US20190345473A1 - Methods and compositions for inhibiting adam10 biological activities - Google Patents

Methods and compositions for inhibiting adam10 biological activities Download PDF

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US20190345473A1
US20190345473A1 US16/149,764 US201816149764A US2019345473A1 US 20190345473 A1 US20190345473 A1 US 20190345473A1 US 201816149764 A US201816149764 A US 201816149764A US 2019345473 A1 US2019345473 A1 US 2019345473A1
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seq
amino acid
adam10
modulating peptide
acid positions
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Marcia L. Moss
Robert Rasmussen
Chris PRINCE
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Verra Therapeutics LLC
Verra Therapeutics Inc
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Verra Therapeutics Inc
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Assigned to Verra Therapeutics LLC reassignment Verra Therapeutics LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MOSS, MARCIA, PRINCE, CHRIS, RASMUSSEN, ROBERT
Priority to US17/748,163 priority patent/US12297469B2/en
Priority to US19/072,106 priority patent/US20250207119A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24081—ADAM10 endopeptidase (3.4.24.81)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the presently disclosed subject matter relates to compositions and methods pertaining to the inhibition of ADAM10 biological activities.
  • the presently disclosed subject matter relates to modified ADAM10 prodomain peptides that, as a result of the modifications, exhibit increased stability to cleavage by furin and furin-like proteases and meprin and meprin-like proteases.
  • the presently disclosed subject matter further relates to the use of said modified ADAM10 prodomain peptides in cellular assays, and to the use of said modified ADAM10 prodomain peptides for treatment of diseases, disorders, and conditions associated with undesirable ADAM10 biological activities such as cancer, inflammation, an allergic response, lupus, asthma, an infectious disease, and fibrosis.
  • ADAM10 activity generates soluble proteins including, but not limited to, EGF ligands, EGF, HB-EGF, and betacellulin (Sabin et al., 2004), Notch, amyloid precursor protein, ephrins, cadherins, protocadherins, chemokines such as CXCL16 and CX3CL1, HER2, AXL, cMET, and CD23, a low affinity receptor for IgE (reviewed in Pruessmeyer & Ludwig, 2009).
  • Inhibition of TACE activity is correlated with beneficial effects in a tumor cell proliferation assay (Witters et al., 2008).
  • the mechanism for this inhibition of tumor cell proliferation is believed to be through prevention of EGF ligand release.
  • EGF ligands such as TGF-alpha, amphiregulin, HB-EGF, EGF and betacellulin, once released, are capable of activating the EGF receptor, which in turn leads to cancer proliferation (Pruessmeyer & Ludwig, 2009).
  • ADAM10 activity would be useful to study the biological functions of the protein, and for the treatment of disorders including but not limited to cancer, asthma, infectious diseases, and allergic responses.
  • ADAM family members are expressed as zymogens with the prodomains maintaining the enzymes in a latent state.
  • the prodomain of TACE suppresses the activity of its catalytic domain with a K i of 50 nM and inhibits TACE activity in vivo (Wong et al., 2016).
  • the wild type prodomain of TACE does not have good pharmacokinetic properties. Mutant prodomains that modified an upstream furin site and cysteine residue stabilized the TACE prodomain for in vivo use (Wong et al., 2016).
  • the wild type prodomain of ADAM10 does not have good pharmacokinetic properties, thereby making it difficult to be used as a drug.
  • the reason for the poor pharmacokinetics could be due to processing by a furin convertase at the upstream cleavage site (amino acids 48-51), and/or processing by Meprin metalloproteinase, which was shown to cleave the prodomain of ADAM10 in vitro between amino acids 109 and 110.
  • the sole cysteine at position 173 may interfere with the ability of the prodomain to have good pharmacokinetic properties as it can undergo oxidation to form a dimeric form of the prodomain.
  • cysteine 151 of SEQ ID NO: 1 or SEQ ID NO: 2 is substituted with alanine, serine, glycine, or threonine, or is pegylated.
  • the ADAM10 modulating peptide comprises, consists essentially of, or consists of an amino acid sequence as set forth in either of SEQ ID NOs: 3 and 4.
