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WO2023034993A1 - Compositions et méthodes de diagnostic, de détection et de traitement de maladies liées aux éosinophiles - Google Patents

Compositions et méthodes de diagnostic, de détection et de traitement de maladies liées aux éosinophiles Download PDF

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Publication number
WO2023034993A1
WO2023034993A1 PCT/US2022/075946 US2022075946W WO2023034993A1 WO 2023034993 A1 WO2023034993 A1 WO 2023034993A1 US 2022075946 W US2022075946 W US 2022075946W WO 2023034993 A1 WO2023034993 A1 WO 2023034993A1
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pro
embpl
eosinophil
piece
subject
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PCT/US2022/075946
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English (en)
Inventor
Gerald J. Gleich
Kristin M. LEIFERMAN
Kathryn A. PETERSON
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Gleich Gerald J
Leiferman Kristin M
Peterson Kathryn A
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Application filed by Gleich Gerald J, Leiferman Kristin M, Peterson Kathryn A filed Critical Gleich Gerald J
Priority to CA3223191A priority Critical patent/CA3223191A1/fr
Publication of WO2023034993A1 publication Critical patent/WO2023034993A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2474/00—Immunochemical assays or immunoassays characterised by detection mode or means of detection
    • G01N2474/20—Immunohistochemistry assay
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7095—Inflammation

Definitions

  • EoE eosinophilic esophagitis
  • an eosinophil in biopsy specimens, can be identified as a cell 12-17 pm in diameter with a bi-lobed nucleus and cytoplasmic granules staining red with acidic dyes, for example, eosin.
  • 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.
  • 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.
  • eMBPl major basic protein- 1
  • pretreatment with antibody to eMBPl prevents airway hyperresponsiveness, demonstrating that blockade of eMBPl has anti-inflammatory effects on targeted tissues.
  • Further studies in the antigen-challenged guinea pig model shows treatment with heparin reverses vagally in-ducedbronchoconstriction.
  • 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.
  • new biologicals to suppress eosinophil-related inflammation by neutralization of IL-5 or by actual destruction of eosinophils using an antibody to the IL-5 receptor a have been approved.
  • 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.
  • 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.
  • eosinophil-related diseases may be a sign of an underlying condition that may not be preventable.
  • Other treatment strategies include diet and dilation of the esophagus.
  • alternative compositions and methods are needed to better diagnose and treat eosinophil-related diseases.
  • pro-eMBPl pro-eosinophil granule major basic protein- 1
  • pro-eMBPl pro-eosinophil granule major basic protein- 1
  • 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-eMBPl) or fragment thereof conjugated to label, wherein the pro-piece of pro-eMBPl 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-eMBPl -labeled protein complex; and b) detecting the pro-piece-eMBPl -labeled protein complex in the tissue, organ, or body part, whereby detecting the pro-piece-eMBPl - labeled protein complex in the tissue, organ, or body part in the subject indicates eosinophil degranulation.
  • pro-eMBPl major basic protein- 1
  • 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-eMBPl) or fragment thereof conjugated to label, wherein the pro-piece of pro- eMBPl 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-eMBPl -labeled protein complex; and b) detecting the pro-piece-eMBPl -labeled protein complex in the tissue, organ, or body part, whereby detecting the pro-piece-eMBPl -labeled protein complex in the tissue, organ, or body part in the subject indicates eosinophil granule protein localization and/or eosinophil
  • pro-eMBPl
  • 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-eMBPl) or fragment thereof conjugated to a supporting element, wherein the pro-piece of pro-eMBPl 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-eMBPl - support complex; and b) detecting the pro-piece-eMBPl complex in the body fluid or tissue, organ, or body part extract, whereby detecting eMBPl -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.
  • pro-eMBPl major basic protein
  • 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- eMBPl) or fragment thereof conjugated to a therapeutic agent to the subject.
  • a composition comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro- eMBPl) or fragment thereof conjugated to a therapeutic agent to the subject.
  • pro- eMBPl pro-eosinophil granule major basic protein-1
  • compositions comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBPl) or fragment thereof as a therapeutic agent to the subject, thereby treating eosinophil-related inflammation in the subject.
  • pro-eMBPl pro-eosinophil granule major basic protein-1
  • eMBPl granule major basic protein- 1
  • methods of neutralizing eMBPl in a subject comprising, administering a therapeutically effective amount of a composition comprising: the pro-piece of pro-eosinophil granule major basic protein- 1 (pro-eMBPl) or fragment thereof as a therapeutic agent to the subject, thereby neutralizing eMBPl in the subject.
  • a composition comprising: the pro-piece of pro-eosinophil granule major basic protein- 1 (pro-eMBPl) or fragment thereof as a therapeutic agent to the subject, thereby neutralizing eMBPl in the subject.
  • compositions comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro- eMBPl) or fragment thereof as a therapeutic agent to the subject, thereby reducing inflammation in the subject.
  • pro-eosinophil granule major basic protein-1 pro- eMBPl
  • compositions comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBPl) or fragment thereof conjugated to a therapeutic agent to the subject, thereby treating eosinophil- related inflammation in the subject.
  • pro-eMBPl pro-eosinophil granule major basic protein-1
  • compositions comprising: the pro-piece of pro-eosinophil granule major basic protein- 1 (pro-eMBPl) or fragment thereof conjugated to a therapeutic agent or a label.
  • pro-eMBPl pro-eosinophil granule major basic protein- 1
  • compositions comprising: the pro-piece of pro-eosinophil granule major basic protein- 1 (pro-eMBPl) or fragment thereof.
  • FIGS. 1A-C show the surface plasmon resonance (SPR) sensorgrams for pro-piece binding to eMBP-1.
  • FIG. 1A shows that triplicate 300 nM to 3.70 nM pro-piece injections over eMBP-1 surfaces do not overlay well.
  • FIG. IB 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 eMBPl by a pro-piece of pro-eosinophil granule major basic protein- 1.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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-eMBPl) 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.
  • a treatment may include administration of a pharmaceutically effective amount of a therapeutic agent.
  • 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.
  • the disease, disorder, and/or condition can be an eosinophoilic-related inflammation or disease.
  • 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.
  • 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).
  • a fragment of the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBPl) binds to and is capable of neutralizing eMBPl or any eosinophil granule proteins.
  • eosinophilic can be used to mean relating to eosinophilia.
  • eosinophilic-related and “eosinophil-related” can be used interchangeably herein.
  • 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.).
  • livestock e.g., cattle, horses, pigs, sheep, goats, etc.
  • laboratory animals e.g., mice, rabbits, rats, gerbils, guinea pigs, possums, etc.
  • 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 are also provided 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-eMBPl) or fragment thereof conjugated to label.
  • the pro-piece of pro-eMBPl 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- eMBPl -labeled protein complex.
  • the method can also comprise detecting the pro-piece- eMBPl -labeled protein complex in the tissue, organ, or body part, whereby detecting the pro- piece-eMBPl -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.
  • the localization of eosinophil granule protein in a tissue, organ, or body part can be microscopically detected.
  • compositions and methods for treating eosinophil-related inflammation can comprise administering to a subject a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBPl) or fragment thereof.
