayi, that it kills bacteria, and that it is toxic for numerous cells, including other leukocytes, skin, intestinal cells, tracheal epithelium, and tumor cells. eMBP1 also activates cells, including other leukocytes such as basophils and neutrophils.
Instillation of eMBP1 into monkey lungs produced bronchospasm and bronchial hyperreactivity;
installation into the lungs of mice produced cough, which are characteristic features of bronchial

- 52 -asthma. Additionally, injection into skin causes vascular permeability and a wheal-and-flare reaction comparable to that produced by histamine; exposure to the mammalian urinary bladder increases membrane permeability. eMBP1 also is extruded with extracellular DNA
traps formed by eosinophils.
Studies of human diseases show that eMBP1 is present in secretions from patients with eosinophil-related diseases, including asthma, chronic rhinosinusitis and gastrointestinal diseases, and is deposited on damaged tissues. It is also present in urine of patients with eosinophil-related diseases. In addition, it is extensively deposited in tissues often in the virtual absence of intact eosinophils and in diseases such as asthma, rhinitis, cutaneous diseases including atopic dermatitis, urticaria and Wells syndrome as well as gastroenteric diseases, including eosinophilic gastroenteritis and eosinophilic esophagitis. In diseases mediated by helminths, eMBP1 is deposited onto damaged microfilaria of Oncocerca volvulus and onto eggs of Schistosoma mansoni. Examination of eosinophil infiltration in eosinophilic esophagitis shows that the majority of eosinophils have lost morphologic integrity with rupture of cytoplasmic membranes and deposition of intact eosinophil granules into the adjacent tissue.
Numerous studies show that the eosinophil mediates its damage to parasites and tissues by depositing its toxin-rich granule proteins onto microbial targets and tissues.
Therefore, neutralization of eMBP1 can be used as a treatment strategy to mitigate tissue damage in eosinophil-related inflammation.
Other eosinophil granule proteins include the eosinophil peroxidase (EPX) , the eosinophil cationic protein (ECP), the eosinophil-derived neurotoxin (EDN) and the homologue of eMBP1, eMBP2; the Charcot-Leyden crystal protein, now known as galectin 10, has been regarded as a granule protein as well. The biologic activities of these molecules contribute further to the damage to microbial targets and tissues caused by eosinophils.
EPX utilizes hydrogen peroxide as a substrate to generate potent oxygen radicals, including hypohalous acids, especially hypobromous acid. EPX also exerts cytotoxic effects as a cationic toxin and, as such, is able to destroy parasites and mammalian cells. ECP is remarkably cationic (PI 10.8), is toxic to bacteria and helminths, can function as an antiviral agent and is more biologically active than EDN. EDN was identified by its ability to cause a neurologic reaction in rabbits (Gordon phenomenon), activates dendritic cells, and has antiviral activities, but, in general, is less biologically active than the other granular proteins. Both ECP and EDN are ribonucleases, and EDN has comparable activity to pancreatic ribonuclease. eMBP2 possesses similar activities to eMBP1 but is less biologically active.

- 53 -Amino acid sequencing of eMBP I revealed 117 amino acids and the absence of any carbohydrate. Surprisingly, sequencing of the eMBP1 cDNA indicated the presence of a precursor molecule, referred to as pre-pro MBP I, and composed of eMBP1 and the pro-piece of pro-eMBP1 sequence. The leader sequence is cleaved off during endoplasmic reticulum translocation. The pre-pro-eMBP1 is the glutamic acid-rich neutralizing pro-piece pro-eMBP1 while the active arginine-rich section is the eMBP1 sequence. More specifically, sequencing of eMBP1 cDNA indicated that eMBP1 is synthesized as a precursor composed of eMBP1 with an exceedingly acidic pro-piece of pro-eMBP1 and referred to as pro-eMBP1.
Developing eosinophils synthesize pro-eMBP1, and the pro-piece of pro-eMBP1 is removed during granule maturation. Studies of pro-piece of pro-eMBP1 established that it can neutralize toxic effects of eMBP1 and, also, of another granule protein, eosinophil cationic protein (ECP). By expressing pro-piece of pro-eMBP1, it was found that it is glycan-rich, and evaluated its binding to eMBP1 by surface plasmon resonance. The characteristics of pro-piece of pro-eMBP1 that maximally neutralize the deleterious effects of eMBP1 and other granule toxins as a therapy for eosinophil-related diseases was also determined.
Expression of pro-eMBP1 in Chinese hamster ovary (CHO) cells showed the presence of a glycosylated molecule devoid of the cytotoxic and cytostimulatory activities of the 14 kDa eMBP1. In fact, the CHO-proMBP1 neutralized the toxicity of eMBP1 itself as demonstrated by inhibition of the basophil histamine release and superoxide anion generation stimulated by eMBP1. Notably, the pro-piece of pro-eMBP1 is remarkably acidic with a strikingly increased content of acidic amino acids, especially glutamic acid, in contrast to the basicity of eMBP1.
Polymers of glutamic and aspartic acids neutralized the toxicity of eMBP1 and ECP. Analyses of human biological fluids have shown that eMBP1 is rarely present in serum, but it can be easily demonstrated in nasal and bronchial fluids from the respiratory tract of patients with eosinophil-related respiratory diseases. Pro MBP1 can also be detected in the blood of patients with eosinophil associated diseases including the hypereosinophilic syndrome, and it is strikingly increased in the blood of pregnant women. Isolation of pro-eMBP1 from pregnancy serum showed that it is heavily glycosylated with a 10 kDa glycosaminoglycan at residue S62 (for example, see SEQ ID NO: 15).
Using expressed pro-piece of pro-eMBP1, the molecular form that optimally binds to and neutralizes eMBP1 will be identified. Also, it will be determined whether glycosylation of pro-piece of pro-eMBP1 importantly alters its binding to eMBP1 by testing whether its glycans and/or glycosaminoglycan modify its activities. By creating overlapping peptides and mutants