  • the ADAM10 modulating peptide comprises, consists essentially of, or consists of an amino acid sequence that comprises an alanine at one or more of positions 26, 28 and 29 of SEQ ID NO: 3 or SEQ ID NO: 4, an alanine at one or more of positions 52, 54 and 55 of SEQ ID NO: 3 or SEQ ID NO: 4, or any combination thereof; and an alanine at one or both of positions 88 and 89, an alanine at one or more of positions 176-178 of SEQ ID NO: 3, or any combination thereof.
  • cysteine 151 of SEQ ID NO: 3 or SEQ ID NO: 4 is substituted with alanine, serine, glycine, or threonine, or is pegylated.
  • the ADAM10 modulating peptide comprises, consists essentially of, or consists of an amino acid sequence that comprises an alanine at one or more of positions 26, 28 or 29 of SEQ ID NO: 3 or SEQ ID NO: 4, an alanine at one or more of positions 52, 54 or 55 of SEQ ID NO: 3 or SEQ ID NO: 4, or any combination thereof.
  • the amino acid at position 151 is selected from the group consisting of serine, glycine, alanine, and threonine.
  • the N-terminus, the C-terminus, the cysteine at position 151, or any combination thereof is pegylated.
  • the ADAM10 modulating peptide comprises, consists essentially of, or consists of one or more modifications that inactivate a furin recognition site at amino acid positions 26-28 of SEQ ID NOs: 1 or SEQ ID NO: 2, a furin recognition site at amino acid positions 52-54 of SEQ ID NOs: 1 or SEQ ID NO: 2, or both.
  • the ADAM10 modulating peptide comprises, consists essentially of, or consists of a plurality of modifications that inactivate the furin recognition sites at amino acid positions 26-28 and 52-54 of SEQ ID NOs: 1 or SEQ ID NO: 2; and the meprin recognition sites at amino acid positions 88 and 89 and 176-178 of SEQ ID NO: 1.
  • the ADAM10 modulating peptide further comprises one or more amino acid substitutions as defined in SEQ ID NO: 3 at one or more of the meprin recognition sites at amino acid positions 34-36, 62/63, 136-138, and 169/170 of SEQ ID NO: 1 or SEQ ID NO: 2 and optionally 176-178 of SEQ ID NO: 1.
  • at least one of the amino acid substitutions is an alanine substitution.
  • the ADAM10 modulating peptide further comprises a PEG moiety at the N-terminus, the C-terminus, amino acid position 151, or any combination thereof.
  • the ADAM10 modulating peptide further comprises a PEG moiety at the N-terminus, the C-terminus, amino acid position 151, or any combination thereof.
  • the ADAM10 modulating peptide comprises an amino acid sequence as set forth in any of SEQ ID NOs: 5-71,814.
  • each of amino acid positions 29 and 55 are substituted with conservative amino acid changes serine, glycine, threonine or serine; at least one of each of amino acid positions 34-36 are substituted are substituted with conservative amino acid changes asparagine, serine, glycine, threonine or serine; at least one of amino acid positions 62 and 63 are substituted with conservative amino acid changes asparagine, serine, glycine, threonine or serine; at least one of amino acid positions 88 and 89 are substituted with conservative amino acid changes asparagine, serine, glycine, threonine or serine; at least one of amino acid positions 136-138 are substituted are substituted with conservative amino acid changes asparagine, serine, glycine, threonine or serine;
  • ADAM10 modulating peptides comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in any one of SEQ ID NOs: 5, 6, 9, 34, 36, and 62-71.
  • the peptide comprises, consists essentially of, or consists of an amino acid sequence as set forth in SEQ ID NO: 62 or SEQ ID NO: 63.
  • ADAM10 modulating peptides comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3 of SEQ ID NO: 4.
  • the amino acid sequence comprises one or more conservative amino acid substitutions at one or more positions selected from the group consisting of positions 39, 48, 73, 107, 115, 126, 135, 171-175, 182, and 183 of SEQ ID NO: 3 or the corresponding positions in SEQ ID NO: 4.
  • the pegylated introduced cysteine is located within the first 20 amino acids of any one of SEQ ID NOs: 5, 6, 9, 34, 36, and 62-71; within the last 20 amino acids of any one of SEQ ID NOs: 5, 6, 9, 34, 36, and 62-71.