  • pro-eMBPl major basic protein-1
  • the pro-piece of pro-eMBPl 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-eMBPl -labeled protein complex, thereby neutralizing the one or more eosinophil granule proteins.
  • the methods can reduce inflammation in a tissue, organ, or body part.
  • a “mucosal tissue” is a tissue lining various cavities within the body.
  • a mucosal tissue include, but are not limited to, mucosal tissue lining the nose, sinuses, bronchi, lungs, conjunctiva, oral cavity, tongue, esophagus, stomach, pylorus, duodenumjejunum, 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.
  • a labeled pro-piece-eMBPl -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.
  • 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.
  • mucosa-associated lymphoid tissue (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.
  • 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.
  • M cells are also present, which sample antigen from the lumen and deliver it to the lymphoid tissue.
  • eosinophil degranulation can include active piecemeal degranulation, cytolytic degranulation, and downstream granule protein deposition.
  • 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.
  • eosinophil granule proteins include, but are not limited to, major basic protein (eMBP), major basic protein- 1 (eMBPl), major basic protein-2 (eMBP2), eosinophil derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO).
  • eMBP major basic protein
  • eMBPl major basic protein- 1
  • eMBP2 major basic protein-2
  • EDN eosinophil derived neurotoxin
  • ECP eosinophil cationic protein
  • EPO eosinophil peroxidase
  • an eosinophil granule protein can be eMBP-1.
  • 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.
  • a radiolabedled heparin can be 99m Tc-heparin.
  • examples of other radiolabels include, but are not limited to, in In, 14 C, 3 H, 13 N, 18 F, 51 Cr, 125 I, 133 , Xe, 81m Kr, and 131 I.
  • Other radiolabels that can be attached to a substance, for example, heparin can be found in Table 1.
  • a radiolabel, for example, 99m Tc can be attached to a substance, for example heparin, using commercially available reagents well known to persons of ordinary skill in the art.
  • 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, eMBPl. eMBPl 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 eMBPl 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.
  • eMBPl is synthesized as a precursor, pro-eMBPl, composed of eMBPl and a remarkably acidic pro-piece sequence. Developing eosinophils synthesize pro-eMBPl, and the pro-piece is removed during granule maturation. Analyses of propiece in different models show that it can neutralize the toxic eMBPl effect and also the toxicity of the eosinophil cationic protein (ECP).
  • ECP eosinophil cationic protein
  • compositions and methods for the neutralization of eMBPl 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- eMBPl and fragments thereof for treatment of eosinophil-mediated diseases by neutralization of eMBPl and other granule toxins. Currently, no therapies for these diseases address the neutralization of granule proteins.
  • Eosinophil granule proteins including eMBPl are toxic and highly basic, and implicated in the pathogenesis of various target tissues including but not limited to the lung, GI tract, sinuses, skin, and the eye.
  • eMBPl is released by activated eosinophils, eMBPl drives inflammation and clinical symptomatology in subjects. It also remains in target tissue much longer than eosinophils and it can be considered a “footprint” of eosinophils.
  • new therapies currently 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 eMBPl.
  • compositions and methods that can be used to neutralize eosinophil granule proteins, and thus, disrupt their cytotoxic effects on tissues.
  • the compositions and methods disclosed herein can be used to treat asthma, and reduce bronchoconstriction.
  • compositions that can be used as therapeutic agents to neutralize the effects of eMBPl and many of the other eosinophil granule proteins.
  • 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.
  • composition can also be utilized as an orally administered medication to treat the eosinophil-mediated inflammation in eosinophilic esophagitis.
  • therapies address the neutralization of the granule proteins (which arguably are the mediators of eosinophil tissue damage).
  • compositions comprising the pro-piece of pro-eosinophil granule major basic protein- 1 (pro-eMBPl) or fragment thereof.
  • Eosinophil granule major basic protein 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).
  • eMBPl 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.
  • SEQ ID NO: 2 a putative signal peptide
  • SEQ ID NO: 3 a middle sequence of amino acids
  • 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 Table 2. Examples of Sequences.
  • the Putative Signal Peptide sequence can be one amino acid shorter than SEQ ID NO: 2.
  • the Putative Signal Peptide sequence can be MKLLLLLALLLGAVS (SEQ ID NO: 9).
  • the Putative Signal Peptide sequence can be one amino acid shorter than SEQ ID NO: 2.
  • the Putative Signal Peptide sequence can be MKLLLLLALLLGAVS AL (SEQ ID NO: 12).
  • the Middle Sequence can be one amino acid longer than SEQ ID NO: 3.
  • the Middle Sequence (Propiece of pro-eMBPl sequence) sequence can be ALHLRSETSTFETPLGAKTLPEDEETPEQEMEETPCRELEEEEEWGSGSEDASKKDGAVE SISVPDMVDKNLTCPEEEDTVKVVGIPGCQ (SEQ ID NO: 10).
  • the the Middle Sequence (Pro-piece of pro-eMBPl sequence) can be one amino acid shorter than SEQ ID NO: 3.
  • the Middle Sequence (Pro-piece of pro- eMBPl sequence) can be HLRSETSTFETPLGAKTLPEDEETPEQEMEETPCRELEEEEEWGSGSEDASKKDGAVESIS VPDMVDKNLTCPEEEDTVKVVGIPGCQ (SEQ ID NO: 11).
  • the Pro-eMBPl sequence can be one amino acid longer than SEQ ID NO: 4.
  • the Pro-eMBPl sequence can be ALHLRSETSTFETPLGAKTLPEDEETPEQEMEETPCRELEEEEEWGSGSEDASKKDGAVE SISVPDMVDKNLTCPEEEDTVKVVGIPGCQTCRYLLVRSLQTFSQAWFTCRRCYRGNLV SIHNFNINYRIQCSVSALNQGQVWIGGRITGSGRCRRFQWVDGSRWNFAYWAAHQPWS RGGHCVALCTRGGHWRRAHCLRRLPFICSY (SEQ ID NO: 13).
  • the Pro- eMBPl sequence can be one amino acid shorter than SEQ ID NO: 4.
  • the Pro- eMBPl sequence can be HLRSETSTFETPLGAKTLPEDEETPEQEMEETPCRELEEEEEWGSGSEDASKKDGAVESIS VPDMVDKNLTCPEEEDTVKVVGIPGCQTCRYLLVRSLQTFSQAWFTCRRCYRGNLVSIH NFNINYRIQCSVSALNQGQVWIGGRITGSGRCRRFQWVDGSRWNFAYWAAHQPWSRG GHCVALCTRGGHWRRAHCLRRLPFICSY (SEQ ID NO: 14).
  • the fragments of pro-piece of pro-eMBPl can comprise a sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% identity to SEQ ID NO: 3.
  • the fragment or portion of pro-eMBPl retains at least 50%, 75%, 80%, 85%, 90%, 95% or 99% of the biological activity of the reference protein described herein.
  • the variants of pro-piece of pro-eMBPl 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.
  • the variants of pro-eMBPl retains at least 50%, 75%, 80%, 85%, 90%, 95% or 99% of the biological activity of the reference protein described herein.