- 54 -with increased amino acid sequences characteristic of active regions, the most active pro-piece of pro-eMBP1 peptide regions for eMBP1 binding and neutralization will be identified. These will also be tested for neutralizing other granule proteins.
As disclosed herein, the ability of the pro-piece of pro-eMBP1 to bind to and neutralize eosinophil granule proteins will be determined. In these experiments, the pro-piece of pro-eMBP1 forms generated using surface plasmon resonance will be tested to determine binding to eMBP1 and using killing of K562 tumor cells and stimulatory activity for histamine release to test neutralization of eMBP1's activities. The pro-piece of pro-eMBP1 will be dissected to determine the effect of removing glycans on its binding and inhibitory activities. If removal of glycans has no effect on, or increases binding and inhibitory activities, we will test overlapping pro-piece of pro-eMBP1 peptides to determine if there are peptide regions with increased binding and neutralizing activities. If glycan removal decreases binding and inhibitory activities, we will remove N-, 0- linked glycans and the glycosaminoglycan at S62 (for example, see SEQ
ID NO: 15), individually and sequentially, to define their contributions to binding and neutralizing activities. An alternative to these enzymatic and chemical approaches is mutation of the eMBP1 plasmid to alter amino acids at glycan binding sites and thus to produce pro-piece of pro-eMBP1 devoid of glycans. It will be further determined if additional modifications of the pro-piece of pro-eMBP1 structure will increase its binding and inhibitory capabilities. If the best binding structure is a peptide, mutants with increased amino acid sequences characteristic of active regions will be generated. If the best binder contains glycans, the glycan composition will be altered to include those glycans associated with strongest binding and engineer pro-piece of pro-eMBP1 forms with increased representation of implicated glycans.
Identification of the optimal chemical composition of pro-piece of pro-eMBP I
for neutralization of eosinophil granule proteins.
Although the pro-piece of pro-eMBP1 as currently expressed in HEK cells may be a useful reagent for neutralization of eMBP1, it is important to determine if the marked quantity of glycans contributes to or detracts from eMBP1 binding. Analyses of pro-eMBP1 isolated from pregnancy serum indicate the presence of 0-linked glycans at residues T23, S24, T25 and T34, a N-linked glycan at N86 and a glycosaminoglycan at S62 (see, for example, SEQ
ID NO: 15).
ProMBP1 produced by CHO cells possessed a glycosaminoglycan at S62 (see, for exampleõ
SEQ ID NO: 15); since this molecule neutralized the cytostimulatory effect of eMBP1 on basophils and neutrophils, these structural features may be important in neutralizing eMBP1.
Pro-eMBP1 from a T-cell hybridoma contained glycosylation at residues S24, T25 and T28 (see,