  • the amino acid sequence comprises at least one introduction of a cysteine into any one of SEQ ID NOs: 5-6,281 internally, at the N-terminus, at the C-terminus, or any combination thereof, and further wherein the at least one introduced cysteine results from a substitution of one or more amino acids of SEQ ID Nos: 5-6281, or any combination thereof.
  • the introduced cysteine is pegylated. In some embodiments, the pegylated introduced cysteine is located within the first 20 amino acids of any one of SEQ ID NOs: 5-6,281; within the last 20 amino acids of any one of SEQ ID NOs: 5-6281.
  • the amino acid sequence comprises at least one introduction of a cysteine into any one of SEQ ID NOs: 6,282-71,814 internally, at the N-terminus, at the C-terminus, or any combination thereof, and further wherein the at least one introduced cysteine results from a substitution of one or more amino acids of SEQ ID Nos: 6,282-71,814, or any combination thereof.
  • the introduced cysteine is pegylated. In some embodiments, the pegylated introduced cysteine is located within the first 20 amino acids of any one of SEQ ID NOs: 6,282-71,814; within the last 20 amino acids of any one of SEQ ID NOs: 5-6281.
  • the amino acid sequence comprises at least one introduction of a cysteine into any one of SEQ ID NOs: 3 and 4 internally, at the N-terminus, at the C-terminus, or any combination thereof, and further wherein the at least one introduced cysteine results from a substitution of one or more amino acids of SEQ ID Nos: 3 and 4, or any combination thereof.
  • the introduced cysteine is pegylated. In some embodiments, the pegylated introduced cysteine is located within the first 20 amino acids of any one of SEQ ID NOs: 3 and 4; within the last 20 amino acids of any one of SEQ ID NOs: 3 and 4.
  • the ADAM10 modulating peptide comprises one or more modifications selected from the group consisting of conservative amino acid substitutions, non-natural amino acid substitutions, D- or D,L-racemic mixture isomer form amino acid substitutions, amino acid chemical substitutions, carboxy- or amino-terminal modifications, addition of one or more glycosyl groups, and conjugation to a molecule selected from the group consisting of a fatty acid, a PEG, a sugar, a fluorescent molecule, a chromophore, a radionuclide, a bioconjugate, a tag that can be employed for purification and/or isolation of the ADAM10 modulating peptide, and an antibody or a paratope-containing fragment or derivative thereof.
  • ADAM10 modulating peptides comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of (i) an amino acid sequence that is at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3 or SEQ ID NO: 4; (ii) an amino acid sequence that is at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 2 that comprises at least one amino acid substitution at one or more of amino acid positions 26-29, 34-36, 52-55, 62/63, 88/89, 136-138, and 169/170 of SEQ ID NO: 1 or SEQ ID NO: 2 and/or amino acid positions 176-178 of SEQ ID NO: 1; (iii)
  • compositions comprising one or more ADAM10 modulating peptides as disclosed herein.
  • compositions comprising one or more ADAM10 modulating peptides as disclosed herein and a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutically composition is pharmaceutically acceptable for use in a human.
  • polypeptides comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in any of SEQ ID NOs: 5-71,814.
  • the polypeptide is pegylated.
  • polypeptides comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in any of SEQ ID NOs: 5-6,281.
  • polypeptide wherein the polypeptide is pegylated.
  • the polypeptide further comprises a tag, optionally a His tag or any other peptide or non-peptide tag that can be employed for purification and/or isolation of the polypeptide.
  • the tag is releasable from the polypeptide by proteolytic cleavage.
  • the polypeptide is adapted for in vitro use. In some embodiments, the polypeptide is adapted for in vivo use.
  • polypeptides comprising, consisting essentially of, or consisting of SEQ ID NO: 1 or SEQ ID NO: 2, wherein at least one meprin cleavage site and at least one furin cleavage site comprises an amino acid substitution as set forth in SEQ ID NO: 3 or SEQ ID NO: 4 that renders the polypeptide less susceptible to cleavage by meprin and by furin.
  • the polypeptide is pegylated.