  • the fragments of pro-eMBPl can comprise a sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% identity to SEQ ID NO: 4.
  • the fragment or portion of pro-eMBPl retains at least 50%, 75%, 80%, 85%, 90%, 95% or 99% of the biological activity of the reference protein described herein.
  • the fragment of the pro-piece of pro-eosinophil granule major basic protein-1 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.
  • the fragment of the pro-piece of pro-eosinophil granule major basic protein-1 can comprise SEQ ID NOs: 16, 17, 18, 19 or 20.
  • the fragment or portion of pro-eMBPl 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.
  • pro-piece of pro-eosinophil granule major basic protein- 1 (pro- eMBPl) 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 modifications of N or C terminal amino acids.
  • unnatural amino acids are, for example but not limited to; disubstituted amino acids, N-alkyl amino acids, lactic acid, 4- hydroxyproline, y-carboxyglutamate, e-N,N,N- trimethyllysine, s-N-acetyllysine, O- phosphoserine, N-acetylserine, N- formylmethionine, 3 -methylhistidine, 5 -hydroxy lysine, o-N- methylarginine.
  • the pro-piece of pro-eosinophil granule major basic protein- 1 (pro- eMBPl) or fragment thereof disclosed herein can further comprise a peptide or polypeptide having one or more amino acid residues with a modified side chain.
  • one or more amino acids of any of the pro-piece of pro-eosinophil granule major basic protein-1 (pro- eMBPl) or fragment thereof disclosed here can have a modified side chain.
  • 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 pridoxal-5-phosphate followed by reduction with NaBEU.
  • 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 pridoxal-5-phosphate followed by reduction with NaBEU.
  • the pro-piece of pro-eosinophil granule major basic protein- 1 contains carbohydrates.
  • one or more of the carbohydrates present in the pro-piece of pro-eosinophil granule major basic protein-1 can be used to label for example, with Technetium" 111 . See, for example, Popken-Harris et al., The Journal of Immunology, 1995, 155:1472-1480, which is incorporated herein by reference.
  • 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.
  • 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.
  • the carbohydrate can be a glycosaminoglycan.
  • the glycosaminoglycan at position 46 of SEQ ID NO: 3 can be associated with a change in the charge.
  • the negative charge can be due to sulfation of the glycosaminoglycan moiety.
  • the carbohydrate is heparan.
  • 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.
  • the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBPl) or fragment thereof disclosed herein can combine binding to eMBPl and a heparin/heparan binding.
  • one or more site(s) of carbohydrate attachment can be at one or more amino acid residues of SEQ ID NO: 3.
  • one or more site(s) of carbohydrate attachment can be at one or more of the amino acid residues 7T, 8S, 9T, 18T, 46S, or 70N of SEQ ID NO: 3.
  • amino acid residue 70N can be the site of an N-linked glycan attachment.
  • amino acid residues 7T, 8S, 9T, and 18T can be the site of an O- linked glycan attachment.
  • amino acid residue 46S can be the site of an O- linked glycosaminoglycan attachment.
  • pro-piece of pro-eosinophil granule major basic protein- 1 (pro- eMBPl) or fragment thereof can be modified.
  • pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBPl) or fragment thereof can be be amidated, acetylated, or both. Such modifications can be at the amino or carboxy terminus of the polypeptide.
  • the propiece of pro-eosinophil granule major basic protein- 1 (pro-eMBPl) or fragment thereof can also include peptidomimetics.
  • the pro-piece of pro-eosinophil granule major basic protein- 1 (pro- eMBPl) or fragment thereof described herein can be further modified to improve stability.
  • any of the amino acid residues of the the pro-piece of pro-eosinophil granule major basic protein- 1 (pro-eMBPl) or fragment thereof described herein can be modified to improve stability.
  • the pro-piece of pro-eosinophil granule major basic protein-1 (pro- eMBPl) 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.
  • an acetyl protective group can be bound to the pro-piece of pro-eosinophil granule major basic protein- 1 (pro-eMBPl) or fragment thereof described herein.
  • the pro-piece of pro-eosinophil granule major basic protein-1 (pro- eMBPl) or fragment thereof can have at least one amino acid substitution (e.g., 2, 3, 4, 5, 6, 7, 8,
  • pro-eMBPl pro-eosinophil granule major basic protein- 1
  • pro-eMBPl pro-eosinophil granule major basic protein- 1
  • amino acid substitutions 10 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.
  • 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-eMBPl) or fragment thereof described herein from the attack of protein cleavage enzymes in vivo.
  • the construction of the pro-piece of pro-eosinophil granule major basic protein- 1 (pro-eMBPl) or fragment thereof can include incorporating one or more D-amino acids into the polypeptide.
  • the construction of the pro-piece of pro-eosinophil granule major basic protein- 1 (pro-eMBPl) or fragment thereof can include incorporating one or more L-amino acids into the polypeptide.
  • 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.
  • functional equivalents of the pro-piece of pro-eosinophil granule major basic protein- 1 (pro-eMBPl) or fragment thereof are included with the scope of the invention described 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-eMBPl) 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-eMBPl) or fragment thereof as described herein.
  • the amino acid substitution can be a conservative substitution.
  • amino acid conservative substitution examples include, for example, aliphatic amino acids (Gly, Ala, and Pro), hydrophobic amino acids (He, Leu, and Vai), 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).
  • 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-eMBPl) or fragment thereof disclosed herein.
  • the amino acid sequence of the pro-piece of pro-eosinophil granule major basic protein- 1 (pro-eMBPl) 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-eMBPl) or fragment thereof disclosed 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 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.
  • the amino acid sequence of the pro-piece of pro-eosinophil granule major basic protein- 1 (pro-eMBPl) 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-eMBPl) 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.
  • the polypeptides 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- eMBPl) 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 proeosinophil granule major basic protein- 1 (pro-eMBPl) or fragment thereof, wherein the propiece of pro-eosinophil granule major basic protein- 1 (pro-eMBPl) or fragment thereof is SEQ ID NO: 3.
  • the pro-piece of pro-eosinophil granule major basic protein-1 (pro- eMBPl) or fragment thereof can be produced by recombinant technology or chemical synthesis.
  • the pro-piece of pro-eosinophil granule major basic protein- 1 (pro-eMBPl) or fragment thereof can be conjugated to a therapeutic agent or a label.
  • the therapeutic agent can be benralizumab.
  • 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.
  • the therapeutic agent can be one that disrupts inflammation.
  • the therapeutic agent can be one that disrupts protein synthesis or reduces cell proliferation.
  • the therapeutic agent can be ruxolitnib.
  • the pro-piece of pro-eosinophil granule major basic protein- 1 (pro-eMBPl) 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.
  • one or more of the cysteine residues present in the Pro-piece of pro- eMBPl or fragment thereof can be a site for conjugation.
  • one or more of the cysteine residues at positions 35, 73, or 88 as identified in SEQ ID NO: 3 in the Pro-piece of pro- eMBPl or fragment thereof can serve as a reactive sulfhydryl group.
  • the reactive sulfhydryl group can be used to label for example, with Technetium" 111 .