- 55 -for example, SEQ ID NO: 15); no information is available on the inhibition ability of this protein.
Therefore, it will be tested if the glycans on the pro-piece of pro-eMBP1 increase or reduce its ability to neutralize eMBP1 and the other granule proteins. Several approaches to testing the role of glycans can be used. First, the pro-piece of pro-eMBP I
will be enzymatically treated to remove glycans from N-linked and 0-linked sugars and to deplete the 0-linked glycosaminoglycan. Second, the pro-piece of pro-eMBP1 will be expressed in HEK293S GnTi-cells engineered to limit glycosylation to further test the importance of the N-linked glycan at N86. Third, the mutant pro-pieces of pro-eMBP I will be expressed to produce product without any carbohydrate attached (by changing the amino acids at sites of glycan binding). If the peptide devoid of sugars binds as well or better than the expressed molecule from HEK
cells, overlapping 25-mers will be synthesized to determine if binding hotspots can be detected. If the pro-piece of pro-eMBP1 binds less avidly than the pro-piece of pro-eMBP1 glycosylated molecule, then the released sugar molecules will be chemically characterized to define their structures with attention to the 0-linked glycosaminoglycan at S62 (see, for example, SEQ ID
NO: 15) suspected of being a glycosaminoglycan, heparin/heparan sulfate-like or chondroitin/dermatan sulfate-like. Next, the pro-piece of pro-eMBP1 peptides will be compared, isolated from HEK and CHO cells, to define how specific glycan structures, which are predicted to be distinct between HEK and CHO cells, contribute to the neutralizing capacity of pro-piece of pro-eMBP1. The 0-linked glycosaminoglycans attached to pro-piece of pro-eMBP I will be isolated and harvested from the HEK and CHO cells, and the structural differences in terms of disaccharide composition and sulfation pattern that may contribute to specificity and differential binding to eMBP1 will be characterized.
Production of pro-piece of pro-eMBP1 in human embryonic kidney cells (HEK):
Sufficient quantities of expressed and purified pro-piece of pro-eMBP I will be generated.
Methods: A synthetic gene complete with IL-5 extracellular leader sequence and C-terminal 6x His tag was assembled from synthetic oligonucleotides and PCR
products, inserted into pcDNA3.4-TOPO to generate pcDNA3.4-eMBP this, and verified by sequencing.
This plasmid will be transfected into Expi293 cells, and the product will be harvested after 6 days, applied to a HisTrap column and eluted using a linear gradient from 20-500 mM
imidazole in Tris-HC1, 500 mM NaCl, pH 7.5. In the prior experiments a peak eluted and fractions combined after SDS-PAGE and western blotting analyses. Using a calculated El% = 5.1, a total of 19.7 mg of pro-piece of pro-eMBP1 is expected to be recovered. Molecular weight of 25 kDa by SDS-

- 56 -PAGE and will be confirmed by mass spectroscopy (MS). FIG. I shows binding of the pro-piece of pro-eMBP1 to eMBP1 by surface plasmon resonance.
Structural studies of pro-piece of pro-eMBP1: Different methods will be utilized to modify pro-piece of pro-eMBP1. N-linked and 0-linked glycans will be removed by three methods and the specific glycosaminoglycan at 562 (of SEQ ID NO: 15) will also be removed.
The pro-piece of pro-eMBP1 will be expressed devoid of glycosylation. With these findings, the peptide regions of propiece of pro-eMBP1 will be identified, including those with attached glycosaminoglycans, that are important for eMBP1 binding. These constructs will be analyzed by SDS-PAGE and MS to determine changes in molecular weights and specific glycosaminoglycan structures and tested to determine binding and neutralization of eMBP1.
Methods: First, pro-piece of pro-eMBP1 will be treated with peptide N-glycosidase F
(PNGase F) to remove the N-linked glycan at residue 96 (see, for example, SEQ
ID NO: 15).
Second, approaches for removal of 0-linked glycans are: a) Treatment with trifluoromethanesulfonic acid (Sigma Kit PP0510), this removes both N- and 0-linked glycans;
b) Treatment with the 0-glycosidase from Streptococcus pneumonia combined with exoglycosidases; and c) Treatment with proprietary kits that remove 0-glycans, including EZGlyco (from S-Bio Company) and OpeRATOR (from Genovis). Third, the glycosaminoglycan at residue 62 (see, for example, SEQ ID NO: 15) will be digested using chondroitinase ABC and heparin lyases I, II and III; prior experiments with pregnancy pro-eMBP1 showed that chondroitinase ABC treatment resulted in an approximately 10 kDa reduction in mass.
Additional approaches to determining the effect of glycans include: a) expression of the pro-piece of pro-eMBP1 devoid of N-linked carbohydrates using HEK293S GnTi-(ATCCD
CRL-3022) cells; b) expression of the pro-piece of pro-eMBP1 using HEK293-GlycoDelete cells (ATCC CRL-1573); and c) expression of mutants at glycan binding sites of the plasmid system described herein. In addition, the pro-piece of pro-eMBP1 will be produced in E. colt; note that pro-piece of pro-eMBP1 is expressed as a soluble protein and spontaneously dimerizes by formation of a disulfide bond. By alkylating the sulfhydryl group, both monomer and dimer propiece can be obtained devoid of glycans for testing. It will be determined if the T-cell hybridoma is available for testing in that the pro-eMBP1 produced by this cell may possess fewer glycans than the CHO and HEK expressed lines.
The polypeptides will be analyzed by SDS-PAGE and MS to determine the change in molecular weight and specific glycosaminoglycan structures in terms of sulfation