  • cysteine 151 is conjugated to one or more moieties selected from the group consisting of a moiety that improves potency, solubility, or a pharmacokinetic property of the polypeptide relative to a wild type ADAM10 polypeptide; a chromophore; a fluorophore; and a radionucleotide; wherein the one or more moieties facilitates study of a pharmacokinetic and/or a pharmacodynamic property of the polypeptide.
  • moieties selected from the group consisting of a moiety that improves potency, solubility, or a pharmacokinetic property of the polypeptide relative to a wild type ADAM10 polypeptide; a chromophore; a fluorophore; and a radionucleotide; wherein the one or more moieties facilitates study of a pharmacokinetic and/or a pharmacodynamic property of the polypeptide.
  • polypeptides comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in one of SEQ ID NOs: 6282-71,814, wherein the polypeptides further comprise a spacer containing one or more charged residues selected from the group consisting of Asp, Glu, Arg, and Lys added at the N-terminus, the C-terminus, or both, and further wherein presence of the added one or more charged residues results in improved solubility of the polypeptide as compared to the polypeptide lacking the added one or more charged residues.
  • polypeptides comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in one of SEQ ID NOs: 5-6,281, wherein the polypeptides further comprise a spacer containing one or more charged residues selected from the group consisting of Asp, Glu, Arg, and Lys added at the N-terminus, the C-terminus, or both, and further wherein presence of the added one or more charged residues results in improved solubility of the polypeptide as compared to the polypeptide lacking the added one or more charged residues.
  • the presently disclosed subject matter also provides in some embodiments methods for modulating ADAM10 biological activities in vitro, ex vivo, or in vivo.
  • the methods comprising contacting a solution or a cell comprising an ADAM10 polypeptide with one or more ADAM10 modulating peptides as disclosed herein or a composition as disclosed herein, wherein the contacting comprises contacting the ADAM10 polypeptide with an amount of the ADAM10 modulating peptide sufficient to modulate a biological activity of the ADAM10 polypeptide.
  • the presently disclosed subject matter also provides in some embodiments methods for inhibiting ADAM10 biological activities in vivo.
  • the methods comprise administering to a subject a composition comprising, consisting essentially of, or consisting of one or more ADAM10 modulating peptides as disclosed herein via a route and in an amount sufficient to inhibit a biological activity of an ADAM10 polypeptide in the subject.
  • the ADAM10 modulating peptide is pegylated.
  • the presently disclosed subject matter also provides in some embodiments methods for inhibiting ADAM10 biological activities associated with a disease, disorder, or condition in a subject.
  • the methods comprise contacting an ADAM10 polypeptide present in a subject with an effective amount of one or more ADAM10 modulating peptides as disclosed herein, wherein the disease, disorder, or condition is selected from the group consisting of cancer, inflammation, an allergic response, lupus, asthma, an infectious disease, and fibrosis, and further wherein the subject has the disease, disorder, or condition or is predisposed thereto.
  • the disease, disorder, or condition results at least in part from excess cell proliferation associated with an ADAM10 biological activity.
  • the disease, disorder, or condition is characterized at least in part by presence of an excess of ADAM10 biological activity or protein.
  • the presently disclosed subject matter also provides in some embodiments methods for inhibiting release of an ADAM10 substrate from a cell.
  • the methods comprise contacting the cell with one or more ADAM10 modulating peptides as disclosed herein in an amount sufficient to inhibit release of the ADAM10 substrate from the cell.
  • the ADAM10 substrate is selected from the group consisting of CD23, IL6-R, EGF, Her-2, HB-EGF, betacellulin, jagged-1, Notch receptor 1, Notch receptor 3, RAGE, fractalkine, MICA A, I-TAC, HGFR, GITR, GM-CSF, an IGF soluble receptors, and TGF beta.
  • the cell is present in or has been isolated from a subject.
  • ADAM10 modulating peptides that comprise modifications at one or more furin sites and/or one or more meprin sites such that the modified ADAM10 peptides are more resistant to cleavage by furin and/or meprin as compared to an identical peptide lacking the modifications.