  • one or more of the lysine residues present in the Pro-piece of pro-eMBPl or fragment thereof can be a site for conjugation.
  • 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.
  • the label can be a radioisotope.
  • the label can be Technetium99m, barium or gadolinium.
  • the radioisotope can be Technetium99m ( 99m Tc). Examples of radiolabels can be found in Table 1.
  • compositions comprising pro-piece of proeosinophil granule major basic protein- 1 (pro-eMBPl) or fragment thereof described herein.
  • pharmaceutical compositions comprising pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBPl) or fragment thereof described herein and a pharmaceutical acceptable carrier.
  • 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.
  • compositions for delivering a therapeutic agent to a diseased tissue, organ, or body part; and treating eosinophilic-related inflammation in a subject.
  • 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-eMBPl) or fragment thereof disclosed herein; and a pharmaceutically acceptable carrier.
  • 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-eMBPl) or fragment thereof.
  • Therapeutic administration encompasses prophylactic applications.
  • 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.
  • 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.
  • the patient can be a human patient.
  • 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).
  • 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.
  • 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.
  • 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-eMBPl) or fragment thereof alone or conjugated to a therapeutic agent.
  • pro-eMBPl pro-eosinophil granule major basic protein-1
  • 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.
  • TGF tumor necrosis factor
  • PDE phosphodiesterase
  • TGF transforming growth factor
  • 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.
  • the term “excipient” means any compound or substance, including those that can also be referred to as “carriers” or “diluents.”
  • the compositions can include a pharmaceutically acceptable adjuvant, carrier or other diluent.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions can be prepared for parenteral administration that includes the the pro-piece of proeosinophil granule major basic protein- 1 (pro-eMBPl) 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.
  • an aqueous carrier such as water, buffered water, saline, buffered saline (e.g., phosphate buffered saline (PBS)), and the like.
  • PBS phosphate buffered saline
  • 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.
  • compositions include a solid component (as they may for oral 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).
  • a binder or filler e.g., for the formulation of a tablet, a capsule, and the like.
  • one or more of the excipients can be a solvent or emulsifier for the formulation of a cream, an ointment, and the like.
  • the compositions described herein can be formulated as a spray.
  • 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.
  • compositions described herein can be pharmaceutical compositions.
  • 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, intraauricle, inner or outer ear, intra-vesicular, per rectum), intra-articularly, or ocularly.
  • the compositions described herein can be administered to a subject via a medical device or tube.
  • the medical device or tube can be a nasal-gastric tube or a radiology- placed cannula.
  • Oral dosing 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-1 (pro-eMBPl) or fragment thereof conjugated to label
  • pro-eMBPl pro-eosinophil granule major basic protein-1
  • sucralose a thickened mixture
  • 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.
  • 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, com syrup, thick juices, maple syrup, coconut oil, and palm oil.
  • RESULTS OF DIAGNOSIS AND DETECTION 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.
  • an eosinophil granule protein e.g., major basic protein 1
  • the methods can comprise administering to a subject a composition comprising the pro-piece of pro-eosinophil granule major basic protein- 1 (pro-eMBPl) or fragment thereof conjugated to label.
  • the pro-piece of pro-eMBPl or fragment thereof can bind to one or more eosinophil granule proteins in the tissue, organ, or body part of the subject.
  • said binding can generate a pro-piece-eMBPl -labeled protein complex.
  • the methods can include detecting the pro-piece-eMBPl -labeled protein complex in the tissue, organ, or body part, whereby detecting the pro-piece-eMBPl - 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.
  • the localization of eosinophil granule protein in a tissue, organ, or body part can be microscopically detected.
  • the methods can comprise detecting the pro-piece-eMBPl -labeled protein complex in the tissue, organ, or body part, whereby detecting the pro-piece-eMBPl - labeled protein complex in the tissue, organ, or body part can indicate eosinophil-related inflammation in the subject.
  • the methods can comprise detecting the pro-piece- eMBPl -labeled protein complex in the tissue, organ, or body part, whereby detecting the pro- piece-eMBPl -labeled protein complex in the tissue, organ, or body part in the subject can indicate eosinophil degranulation.
  • the methods can comprise: administering to a subject a composition comprising the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBPl) or fragment thereof conjugated to label.
  • pro-eMBPl pro-eosinophil granule major basic protein-1
  • the pro-piece of pro-eMBPl or fragment thereof can bind to one or more eosinophil granule proteins in a tissue, organ, or body part of the subject.
  • the binding can generate a pro-piece- eMBPl -labeled protein complex.
  • the methods can further comprise detecting the pro-piece-eMBPl -labeled protein complex in the tissue, organ, or body part, whereby detecting the pro-piece-eMBPl -labeled protein complex in the tissue, organ, or body part in the subject can indicate eosinophil granule protein localization and/or eosinophil degranulation.
  • eosinophil granule proteins in a subject, the methods comprising: utilizing a composition comprising the pro-piece of proeosinophil granule major basic protein- 1 (pro-eMBPl) or fragment thereof conjugated to a supporting element, wherein the pro-piece of pro-eMBPl 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-eMBPl -support complex; and b) detecting the pro-piece-eMBPl -support complex in the body fluid or tissue, organ, or body part extract, whereby detecting the pro-piece-eMBPl -support complex in the body fluid, tissue, organ, or body part extract from the subject indicates eosinophil granule protein localization.
  • pro-eMBPl major basic protein- 1
  • the pro-piece-eMBPl-support complex can be 99m Tc-pro-eMBPl/MBP-l, 99m Tc- pro-eMBPl/MBP, 99m Tc- pro-eMBPl/MBP-2, 99m Tc- pro-eMBPl /EDN, 99m Tc- pro-eMBPl /ECP, and 99m Tc- pro- eMBPl/EPX.
  • a support element can be 99m Tc-heparin, in In-heparin, or deheparin.
  • a labeled pro-piece-eMBPl -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.
  • 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-eMBPl) or fragment thereof conjugated to a supporting element, wherein the pro-piece of pro-eMBPl or fragment thereof binds to one or more eosinophil granule proteins in the sample, wherein said binding generates a pro-piece-eMBPl -support complex; and detecting the pro-piece-eMBPl -support complex in the sample, whereby detecting the pro-piece- eMBPl -support complex in the sample thereby detecting a eosinophil granule protein in the sample.
  • pro-eMBPl major basic protein- 1
  • eosinophil granule proteins in a sample, the methods comprising: contacting a sample with a composition comprising the propiece of pro-eosinophil granule major basic protein- 1 (pro-eMBPl) or fragment thereof conjugated to a supporting element under conditions to allow the pro-piece of pro-eMBPl or fragment to bind to one or more eosinophil granule proteins in a sample, wherein said binding generates a pro-piece-eMBPl -support complex; and detecting the pro-pi ece-eMBPl -support complex in the sample, thereby detecting a eosinophil granule protein in the sample.