- 57 -pattern/disaccharide composition, respectively. Binding to eMBP I will be measured by surface plasmon resonance and the ability to inhibit the biological effects of eMBP1 will be performed.
An alternative to the enzymatic and chemical approaches to delineate the role of glycans in binding to eMBP1 is mutation of the eMBP1 plasmid to alter amino acids at glycan binding sites and thus produce pro-piece of pro-eMBP1 devoid of glycans at specific sites. While both N-and 0-linked glycans will be analyzed for their contributions to the binding of pro-piece of pro-eMBP1 to eMBP1, experiments testing whether the removal of the glycosaminoglycan at S62 (for example, see SEQ ID NO: 15) will be carried out, and is particularly important in view of the information that heparin and heparan bind to eMBP1 by surface plasmon resonance and that heparin neutralizes eMBP1's killing.
Modified pro-piece of pro-eMBP I analyses for neutralization of eMBP I and other granule proteins These experiments will test the ability of pro-piece of pro-eMBP1 to bind to and neutralize eMBP1. First, pro-piece of pro-eMBP1 will be analyzed by surface plasma resonance to determine which molecules have the greatest affinity for immobilized eMBP1.
These experiments will test whether the molecules with greater affinity will be most effective at neutralization of its biologic activities. The binding activity of the pro-piece of pro-eMBP1 peptides will also be analyzed for other eosinophil granule proteins utilizing both EPX and ECP
as targets. Therefore, surface plasma resonance will be used as a first screening method to detect optimal pro-piece of pro-eMBP1 peptides. Second, it the pro-piece of pro-eMBP1 peptides will further be tested for their ability to inhibit eMBP1 killing of K562 cells and to inhibit eMBP1's activation of mast cells.
Measurement of the avidity of modified pro-piece of pro-eMBP1 for eosinophil granule proteins by surface plasmon resonance: These experiments will test whether the modified molecules will have greater avidities for eMBP1, ECP and the eosinophil peroxidase (EPX) using surface plasmon resonance,and will also be the most effective at neutralization of their biologic activities.
Because surface plasmon resonance can be utilized without additional labeling of the tested molecules, it will be employed for analyses of the pro-piece of pro-eMBP1. Experiments demonstrate that pro-piece of pro-eMBP1 binds to immobilized eMBP1 (FIG. 1).
In these experiments, the HEK derived pro-piece of pro-eMBP1 and the differing forms generated will be tested.

- 58 -Methods: Surface plasmon resonance will be performed on a Sierra SPR- I 6 (Bruker). A
low-charge density polycarboxylate hydrogel surface (HLC200M, Xantec Bioanalytics) decreases non-specific interactions with these highly charged molecules.
Approximately 1500 -3000 response units of eMBP1 will be immobilized on the surface via thiol coupling. First, the carboxylate surface is activated with sulfo-NHS (N-hydroxysulfosuccinimide)/EDC (1-ethy1-3-(3-dimethylamino propyl) carbodiimide hydrochloride) followed by PDEA (2-(2-Pyridinyldithio)ethaneamine hydrochloride) to produce a reactive mixed disulfide. eMBP1 is then immobilized by its free cysteines. With eight flow channels, each with an in-line control (background binding), eight simultaneous binding assays can be performed.
Binding of the eMBP1 ligands (the peptides described herein, including the overlapping peptides) will be tested in triplicate at equal molar concentrations. Surface regeneration will be performed with two 5-second pulses of 6M guanidine-HC1. Data will be corrected by subtracting the in-line blank surface and double-referenced using running buffer injections performed before each ligand injection. By immobilizing EPX and ECP, the binding of the pro-piece of pro-eMBP1 peptides for these targets in addition to eMBP1 will be determined. Synthetic peptides from eMBP1 residues 17-45 and 89-117 (see, for example, SEQ ID NO: 1) can be used in certain experiments as a substitute for 14 kDa eMBP1.
Inhibition of the toxicity of eosinophilic granule proteins for 1(562 cells:
eMBP1 and other eosinophil granule proteins kill tumor cells including 1(562 cells. The ability of the modified propiece of pro-eMBP1 constructs to neutralize killing of 1(562 cells by eMBP I, ECP
and EPX will be tested.
eMBP1 and other eosinophil granule proteins killed tumor cells utilizing K562 cells;
eMBP1 produces dose dependent killing, and the cytotoxi city is measured by trypan blue exclusion.
Methods: 1(562 cells, purchased from ATCC, will be maintained in RPMI 1640 (Sigma) supplemented with L-glutamine and 10% defined calf serum (Hyclone Laboratories Logan Utah). Before testing with eMBP1 or other granule proteins, 1(562 cells will be washed thrice with RPMI 1640 without serum, centrifuged at 200 X G at room temperature for 10 minutes, and suspended at 1x106 cells/mL in serum free RPMI 1640. For measurement of cytotoxicity, sterile 0.4% trypan blue (Sigma) in PBS filtered through a 0.45 p.m filter will be used. Aliquots of a suspension of 1x106 1(562 cells/mL, 90 pL/well, diluted with RPMI 1640 without serum will be intubated with eMBP1 or other granule proteins. Incubations with 1(562 and eMBP1 or other granule proteins will be performed in 96 well cell culture plates at 37 C
under 5% CO2 and