  • FIG. 1 is a image of a Coomassie-stained SDS-PAGE gel showing the results of furin cleavage of C-terminal His-tagged wild type (WT) and pegylated (Peg) human ADAM10 peptides corresponding to amino acids 23-211 of the wild type human ADAM10 prodomain (i.e., SEQ ID NO: 1) at 0, 13, 26, and 60 minutes.
  • the left-most lane shows the positions of various molecular weight markers.
  • the uncleaved peptide migrated at about 25 kiloDaltons (kDa) and its position can be seen between the top and the middle arrows on the right of the Figure.
  • the arrows on the right side of the Figure show the positions of the cleaved pegylated peptide (top arrow), the 19.7 kDa product produced by cleavage between amino acids 51 and 52 of the non-pegylated wild type human ADAM10 prodomain (i.e., between amino acids 29 and 30 of SEQ ID NO: 1; middle arrow), and 16.5 kDa product produced by cleavage between amino acids 77 and 78 of the non-pegylated wild type human ADAM10 prodomain (i.e., between amino acids 55 and 56 of SEQ ID NO: 1; lower arrow) after cleavage and recovery of the C-terminal cleavage products using an Ni-NTA resin to bind the His tag.
  • FIG. 2 is a image of a Coomassie-stained SDS-PAGE gel showing the results of furin cleavage of non-pegylated peptides of SEQ ID NO: 6 (also referred to herein as mutt) and SEQ ID NO: 1 (i.e., wild type).
  • Lane 1 shows a SEQ ID NO: 6 negative control (i.e., no furin).
  • Lane 2 shows SEQ ID NO: 6 plus furin.
  • Lane 3 shows a wild type negative control (i.e., no furin).
  • Lane 4 shows SEQ ID NO: 1 plus furin.
  • FIG. 3 is a image of a Coomassie-stained SDS-PAGE gel showing the results of meprin cleavage of SEQ ID NO: 1.
  • Lane 1 is SEQ ID NO: 1 cleaved with meprin.
  • Lane 2 is a negative control that includes activation buffer with furin used to activate the meprin.
  • Lane 3 is the activation buffer with meprin.
  • Lane 4 is blank.
  • Lane 5 shows the positions of various molecular weight markers. There are multiple meprin recognition sites in SEQ ID NO: 1, resulting in a cleavage pattern as set forth in Lane 1.
  • the cleaved fragments corresponded to uncleaved SEQ ID NO: 1 (uncleaved), and fragments that included amino acids 1-177 of SEQ ID NO: 1 (1-177), amino acids 1-169 of SEQ ID NO: 1 (1-169), amino acids 34/35-189 of SEQ ID NO: 1 (-34-189), amino acids 1-136 of SEQ ID NO: 1 (1-136), amino acids 88-189 of SEQ ID NO: 1 (88-189), and several smaller fragments that presumably derived from cleavage at multiple cleavage sites within SEQ ID NO: 1 (small cleavage products).
  • FIG. 4 is a image of a Coomassie-stained SDS-PAGE gel showing the results of meprin cleavage of SEQ ID NO: 1 or a peptide of SEQ ID NO: 9.
  • Lane 1 is SEQ ID NO: 1 without meprin
  • Lane 2 is SEQ ID NO: 1 plus meprin
  • Lane 3 is SEQ ID NO: 7 without meprin.
  • Lane 4 is SEQ ID NO: 7 plus meprin.
  • FIG. 5A is a bar graph of percent inhibition of MDA-MB-468 cell proliferation in the presence of SEQ ID NO: 1 (Wt), SEQ ID NO: 1 pegylated at cysteine 151 (WT PEG), SEQ ID NO: 5 (MUT1), SEQ ID NO: 6 (MUT2), or pegylated SEQ ID NO: 5 (MUT1 PEG).
  • Error bars are ⁇ standard error of the mean (SEM) calculated from samples run in quadruplicate.
  • FIG. 5B is a bar graph of the rate of cell growth of MDA-MB-468 in the presence of vehicle (20 mM Tris, 40 mM NaCl, 10% glycerol) or SEQ ID NO: 62. P ⁇ 0.0001. Error bars are ⁇ standard error of the mean (SEM) calculated from samples run in quadruplicate.