  • pro-eMBPl major basic protein- 1
  • eosinophil granule proteins in a sample, the methods comprising: utilizing a composition comprising the pro-piece of proeosinophil granule major basic protein- 1 (pro-eMBPl) or fragment thereof conjugated to a supporting element, wherein the pro-piece of pro-eMBPl or fragment thereof binds to one or more eosinophil granule proteins in a sample, wherein said binding generates a pro-piece- eMBPl -support complex; and detecting the pro-piece-eMBPl -support complex in the sample, whereby detecting the pro-piece-eMBPl -support complex in the sample indicates eosinophil granule protein localization.
  • pro-eMBPl major basic protein- 1
  • 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-eMBPl complex in the sample indicates eosinophil degranulation.
  • the pro-piece-eMBPl -support complex can be 99m Tc- pro-eMBPl/MBP-1, 99m Tc- pro-eMBPl /MBP, 99m Tc- pro-eMBPl /MBP-2, 99m Tc- pro- eMBPl/EDN, 99m Tc- pro-eMBPl /ECP, and 99m Tc- pro-eMBPl /EPX.
  • a support element can be 99m Tc-heparin, 111 In-heparin, or 14 C-heparin.
  • the pro-piece-eMBPl can be used as a reagent.
  • a labeled pro-piece-eMBPl -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.
  • the subject can be diagnosed with an eosinophil-related disease.
  • 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.
  • the method can comprise administering to a subject a composition comprising the pro-piece of proeosinophil granule major basic protein-1 (pro-eMBPl) or fragment thereof conjugated to label.
  • pro-eMBPl major basic protein-1
  • the pro-piece of pro-eMBPl 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-eMBPl -labeled protein complex. In some aspects, the method 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-eMBPl) or fragment thereof conjugated to label.
  • pro-eMBPl major basic protein- 1
  • the pro-piece of pro-eMBPl 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-eMBPl -labeled protein complex.
  • 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.
  • the eosinophil granule protein can be major basic protein 1 (eMBPl). 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).
  • eMBPl major basic protein 1
  • ECP eosinophil cationic protein
  • EPX eosinophil peroxidase
  • 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.
  • fluorochromes include fluorescent dyes.
  • enzymes and substrates include peroxidases and/or peroxidase substrates).
  • glycoproteins include avidin and biotin.
  • the label can be a radioisotope.
  • the radioisotope can be Technetium99m ( 99m Tc).
  • 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-eMBPl) 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.
  • a composition can be conjugated to 99m Tc- heparin, ni In-heparin, or 14 C-heparin, or any combination thereof.
  • such a composition can be conjugated to a radioisotope or label as described herein without using heparin.
  • a label (or radioisotope) can be Technetium99m ( 99m Tc).
  • Examples of a pro-piece-eMBPl -labeled protein complex include, but are not limited to, 99m Tc-pro- eMBPl/MBP-1, 99m Tc- pro-eMBPl/MBP, 99m Tc- pro-eMBPl /MBP-2, 99m Tc- pro-eMBPl /EDN, " m Tc- pro-eMBPl /ECP, and 99m Tc- pro-eMBPl /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, 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.
  • 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.
  • the volume of the composition administered to a subject can be from about 0.1 mL to about 5 mL.
  • 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.
  • the volume of the composition administered to a subject can be about 0.1 mL to about 1 mL.
  • 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.
  • the volume of the composition administered to a subject can be about 1 mL to about 5 mL.
  • 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.
  • the volume of the composition administered to a subject can be from about 5 mL to about 250 mL.
  • the volume of the composition administered to a subject can be from about 10 mL to about 125 mL.
  • the volume of the composition administered to a subject can be from about 15 mL to about 100 mL.
  • 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,
  • pro-eMBPl pro-eosinophil granule major basic protein-1
  • a person of skill can use one or more technologies and processes to detect pro- piece-eMBPl -labeled protein complexes in a tissue, organ, or body part in a subject.
  • pro-pi ece-eMBPl -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.
  • SPECT single photon emission computed tomography
  • PET positron emission
  • CT computed tomography
  • MRI magnetic resonance imaging
  • 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.
  • 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.
  • any of the methods disclosed herein can comprise one or more of the following steps.
  • the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBPl) or fragment thereof can be conjugated to reduced Technetium-99m (Tc99m) using a cysteine residue as the amino acid for attachment.
  • Tc99m Technetium-99m
  • 3 mg pro-piece of pro- eMBPl can be labeled with 3 mCi Tc99m.
  • the pro-piece of pro-eMBPl -Tc99m (3 mCi and 3 mg pro-piece of pro-eMBPl) can be present in 15 mL of water.
  • the methods can comprise a subject swallowing the pro-piece of pro-eMBPl -Tc99m in 1 mL sips over a 15 minute period (start at zero time).
  • SPECT/CT can be performed.
  • a subject can swallow 100 mL of water (as a wash) in 15 swallows of about 6.5 mL over a 15 minute period.
  • the SPECT/CT can be repeated.
  • the radiolabeled pro-piece of pro-eMBPl or fragment thereof disclosed herein can be prepared at various doses.
  • the radiolabeled pro-piece of pro-eMBPl 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.
  • the dose of radiolabeled propiece of pro-eMBPl or fragment thereof can be 0.3 mCi.
  • the dose of radiolabeled pro-piece of pro-eMBPl or fragment thereof can be 1.0 mCi. In some aspects, the dose of radiolabeled pro-piece of pro-eMBPl or fragment thereof can be 10 mCi. In some aspect, the radiolabeled pro-piece of pro-eMBPl or fragment thereof can be Tc-99m-pro-piece of pro- eMBPl.
  • the doses of Tc-99m-pro-piece of pro-eMBPl 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.
  • the dose of Tc-99m-pro-piece of pro-eMBPl can be 0.3 mCi.
  • the dose of Tc-99m-pro-piece of pro-eMBPl can be 1.0 mCi.
  • the dose of Tc-99m-pro-piece of pro-eMBPl can be 10 mCi.
  • 1-100 mg of propiece of pro-eMBPl can be labeled with 0.1 to 30 mCi of a radiolabel (e.g. technetium-99M).
  • the ratio of pro-piece of pro-eMBPl to Tc-99m can be 1:1.
  • 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.
  • 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.
  • the eosinophil- related inflammation or eosinophil-related disease can be an eosinophilic pancreatitis.
  • 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.
  • 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.
  • the eosinophil-related inflammation or eosinophil-related disease can be an eosinophilic ocular disorder.
  • eosinophilic ocular disorder include but are not limited to allergic conjunctivitis (e.g., seasonal and perennial), giant papillary conjunctivitis, and keratoconjunctivitis (atopic and vernal)).
  • the eosinophil-related inflammation or eosinophil-related disease can be an eosinophilic nephritis.
  • the eosinophil- related inflammation or eosinophil-related disease can be an eosinophilic cystitis.
  • the eosinophil-related inflammation or eosinophil-related disease can be an eosinophilic 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.
  • 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.
  • 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.
  • 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
  • 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.
  • a parasitic disease can include but is not limited to helminthic infections and ectoparasites.
  • 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).
  • the eosinophil-related inflammation can be caused by a solid tumor (e.g., a malignancy), a lymphoma or a leukemia.
  • the activating factor can be a marker for a cancer.
  • eosinophils can indicate a gastrointestinal cancer.
  • the eosinophil-related inflammation or eosinophil-related disease can be eosinophil-related syndrome.