- 59 -under humid conditions and a final volume of 100 ut. Cells will be incubated for 1 hour or 4 hours depending on the initial potency of the granule proteins and 5 JAL of trypan blue will be added, and the cells will be thoroughly mixed. Then, 10 mL of cell suspension will be transferred to a hemocytometer and counted. Live (unstained) and damaged (stained blue) cells will be counted.
Inhibition of granule protein stimulated mast cell histamine release by modified pro-piece of pro-eMBP1 constructs and analogs: The modified pro-piece of pro-eMBP1 analogs will be measured for their abilities to inhibit the activity of eMBP1, ECP and EPX in non-cytotoxic, stimulated histamine release from fresh rat mast cells.
eMBP1 is toxic to numerous biological targets, and it also has the capability to activate cells in a non-cytotoxic manner. Therefore, it will be tested whether pro-piece of pro-eMBP1 can inhibit non-cytotoxic eMBP1 activity. Rat mast cells will be used because both eMBP1 and ECP
stimulate histamine release in a non-cytotoxic reaction whereas human basophils are activated by eMBP1 (and also eMBP2).
Methods: Male 250-300 g Sprague-Dawley rats will be sacrificed, and mast cells collected by rinsing the peritoneal cavity with 20 mL of 25 mM piperazine-N,M-bis-(2-ethanesulfonic acid) (PIPES), 150 mM NaCl, 5 mM KC1, 40 mM NaOH and 0.003%
human serum albumin, pH 7.4 (PA buffer) containing 10 units/mL of heparin. Mast cells will be purified by centrifugation through a 38% (weight/volume) bovine serum albumin layer, and the cell pellet washed thrice in PA before use. These cell preparations routinely contained greater than 90%
mast cells by toluidine blue staining. Aliquots containing 1.5x104 mast cells and PA will be added to a 12 x 75 mm plastic tubes, and eosinophil granule proteins added to a final volume of 0.25 mL. Compound 48/80 will be used as a positive control. After addition of 0.6 mM CaCl2 and 1 mM MgCl2 the cells are incubated for 10 minutes, the reaction stopped by addition of 0.25 mL PA buffer and cells are centrifuged for 2 minutes at 1000 G. Histamine will be measured using a colorimetric competitive immunoassay (Enzo Biochem). Inhibition by pro-piece of pro-eMBP1 will be tested by pre-incubation with granule proteins for 30 minutes at 20 C.
The pro-piece of pro-eMBP1 was expressed in human embryonic kidney (HEK) cells. It is a 25 kDa heterogeneous glycoprotein able to bind to eMBP1 by surface plasma resonance (FIG. 1); the peptide part of the pro-piece of pro-eMBP1 accounts for about 10 kDa indicating that the HEK expressed molecule is rich in glycan structures.
For these experiments, biosensor analysis was conducted at 25 C in an HBS
buffer system (10 mM HEPES, pH 7.4 and 150 mM NaCl) using a Biacore 3000 optical biosensor

- 60 -(Cytiva Life Sciences; Marlborough, MA) equipped with a CM4 sensor chip.
During data collection, the autosampler was maintained at 8 C.
Using thiol coupling chemistry, eMBP-1 was immobilized to the chip surface to a level of 4000 RU. The thiol coupling kit (Cytiva Life Sciences; Marlborough, MA) was followed by first activating the surface with 0.2 M EDC and 0.05 M NHS for 2 minutes followed by a 4 minute injection of 80 mM PDEA in 50 mM sodium borate buffer pH8.5, at a flow rate of 10 eMPB-1 was diluted to 0.6 RM in 10 mM sodium acetate, pH 5.25 and injected for minutes at 10 RL/min. Finally, the remaining free cysteines were blocked with the injection of 50 mM L-cysteine in 0.1 M sodium acetate, 1.0 M sodium chloride, pH 4.0 for 4 minutes at 10 L/min. The reference flow cell was produced using the same immobilization procedure but without the addition of eMBP-1.
Five, pro-piece of pro-eMBP1 concentrations were prepared in running buffer ranging from 300 nM to 3.70 nM (3-fold dilutions). Each of the five analyte sample concentrations were run in triplicate and in a mixed order, as a means of assessing the reproducibility of binding and mitigating the potential for bias arising from the order of injection.
Multiple blank (buffer) injections were run to allow system-related artifacts specific to each flow cell to be corrected for by subtraction. The association phases were monitored for 600 s at a flow rate of 25 RL/min, the dissociation phases for 300 s at the same flow rate. A long dissociation phase experiment was run using 300 nM pro-piece of pro-eMBP1 and extending the dissociation time for 1800 s. At the completion of each dissociation phase, the surface was regenerated using two, 5 s injections of 6 M guanidine-HCl.
The data were aligned, double referenced, and fit using Scrubber v2.0 software (BioLogic Software Pty Ltd, Campbell, Australia), which is an surface plasmon resonance (SPR) data processing and non-linear least squares regression fitting program_ Since the data demonstrated complex binding from an unknown origin, and the data did not fit a simple 1:1 binding model, no kinetic rate constants were recovered.
The results are shown in FIGs. IA-C.
Because pro-eMBP1 neutralized the cytotoxic and cyto-stimulatory activities of eMBP1 itself, and because the structural difference between toxic eMBP1 and pro-eMBP1 is the presence of the pro-piece of pro-eMBP1, these results show that the pro-piece of pro-eMBP1 is able to neutralize the biologic activities of eMBP1. The properties of the eMBP1 pro-piece of pro-eMBP1 will be determined for the strongest binding to eMBP1 and to test if it is able to neutralize the biological effects of eMBP1; the pro-piece of pro-eMBP1 will also be tested for