  • FIGS. 6A-6D are bar graphs presenting the results of experiments testing the pharmacokinetic properties of various modified ADAM10 peptides as measured by the abilities of sera isolated from mice that had received a single intraperitoneal dose of those various modified ADAM10 peptides to inhibit ADAM10 activity.
  • FIG. 6A is a bar graph showing serum levels (in ⁇ M) of modified ADAM10 peptides.
  • FIG. 6B is a bar graph showing serum levels (in nM) of modified ADAM10 peptides.
  • FIG. 6C is a bar graph showing serum concentrations (in nM) of modified ADAM10 peptides.
  • FIG. 6D is a bar graph showing serum concentrations (in nM) of modified ADAM10 peptides.
  • FIGS. 8A-8C are images of Coomassie-stained SDS-PAGE gels showing the results of testing the noted SEQ ID NOs. for resistance to cleavage by furin.
  • FIG. 8A shows the result of testing SEQ ID NOs: 62, 63, 34, and a pegylated version of SEQ ID NO: 71 at 3, 10, 20, and 120 minutes after adding furin.
  • FIG. 8B shows the result of testing SEQ ID NOs: 62, 65, and 68 at 0, 20, 40, and 120 minutes after adding furin.
  • FIG. 8C shows the result of testing SEQ ID NOs: 63 and pegylated versions of SEQ ID NOs: 69, 70, and 71 at 0, 30, and 120 minutes after adding furin.
  • FIGS. 9A-9C are images of Coomassie-stained SDS-PAGE gels showing the results of testing the noted SEQ ID NOs. for resistance to cleavage by meprin.
  • FIG. 9A shows the result of testing SEQ ID NOs: 5, 62, 63, and 34 at 0 and 10 minutes after adding meprin.
  • FIG. 9B shows the result of testing SEQ ID NOs: 34, 66, 67, and 68 at 0 and 17 minutes after adding meprin.
  • FIG. 9C shows the result of testing SEQ ID NOs: 63 and pegylated versions of SEQ ID NOs: 69 (69P), 70 (70P), and 71 (71P) at 2 and 10 minutes after adding meprin.
  • FIG. 10 is a bar graph showing the results of a Hollow Fiber Model test of SEQ ID NO: 62 versus a negative vehicle control. *: P ⁇ 0.05. Error bars are ⁇ standard error of the mean (SEM) calculated from samples run in sextuplicate.
  • the Sequence Listing associated with the instant disclosure has been submitted as a 123.3 MB file on a compact disc.
  • the compact disc is marked in indelible ink to identify the Applicants, Title, File Name (FINAL_3217-2-2_ST25.txt), Creation Date (Oct. 2, 2018), Computer System (IBM-PC/MS-DOS/MS-Windows), Docket No. (3217/2/2), and the Serial No. of the corresponding U.S. utility application.
  • the Sequence Listing submitted on compact disc is hereby incorporated by reference into the instant disclosure.
  • SEQ ID NO: 1 is the amino acid sequence of a wild type human ADAM10 prodomain peptide. It corresponds to amino acids 23-211 of Accession No. NP_001101.1 of the GENBANK® biosequence database
  • SEQ ID NO: 2 is the amino acid sequence of a wild type murine ADAM10 prodomain peptoide. It corresponds to amino acids 23-212 of Accession No. NP_031425.2 of the GENBANK® biosequence database.
  • SEQ ID NO: 3 is a consensus amino acid sequence for the ADAM10 modulating peptides of the presently disclosed subject matter that are based on SEQ ID NO: 1. As compared to SEQ ID NO: 1, SEQ ID NO: 3 includes 24 amino acid positions that can be substituted individually or in any combination or subcombination. The 24 amino acid positions correspond to two (2) furin recognition sites, six (6) meprin recognition sites, and the single cysteine present in SEQ ID NO: 1 at position 151.
  • SEQ ID NO: 4 is a consensus amino acid sequence for the ADAM10 modulating peptides of the presently disclosed subject matter that are based on SEQ ID NO: 2. As compared to SEQ ID NO: 2, SEQ ID NO: 4 includes 21 amino acid positions that can be substituted individually or in any combination or subcombination. The 21 amino acid positions correspond to two (2) furin recognition sites, five (5) meprin recognition sites, and the single cysteine present in SEQ ID NO: 2 at position 151.