  • eosinophil-related syndromes can include eosinophilia myalgia syndrome (EMS) and toxic oil syndrome (TOS).
  • Eosinophilia myalgia syndrome and toxic oil syndrome include but are not limited to severe myalgia 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.
  • eosinophil-related syndromes can include eosinophilic granulomatosis with polyangiitis (Churg- Strauss syndrome).
  • Symptoms of eosinophilic granulomatosis with polyangiitis include but are not limited necrotizing vasculitis with hypereosinophilia; antineutrophil cytoplasmic antibodies (e.g., ANCA1 and ANCA2 subvariants); 4 of 6 criteria including asthma, eosinophilia, history of allergy, nonfixed pulmonary infiltrates, paranasal sinus abnormalities, and extravascular eosinophils.
  • eosinophil-related syndromes can include episodic angioedema with eosinophilia (Gleich syndrome).
  • Episodic angioedema with eosinophilia 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.
  • 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.
  • STAT3 signal transducer and activator of transcription 3
  • DOCK8 dedicatedor of cytokinesis 8
  • 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.
  • 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., RAG I or RAG2).
  • the eosinophil-related inflammation or eosinophil-related disease can be an eosinophil-related dermatoses.
  • eosinophil-related dermatoses can be an eosinophil-related dermatoses.
  • diseases including but not limited to: epidermis eosinophilic spongiosis; dermis, connective tissue eosinophilic cellulitis; dermis, blood vessels eosinophilic vasculitis; hair follicles eosinophilic folliculitis; subcutaneous fat eosinophilic panniculitis; fascia eosinophilic fasciitis; muscle eosinophilic myositis; and nerve eosinophilic neuritis.
  • 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 his
  • 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.
  • eosinophil-related diseases 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.
  • Table 8 Eosinophil-related cutaneous disease; Biopsy specifications for testing and site selection. Table 9. Definition and gradations of peripheral blood eosinophilia and tissue hypereosinophilia in eosinophil-related diseases. Table 10. Histopathological patterns with eosinophil-related inflammation in different cutaneous compartments.
  • 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.
  • an organ can include one or more glands.
  • the gland can be thymus.
  • an organ can include one or more organ systems.
  • an organ can include the nervous system.
  • the 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.
  • an organ can include the respiratory system.
  • the respiratory system includes mouth, nose, pharynx, larynx, trachea, bronchi, lungs and diaphragm.
  • an organ can include the biliary system.
  • the biliary 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.
  • the tissue can be epithelial, connective, muscular or nervous.
  • 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.
  • the tissue can be subcutaneous fat, fascia, muscle, endomysium, fibrous tissue, mesentery, or diaphragm.
  • 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.
  • the body part can be an extremity, including for example, an arm, a leg, neck, ear or nose.
  • ultrasound can be used to aid the diagnosis of eosinophilic fasciitis; it is not precise.
  • the compositions and methods disclosed herein can be used to precisely detect the location of eosinophilic fasciitis inflammation, for instance, in a leg.
  • the subject can be a mammal.
  • the mammal can be a human, cat, a dog, a pig, or a cow.
  • the subject can be a human.
  • the subject can be a dog.
  • the pro-piece-eMBPl -labeled protein complex resulting from the pro-piece of pro-eMBPl 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 (MRI), ultrasound, laser, and light.
  • SPECT single-photon emission computed tomography
  • PET positron emission tomography
  • CT conventional or computed tomography
  • MRI magnetic resonance imaging
  • ultrasound laser
  • the light can be visible, ultraviolet, or infrared (with and without magnification).
  • 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-eMBPl) or fragment thereof conjugated to a therapeutic agent to a subject.
  • 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- eMBPl) or fragment thereof as a therapeutic agent to a subject.
  • 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- eMBPl) or fragment thereof conjugated to a therapeutic agent to a subject.
  • a composition comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro- eMBPl) or fragment thereof conjugated to a therapeutic agent to a subject.
  • 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-eMBPl) or fragment thereof as a therapeutic agent to the subject, thereby reducing inflammation in the subject.
  • a composition comprising: the pro-piece of pro-eosinophil granule major basic protein-1 (pro-eMBPl) or fragment thereof as a therapeutic agent to the subject, thereby reducing inflammation in the subject.
  • pro-eMBPl pro-eosinophil granule major basic protein-1
  • 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-eMBPl) or fragment thereof conjugated to a therapeutic agent to a subject.
  • a composition comprising: the pro-piece of pro-eosinophil granule major basic protein- 1 (pro-eMBPl) or fragment thereof conjugated to a therapeutic agent to a subject.
  • the diseased tissue, organ or body part can be any tissue, organ or body part disclosed herein.
  • 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.
  • 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.
  • the calcineurin inhibitor can be sirolimus, thiopurine, cyclosporine or tacrolimus.
  • the glucocorticoid can be mometasone, fluticasone, budesonide, prednisone or solumedrol.
  • the therapeutic agent can be CellCept®.
  • the therapeutic agent can be benralizumab.
  • the dose of benralizumab can be lower than the dose currently administered.
  • other therapeutic agents include but are not limited to IL-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).
  • a step comprising identifying the subject in need thereof.
  • the subject in need thereof can be identified by any of the methods disclosed herein.
  • Example 1 Pro-piece of pro-eMBPl (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 pl values of 11. Among these, the most abundant on a molar basis is eMBP 1 , which is exceedingly cationic with a calculated pl of 11.6, is rich in arginine and has a molecular weight of 14 kDa. The eMBPl crystal structure reveals an atypical lectin that binds heparin.
  • eMBPl is the most highly expressed gene at 8% of transcripts; these analyses also revealed a homologue of eMBPl with a reduced isoelectric point, referred to as eMBP2.
  • eMBPl is toxic to helminths, bacteria and numerous cells, such as respiratory epithelium, and is stimulatory to other cells, including basophils and mast cells.
  • eMBPl BacteMBPl kills parasites including schistosomules of Schistosoma mansoni, bloodstream forms of Trypanosoma cruzi, newborn larvae of Trichinella spiralis and microfilariae of Brugia pahangi and Brugia malayi, that it kills bacteria, and that it is toxic for numerous cells, including other leukocytes, skin, intestinal cells, tracheal epithelium, and tumor cells.
  • eMBPl also activates cells, including other leukocytes such as basophils and neutrophils.
  • eMBPl Instillation of eMBPl into monkey lungs produced bronchospasm and bronchial hyperreactivity; installation into the lungs of mice produced cough, which are characteristic features of bronchial 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. eMBPl also is extruded with extracellular DNA traps formed by eosinophils.
  • eMBPl 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.
  • eMBPl In diseases mediated by helminths, eMBPl 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 eMBPl can be used as a treatment strategy to mitigate tissue damage in eosinophil-related inflammation.
  • eosinophil granule proteins include the eosinophil peroxidase (EPX) , the eosinophil cationic protein (ECP), the eosinophil-derived neurotoxin (EDN) and the homologue of eMBPl, eMBP2; the Charcot-Leyden crystal protein, now known as galectin 10, has been regarded as a granule protein as well.