- 61 -neutralizing activity against EPX and ECP in these same assays. The composition developed through these studies can be used as a therapy to mitigate tissue damage in eosinophil-related diseases by combining (e.g., binding) with eosinophil granule proteins, especially eMBP1, but also with ECP and EPX.
Example 2: Inhibition of eMBP1 by WT ProPiece The MBP neutralization assay can quantify the neutralizing ability of inhibitors for the toxic eosinophil granule protein, eMBP1. The tumor cell line, K562 is used as a model for cytotoxicity. 1(562 cells are grown in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% Fetal Bovine Serum (FBS) and incubated in a humidified incubator at 37 C and 5% CO2. The cells are maintained at log-phase growth at densities of 1x105 to 1 x106 cells/mL. In preparation for the assay, samples of approximately 1 x105 cells are washed to remove serum proteins by repeated centrifugation (100 x g, 3 min) and resuspension in 1 mL
quantities of IMDM. Three washes are adequate to remove serum proteins without affecting the cells. Washed cells are diluted to 1><i05 cells/mL for use in the assay.
Exposure to eMBP1 takes place in 384-well microtiter plates over the course of an 18-hour incubation at 37 C and 5%
CO2.
First, 5 pl of eMBP1 in Phosphate-buffered saline (PBS) is added to each microtiter well followed by 5 pl of the inhibitor in PBS. Multiple replicates and the appropriate vehicle controls are also prepared for use in analyses. A concentration range of each inhibitor is selected such that an IC50 can be determined. The IC50 is the theoretical concentration of an inhibitor that neutralizes half of the toxin activity in an assay. An eMBP1 concentration of 5x10-6M was chosen to be neutralized as it reproducibly kills most (e.g., 90-99%) K562 cells in the K562 cytotoxi city assay. A series of toxin concentrations are tested in the assay to determine the 1050.
Next, 10 pl of washed cells are added to each well (approx.1000 cells), and the microtiter plate is incubated for 18 hours. After incubation, the viability of the 1(562 cells is determined using the CellTiter-GloTm 2.0 Assay from Promega. The CellTiter-Glo reagent (CTiGo) allows the determination of viability by quantitating adenosine 51-triphosphate (ATP) in a culture. The CTiGo reagent lyses the cells in solution, liberating ATP from cell membranes.
ATP acts as a substrate for a luciferin-based reaction producing light. The amount of luminescence produced is proportional to the concentration of ATP, and, thus, the number of metabolically active cells in the culture. Both the microliter plate and CTiGo reagent are equilibrated at room temperature for 20-30 minutes. The culture volume of each well is diluted 1:1 with CTiGo (20 pl/well), and the plate is loaded into a Thermo ScientificTM VarioskanTM LUX for measurement of luminescence.