  • SEQ ID NOs: 5-71,814 are the amino acid sequences of exemplary ADAM10 modulating peptides of the presently disclosed subject matter that correspond to embodiments of SEQ ID NO: 3 or SEQ ID NO: 4.
  • the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims.
  • the phrase “an antibody” refers to one or more antibodies, including a plurality of the same antibody.
  • the phrase “at least one”, when employed herein to refer to an entity refers to, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, or more of that entity, including but not limited to whole number values between 1 and 100 and greater than 100.
  • amino acid refers to a-amino acids that can be employed in producing the ADAM10 modulating peptides of the presently disclosed subject matter.
  • standard amino acids There are twenty “standard” amino acids that naturally occur in polypeptides, and these are summarized in Table 1.
  • the phrase “A, B, C, and/or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.
  • the phrase “consisting of” excludes any element, step, or ingredient not specifically recited. It is noted that, when the phrase “consists of” appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.
  • a pharmaceutical composition can “consist essentially of” a pharmaceutically active agent or a plurality of pharmaceutically active agents, which means that the recited pharmaceutically active agent(s) is/are the only pharmaceutically active agent(s) present in the pharmaceutical composition. It is noted, however, that carriers, excipients, and other inactive agents can and likely would be present in the pharmaceutical composition.
  • subject refers to a member of any invertebrate or vertebrate species. Accordingly, the term “subject” is intended to encompass any member of the Kingdom Animalia including, but not limited to the phylum Chordata (e.g., members of Classes Osteichythyes (bony fish), Amphibia (amphibians), Reptilia (reptiles), Ayes (birds), and Mammalia (mammals)), and all Orders and Families encompassed therein.
  • phylum Chordata e.g., members of Classes Osteichythyes (bony fish), Amphibia (amphibians), Reptilia (reptiles), Ayes (birds), and Mammalia (mammals)
  • compositions and methods of the presently disclosed subject matter are particularly useful for warm-blooded vertebrates.
  • the presently disclosed subject matter concerns mammals and birds. More particularly provided are compositions and methods derived from and/or for use in mammals such as humans and other primates, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economic importance (animals raised on farms for consumption by humans) and/or social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), rodents (such as mice, rats, and rabbits), marsupials, and horses.
  • carnivores other than humans such as cats and dogs
  • swine pigs, hogs, and wild boars
  • domesticated fowl e.g., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans.
  • livestock including but not limited to domesticated swine (pigs and hogs), ruminants, horses, poultry, and the like.
  • genes, gene names, and gene products disclosed herein are intended to correspond to orthologs from any species for which the compositions and methods disclosed herein are applicable.
  • the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates.
  • the human amino acid sequences disclosed are intended to encompass homologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds. Also encompassed are any and all nucleotide sequences that encode the disclosed amino acid sequences, including but not limited to those disclosed in the corresponding GENBANK® biosequence database entries.
  • cancer and “tumor” are used interchangeably herein and can refer to both primary and metastasized solid tumors and carcinomas of any tissue in a subject, including but not limited to breast; colon; rectum; lung; oropharynx; hypopharynx; esophagus; stomach; pancreas; liver; gallbladder; bile ducts; small intestine; urinary tract including kidney, bladder, and urothelium; female genital tract including cervix, uterus, ovaries (e.g., choriocarcinoma and gestational trophoblastic disease); male genital tract including prostate, seminal vesicles, testes and germ cell tumors; endocrine glands including thyroid, adrenal, and pituitary; skin (e.g., hemangiomas and melanomas), bone or soft tissues; blood vessels (e.g., Kaposi's sarcoma); brain, nerves, eyes, and meninge
  • a cancer or a tumor comprises a cancer or tumor of an epithelial tissue such as, but not limited to a carcinoma.
  • a tumor is an adenocarcinoma, which in some embodiments is an adenocarcinoma of the pancreas, breast, ovary, colon, or rectum, and/or a metastatic cell derived therefrom.