  • ECP eosinophil cationic protein
  • EDN eosinophil-derived neurotoxin
  • galectin 10 the Charcot-Leyden crystal protein
  • 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 eMBPl but is less biologically active.
  • Amino acid sequencing of eMBPl revealed 117 amino acids and the absence of any carbohydrate.
  • sequencing of the eMBPl cDNA indicated the presence of a precursor molecule, referred to as pre-pro MBP1, and composed of eMBPl and the pro-piece of pro-eMBPl sequence.
  • the leader sequence is cleaved off during endoplasmic reticulum translocation.
  • the pre-pro-eMBPl is the glutamic acid-rich neutralizing pro-piece pro-eMBPl while the active arginine-rich section is the eMBPl sequence.
  • sequencing of eMBPl cDNA indicated that eMBPl is synthesized as a precursor composed of eMBPl with an exceedingly acidic pro-piece of pro-eMBPl and referred to as pro-eMBPl .
  • pro-eMBPl Developing eosinophils synthesize pro-eMBPl, and the pro-piece of pro-eMBPl is removed during granule maturation.
  • pro-piece of pro-eMBPl established that it can neutralize toxic effects of eMBPl and, also, of another granule protein, eosinophil cationic protein (ECP).
  • ECP eosinophil cationic protein
  • pro-piece of pro-eMBPl By expressing pro-piece of pro-eMBPl, it was found that it is gly can-rich, and evaluated its binding to eMBPl by surface plasmon resonance. The characteristics of pro-piece of pro-eMBPl that maximally neutralize the deleterious effects of eMBPl and other granule toxins as a therapy for eosinophil- related diseases was also determined.
  • pro-eMBPl in Chinese hamster ovary (CHO) cells showed the presence of a glycosylated molecule devoid of the cytotoxic and cytostimulatory activities of the 14 kDa eMBPl.
  • the CHO-proMBPl neutralized the toxicity of eMBPl itself as demonstrated by inhibition of the basophil histamine release and superoxide anion generation stimulated by eMBPl.
  • the pro-piece of pro-eMBPl is remarkably acidic with a strikingly increased content of acidic amino acids, especially glutamic acid, in contrast to the basicity of eMBPl. Polymers of glutamic and aspartic acids neutralized the toxicity of eMBPl and ECP.
  • eMBPl 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-eMBPl from pregnancy serum showed that it is heavily glycosylated with a 10 kDa glycosaminoglycan at residue S62 (for example, see SEQ ID NO: 15).
  • pro-piece of pro-eMBPl the molecular form that optimally binds to and neutralizes eMBPl will be identified. Also, it will be determined whether glycosylation of propiece of pro-eMBPl importantly alters its binding to eMBPl by testing whether its gly cans and/or glycosaminoglycan modify its activities. By creating overlapping peptides and mutants with increased amino acid sequences characteristic of active regions, the most active pro-piece of pro-eMBPl peptide regions for eMBPl binding and neutralization will be identified. These will also be tested for neutralizing other granule proteins.
  • the ability of the pro-piece of pro-eMBPl to bind to and neutralize eosinophil granule proteins will be determined.
  • the pro-piece of pro- eMBPl forms generated using surface plasmon resonance will be tested to determine binding to eMBPl and using killing of K562 tumor cells and stimulatory activity for histamine release to test neutralization of eMBPl’s activities.
  • the pro-piece of pro-eMBPl will be dissected to determine the effect of removing glycans on its binding and inhibitory activities.
  • pro-eMBPl peptides If removal of glycans has no effect on, or increases binding and inhibitory activities, we will test overlapping pro-piece of pro-eMBPl 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-, O- 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 eMBPl plasmid to alter amino acids at glycan binding sites and thus to produce pro-piece of pro-eMBPl devoid of glycans.
  • pro-piece of pro-eMBPl 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-eMBPl forms with increased representation of implicated glycans.
  • pro-piece of pro-eMBPl as currently expressed in HEK cells may be a useful reagent for neutralization of eMBPl, it is important to determine if the marked quantity of glycans contributes to or detracts from eMBPl binding.
  • Analyses of pro-eMBPl isolated from pregnancy serum indicate the presence of O-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).
  • ProMBPl produced by CHO cells possessed a glycosaminoglycan at S62 (see, for example, , SEQ ID NO: 15); since this molecule neutralized the cytostimulatory effect of eMBPl on basophils and neutrophils, these structural features may be important in neutralizing eMBPl.
  • Pro-eMBPl from a T-cell hybridoma contained glycosylation at residues S24, T25 and T28 (see, for example, SEQ ID NO: 15); no information is available on the inhibition ability of this protein.
  • the pro-piece of pro-eMBPl will be enzymatically treated to remove glycans from N-linked and O-linked sugars and to deplete the O-linked glycosaminoglycan.
  • the pro-piece of pro-eMBPl will be expressed in HEK293S GnTi- cells engineered to limit glycosylation to further test the importance of the N-linked glycan at N86.
  • the mutant pro-pieces of pro-eMBPl 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.
  • pro-piece of pro-eMBPl binds less avidly than the pro-piece of pro-eMBPl glycosylated molecule, then the released sugar molecules will be chemically characterized to define their structures with attention to the O-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.
  • pro-piece of pro-eMBPl 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-eMBPl .
  • the O-linked glycosaminoglycans attached to pro-piece of pro-eMBPl 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 eMBPl will be characterized.
  • 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-eMBPlhis, 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-HCl, 500 mM NaCl, pH 7.5. In the prior experiments a peak eluted and fractions combined after SDS-PAGE and western blotting analyses.
  • FIG. 1 shows binding of the pro-piece of pro-eMBPl to eMBPl by surface plasmon resonance.
  • pro-piece of pro-eMBPl 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).
  • PNGase F peptide N-glycosidase F
  • approaches for removal of O-linked glycans are: a) Treatment with trifluoromethanesulfonic acid (Sigma Kit PP0510), this removes both N- and O-linked glycans; b) Treatment with the O-glycosidase from Streptococcus pneumonia combined with exoglycosidases; and c) Treatment with proprietary kits that remove O-glycans, including EZGlyco (from S-Bio Company) and OpeRATOR (from Genovis).
  • EZGlyco from S-Bio Company
  • OpeRATOR from Genovis
  • glycosaminoglycan at residue 62 will be digested using chondroitinase ABC and heparin lyases I, II and III; prior experiments with pregnancy pro- eMBPl 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-eMBPl devoid of N-linked carbohydrates using HEK293S GnTi- (ATCCD CRL-3022) cells; b) expression of the pro-piece of pro-eMBPl using HEK293-GlycoDelete cells (ATCC CRL-1573); and c) expression of mutants at glycan binding sites of the plasmid system described herein.
  • the pro-piece of pro-eMBPl will be produced in E. coll; note that pro-piece of pro-eMBPl is expressed as a soluble protein and spontaneously dimerizes by formation of a disulfide bond.
  • 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-eMBPl 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 pattem/disaccharide composition, respectively. Binding to eMBPl will be measured by surface plasmon resonance and the ability to inhibit the biological effects of eMBPl will be performed.