- 62 -The Varioskan is pre-programmed for 3 minutes of shaking at 600 rpm to assist in cell lysis, a 10-minute incubation at room temperature to equilibrate the luciferin reaction, and a 500 ms exposure of each well to measure luminescence.
Raw luminescence values are carried through a series of transformations to quantify inhibition. First, raw luminescence data are imported into GraphPad Prism from the plate-reading software, SkanIt (Thermo Scientific). Next, a blank signal is subtracted from the data points in each trial. In this case, a "blank" corresponds to a well of cells incubated with 5 x10-6 M eMBP1 (killing 90-99% of the cells). The blank-subtracted signals are normalized to generate %
inhibition quantities using 0 signal as 0% and the average signal of the highest inhibitor concentration as 100%. The inhibitor concentrations used, and corresponding %
inhibition quantities are used to fit an [Inhibitor] response curve. The IC50 parameter from the curve fit is used to compare one inhibitor to another in their ability to neutralize eMBP1.
FIG. 2 shows the percent inhibition of eMBP1 by a pro piece of pro-eosinophil granule major basic protein-1.
Example 3: Inhibition of eMBP1 by WT ProPiece of Pro-eMBP1 The pro-piece of pro-eMBP1 will be expressed and associated glycans will be modified to identify the optimal form of the molecule for binding to and neutralizing eMBP1. These tests will determine whether glycosylation of pro-piece of pro-eMBP1 importantly alters its binding to eMBP1 by analyzing whether N- or 0- glycans and the glycosaminoglycan at S62 modify its activities. This study will identify the most important pro-piece of pro-eMBP1 regions for binding eMBP1 and neutralizing it by creating overlapping peptides and creating mutants with increased amino acid sequences characteristic of active regions. Evaluation of the pro-piece of pro-eMBP1 panel for binding to and inhibiting eMBP1 will be evaluated in several in vitro models. This study will also identify an active form of the pro-piece of pro-eMBP1 with optimal inhibition effects on eMBP1 and other granule toxic proteins.
It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention.
Other aspects of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

- 63 -

Claims

What is claimed is:

1. A method of producing a medical image of a tissue, organ, or body part, the method comprising:
a) administering to a subject a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to label, wherein the pro-piece of pro-eMBP1 or fragment thereof binds to one or more eosinophil granule proteins in the tissue, organ, or body part of the subject, wherein said binding generates a pro-piece-eMBP1-labeled protein complex; and b) detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part, whereby detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part produces a medical image of the eosinophil granule protein localization in the tissue, organ, or body part.

2. A method of diagnosing eosinophil-related inflammation in a subject, the method comprising:
a) administering to a subject a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to label, wherein the pro-piece of pro-eMBP1 or fragment thereof binds to one or more eosinophil granule proteins in a tissue, organ, or body part of the subject, wherein said binding generates a pro-piece-eMBP-1-labeled protein complex; and b) detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part, whereby detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part indicates eosinophil-related inflammation in the subject.

3. A method of detecting a change in an eosinophil-related disease in a subject diagnosed with an eosinophil-related disease, the method comprising:

a) producing a first medical inlage of a tissue, organ, or body part in a subject diagnosed with an eosinophil-related disease according to the method of claim 1;
b) producing a second medical image of the tissue, organ, or body part in the subject of step (a), according to the method of claim 1; and c) comparing the medical image of step (b) with the medical image of step (a), whereby detecting a change in the medical image of step (b) compared to the medical image of step (a) detects a change in the eosinophil-related disease in the subject.

4. A method of detecting eosinophil degranulation in a subject, the method comprising:
a) administering to a subject a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to label, wherein the pro-piece of pro-eMBP1 or fragment thereof binds to one or more eosinophil granule proteins in a tissue, organ, or body part of the subject, wherein said binding generates a pro-piece-eMBP1-labeled protein complex; and b) detecting the pro-piece-eMBP1 -labeled protein complex in the tissue, organ, or body part, whereby detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part in the subject indicates eosinophil degranulation.

5. The method of claim 1, 2, or 4, wherein the label is a fluorochrome, an enzyme, a substrate, a glycoprotein, a solid-phase immobilizing agent, an ultrasound signal-detection agent, a laser-induced ultrasound optoacoustic signal-detection agent, a radioisotope, or a radiolabel.

6. The method of claim 5, wherein the label is a radioisotope.

7. The method of claim 6, wherein the radioisotope is Technetium99m (99"Tc).

8. The method of claim 2, wherein the eosinophil-related inflammation is caused by a parasitic disease; an allergic reaction; asthma; an autoimmune disease; a drug reaction; an environmental exposure; a topical contact; a genetic disease; a transplant rejection, a hematologic or lymphocytic disease, or an inflammatory or immunological reaction with expression of eosinophil differentiating, chemoattracting, activating factors or a combination thereof

9. The method of claim 3, wherein the eosinophil-related disease is eosinophilic cystitis, eosinophilic fasciitis, eosinophilic colitis, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic granulomatosis with polyangiitis, eosinophilic pneumonia, hypereosinophilic syndrome, vernal conjunctivitis, giant papillary conjunctivitis, atopic dermatitis, chronic rhinosinusitis or transplant rejection.

10. The method of claims 1, 2, 3, or 4, wherein the organ is an ovary, a breast, a brain, a muscle, a heart, a lung, a stomach, a proximal large intestine, a distal large intestine, a small intestine, a pancreas, a thyroid, skin, an eye, a testicle, a thymus, a gallbladder, a uterus, a liver, a spleen, a kidney, an esophagus, or a bladder.

11. The method of claims 1, 2, 3, or 4, wherein the eosinophil granule protein is major basic protein 1 (eMBP1).

12. The method of claims 1, 2, 3, or 4, wherein the composition is administered to the subject orally, intravenously, topically, intra-orificially, intra-articularly, or via a medical tube or medical devices.