  • An alternative to the enzymatic and chemical approaches to delineate the role of glycans in binding to eMBPl is mutation of the eMBPl plasmid to alter amino acids at glycan binding sites and thus produce pro-piece of pro-eMBPl devoid of glycans at specific sites.
  • pro-piece of pro-eMBPl will be analyzed by surface plasma resonance to determine which molecules have the greatest affinity for immobilized eMBPl. 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-eMBPl 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-eMBPl peptides.
  • the pro-piece of pro-eMBPl peptides will further be tested for their ability to inhibit eMBPl killing of K562 cells and to inhibit eMBPl’s activation of mast cells.
  • eMBPl Approximately 1500 - 3000 response units of eMBPl will be immobilized on the surface via thiol coupling.
  • carboxylate surface is activated with sulfo-NHS (N-hydroxysulfosuccinimide)ZEDC (l-ethyl-3- (3 -dimethylamino propyl) carbodiimide hydrochloride) followed by PDEA (2-(2- Pyridinyldithio)ethaneamine hydrochloride) to produce a reactive mixed disulfide.
  • eMBPl is then immobilized by its free cysteines.
  • eight flow channels each with an in-line control (background binding), eight simultaneous binding assays can be performed.
  • Binding of the eMBPl ligands will be tested in triplicate at equal molar concentrations. Surface regeneration will be performed with two 5- second pulses of 6M guanidine-HCl. 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-eMBPl peptides for these targets in addition to eMBPl will be determined. Synthetic peptides from eMBPl 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 eMBPE
  • eMBPl and other eosinophil granule proteins kill tumor cells including K562 cells.
  • the ability of the modified propiece of pro-eMBPl constructs to neutralize killing of K562 cells by eMBPl, ECP and EPX will be tested.
  • eMBPl and other eosinophil granule proteins killed tumor cells utilizing K562 cells; eMBPl produces dose dependent killing, and the cytotoxicity is measured by trypan blue exclusion.
  • K562 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 eMBPl or other granule proteins, K562 cells will be washed thrice with RPMI 1640 without serum, centrifuged at 200 X G at room temperature for 10 minutes, and suspended at 1 *10 6 cells/mL in serum free RPMI 1640. For measurement of cytotoxicity, sterile 0.4% trypan blue (Sigma) in PBS filtered through a 0.45 pm filter will be used.
  • modified pro-piece of pro-eMBPl constructs and analogs will be measured for their abilities to inhibit the activity of eMBPl, ECP and EPX in non-cytotoxic, stimulated histamine release from fresh rat mast cells.
  • eMBPl is toxic to numerous biological targets, and it also has the capability to activate cells in anon-cytotoxic manner. Therefore, it will be tested whether pro-piece of pro-eMBPl can inhibit non-cytotoxic eMBPl activity.
  • Rat mast cells will be used because both eMBPl and ECP stimulate histamine release in a non-cytotoxic reaction whereas human basophils are activated by eMBPl (and also eMBP2).
  • the pro-piece of pro-eMBPl was expressed in human embryonic kidney (HEK) cells. It is a 25 kDa heterogeneous glycoprotein able to bind to eMBPl by surface plasma resonance (FIG. 1); the peptide part of the pro-piece of pro-eMBPl accounts for about 10 kDa indicating that the HEK expressed molecule is rich in glycan structures.
  • HEK human embryonic kidney
  • 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 (Cytiva Life Sciences; Marlborough, MA) equipped with a CM4 sensor chip. During data collection, the autosampler was maintained at 8°C.
  • 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 pL/min.
  • eMPB-1 was diluted to 0.6 pM in 10 mM sodium acetate, pH 5.25 and injected for 6 minutes at 10 pL/min.
  • pro-piece of pro-eMBPl 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 pL/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-eMBPl and extending the dissociation time for 1800 s.
  • the surface was regenerated using two, 5 s injections of 6 M guanidine-HCl.
  • pro-eMBPl neutralized the cytotoxic and cyto-stimulatory activities of eMBPl itself, and because the structural difference between toxic eMBPl and pro-eMBPl is the presence of the pro-piece of pro-eMBPl, these results show that the pro-piece of pro-eMBPl is able to neutralize the biologic activities of eMBPl.
  • the properties of the eMBPl pro-piece of pro-eMBPl will be determined for the strongest binding to eMBPl and to test if it is able to neutralize the biological effects of eMBPl; the pro-piece of pro-eMBPl will also be tested for neutralizing activity against EPX and ECP in these same assays.
  • 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 eMBPl, but also with ECP and EPX.
  • the MBP neutralization assay can quantify the neutralizing ability of inhibitors for the toxic eosinophil granule protein, eMBPl.
  • the tumor cell line, K562 is used as a model for cytotoxicity. K562 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 l*10 5 to l *10 6 cells/mL.
  • IMDM Iscove's Modified Dulbecco's Medium
  • FBS Fetal Bovine Serum
  • samples of approximately I / 10 s 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 *10 5 cells/mL for use in the assay. Exposure to eMBPl takes place in 384-well microtiter plates over the course of an 18-hour incubation at 37°C and 5% CO2.
  • eMBPl in Phosphate-buffered saline is added to each microtiter well followed by 5 pl of the inhibitor in PBS.
  • PBS Phosphate-buffered saline
  • 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 eMBPl concentration of 5*10' 6 M was chosen to be neutralized as it reproducibly kills most (e.g., 90-99%) K562 cells in the K562 cytotoxicity assay.
  • a series of toxin concentrations are tested in the assay to determine the IC50.
  • the CellTiter-Glo reagent allows the determination of viability by quantitating adenosine 5'-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 microtiter 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.
  • 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.
  • a blank signal is subtracted from the data points in each trial.
  • a ”blank“ corresponds to a well of cells incubated with 5xl0 -6 M eMBPl (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 eMBPl.
  • FIG. 2 shows the percent inhibition of eMBPl by a pro piece of pro-eosinophil granule major basic protein- 1.
  • Example 3 Inhibition of eMBPl by WT ProPiece of Pro-eMBPl
  • pro-piece of pro-eMBPl will be expressed and associated glycans will be modified to identify the optimal form of the molecule for binding to and neutralizing eMBPl.
  • These tests will determine whether glycosylation of pro-piece of pro-eMBPl importantly alters its binding to eMBPl by analyzing whether N- or O- glycans and the glycosaminoglycan at S62 modify its activities.
  • This study will identify the most important pro-piece of pro-eMBPl regions for binding eMBPl and neutralizing it by creating overlapping peptides and creating mutants with increased amino acid sequences characteristic of active regions.

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Abstract

L'invention concerne des compositions et des méthodes de diagnostic, de détection et de traitement d'une maladie liée aux éosinophiles ou d'une inflammation liée aux éosinophiles chez un sujet. L'invention concerne également des compositions comprenant la pro-pièce de protéine basique majeur de granule pro-éosinophile-1 ou un fragment de celle-ci et leurs procédés d'utilisation.
PCT/US2022/075946 2021-09-03 2022-09-02 Compositions et méthodes de diagnostic, de détection et de traitement de maladies liées aux éosinophiles WO2023034993A1 (fr)

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