13. The method of claims 1, 2, 3, or 4, wherein the subject is a mammal.

14. The method of claim 13, wherein the mammal is a cat, a dog, a pig, or a cow.

15. The method of claim 13, wherein the mammal is a human.

16. The method of claims 1, 2, 3, or 4, wherein the pro-piece-eMBP1-labeled protein complex resulting from the pro-piece of pro-eMBP1 or fragment thereof binding to one or more eosinophil granule proteins in the tissue, organ, or body part of the subject is detected using one or more of single-photon emission computed tomography (SPECT), positron emission tomography (PET), conventional or computed tomography (CT), a combination of SPECT and CT, magnetic resonance imaging (MRI), ultrasound, laser, and light

17. A method of delivering a therapeutic agent to a diseased tissue, organ, or body part, the method comprising, administering a therapeutically effective amount of a composition comprising: the pro-piece of pro-eosinophil granule maj or basic protein-1 (pro-eMBP1) or fragment thereof conjugated to a therapeutic agent to a subject.

18. A method of treating eosinophil-related inflammation in a subject, the method comprising, administering a therapeutically effective amount of a composition comprising: the pro-piece of pro-eosinophil granule maj or basic protein-1 (pro-eMBP1) or fragment thereof as a therapeutic agent to a subject.

19. A method of treating eosinophil-related inflammation in a subject, the method comprising, administering a therapeutically effective amount of a composition comprising: the pro-piece of pro-eosinophil granule maj or basic protein-1 (pro-eMBP1) or fragment thereof conjugated to a therapeutic agent to a subject.

20. The method of claim 18 or 19, wherein the therapeutic agent is a glucocorticoid, a tumor necrosis factor (TNF) inhibitor, a phosphodiesterase (PDE) inhibitor, a calcineurin inhibitor, a thiopurine, a integrin inhibitor, eotaxin inhibitor, a JAK kinase inhibitor, IL13 inhibitor, a IL4/13 inhibitor, or a transforming growth factor (TGF) modulator.

21. The method of claim 20, wherein the glucocorticoid is mometasone, fluticasone, budesonide, prednisone or solumedrol.

22. The method of claim 18 or 19, wherein the therapeutic agent is benralizumab.

23. The method of claim 17, wherein the diseased tissue or organ or body part is subcutaneous fat, fascia, muscle, endomysium, fibrous tissue, mesentery, an ovary, a breast, a brain, a muscle, a heart, a lung, a stomach, a proximal large intestine, a distal large intestine, a small intestine, a pancreas, a thyroid, skin, mucous membrane, an eye, a testicle, a thymus, a gallbladder, a uterus, a liver, a spleen, a kidney, an esophagus, or a bladder.

24. The method of claim 17, 18, or 19, further comprising identifying the subject in need thereof

25. The method of claim 24, wherein the subject in need thereof is identified by any one of the methods of claims 2, 3, or 4.

26. The method of claims 17, 18, or 19, wherein the composition is administered to the subject orally, intravenously, topically, intra-orificially, intra-articularly, or via a medical tube or medical devices.

27. A composition comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to a therapeutic agent or a label.

28. A composition comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof

29. The composition of claim 26, wherein the therapeutic agent is benralizumab.

30. The composition of claim 27, wherein the label is Technetium99m, barium or Gadolinium.

31. A method of detecting eosinophil granule protein localization including from eosinophil degranulation in a subject, the method comprising: a) administering to the subject a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to alabel, wherein the pro-piece of pro-eMBP1 or fragment thereof binds to one or more eosinophil granule proteins in a tissue, organ, or body part of the subject, wherein said binding generates a pro-piece-eMBP1-labeled protein complex; and b) detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part, whereby detecting the pro-piece-eMBP1-labeled protein complex in the tissue, organ, or body part in the subject indicates eosinophil granule protein localization and/or eosinophil degranulation.

32. A method of detecting eosinophil granule proteins including from eosinophil degranulation in a subject, the method comprising: a) utilizing a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof conjugated to a supporting element, wherein the pro-piece of pro-eMBP1 or fragment thereof binds to one or more eosinophil granule proteins in a body fluid or tissue, organ, or body part extract from the subject, wherein said binding generates a pro-piece-eMBP I -support complex; and b) detecting the pro-piece-eMBP I complex in the body fluid or tissue, organ, or body part extract, whereby detecting eMBP1-support element complex in the body fluid, tissue, organ, or body part extract from the subject indicates eosinophil granule protein localization and/or eosinophil degranulation.

33. A method of reducing inflammation in a subject with eosinophil-related inflammation, the method comprising, administering a therapeutically effective amount of a composition comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBP1) or fragment thereof as a therapeutic agent to the subject, thereby reducing inflammation in the subject.