  • ADAM10 Modulating Peptides and Conjugates Thereof, Compositions, Pharmaceutical Compositions, and Polypeptides
  • the first (i.e., N-terminal) furin recognition sequence corresponds to amino acids 26-29 of SEQ ID NO: 1
  • the second (i.e., C-terminal) furin recognition sequence corresponds to amino acids 52-55 of SEQ ID NO: 1. It has been determined that modifications the amino acid sequence of SEQ ID NO: 1 at one or more of amino acids 26-29 and/or amino acids 52-55 can reduce the ability of furin to cleave the ADAM10 modulating peptides of the presently disclosed subject matter.
  • the presently disclosed subject matter provides ADAM10 modulating peptides, wherein the ADAM10 modulating peptide comprises, consists essentially of, or consists of an amino acid sequence comprising at least one amino acid substitution of at least one furin recognition site such as but not limited to amino acids 26-29 and/or amino acids 52-55 of SEQ ID NO: 1, wherein the at least one amino acid substitution of at least one furin recognition site renders the ADAM10 modulating peptide less sensitive to cleavage by furin.
  • Exemplary, non-limiting amino acid modifications to the furin recognition sites are presented in Table 2:
  • amino acid positions 26-29 and 52-54 one, two, three, four, five, six, seven, or all eight amino acid positions can be modified in order to enhance the stability of the ADAM10 modulating peptides of the presently disclosed subject matter, and that in some embodiments at each of these positions any of the amino acid substitutions listed in Table 2 can be employed.
  • the presently disclosed subject matter provides ADAM10 modulating peptides wherein the ADAM10 modulating peptides comprise, consist essentially of, or consist of an amino acid sequence comprising at least one amino acid substitution of at least one meprin recognition site of SEQ ID NO: 1 or SEQ ID NO: 2, wherein the at least one amino acid substitution of at least one meprin recognition site renders the ADAM10 modulating peptide less sensitive to cleavage by meprin.
  • Exemplary, non-limiting amino acid modifications to the meprin recognition sites are presented in Table 3.
  • an ADAM10 modulating peptide of the presently disclosed subject matter comprises, consists essentially of, or consists of an amino acid sequence comprising at least one amino acid substitution of at least one furin recognition site and optionally comprises at least one amino acid substitution of at least one meprin recognition site of SEQ ID NO: 1 or SEQ ID NO: 2, and combinations thereof, such that the ADAM10 modulating peptide is less sensitive to cleavage by furin and optionally by both furin and meprin.
  • non-amino acid substitution modifications can also be included in the ADAM10 modulating peptides of the presently disclosed subject matter.
  • the ADAM10 modulating peptides of the presently disclosed subject matter can be conjugated to a polyethylene glycol (PEG) moiety (i.e., they can be pegylated).
  • PEG polyethylene glycol
  • the ADAM10 modulating peptides of the presently disclosed subject matter can be pegylated at or near the N-terminus and/or at or near the C-terminus.
  • Cys151 can also be modified by amino acid substitution.
  • Exemplary substitutions at Cys151 include serine, glycine, alanine, and threonine.
  • an ADAM10 modulating peptide of the presently disclosed subject matter can in some embodiments include modifications at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or all 23 amino acid positions listed in Tables 2-3 in addition to position 151.
  • an ADAM10 modulating peptide of the presently disclosed subject matter comprises 1-24 amino acid substitutions at positions 26-29, 34-36, 52-55, 62, 63, 88, 89, 136-138, 151, 169, 170, and 176-178 of SEQ ID NO: 1.
  • SEQ ID NOs: 3 and 4 provide consensus sequences based on the human and mouse ADAM10 amino acid sequences, respectively, with these amino acid positions defined as in Tables 2-3 in addition to position 151.
  • amino acid substitutions at these positions are selected from those in Tables 2-3 in addition to serine, glycine, alanine, or threonine at position 151.
  • amino acid substitutions are selected from those in Tables 2-3 in addition to serine, glycine, alanine, or threonine at position 151.
  • SEQ ID NOs: 5-71,814. Additional non-limiting examples of an ADAM10 modulating peptides of the presently disclosed subject matter are as follows:
  • the presently disclosed subject matter provides ADAM10 modulating peptides.
  • the ADAM10 modulating peptides comprise, consist essentially of, or consist of an amino acid sequence comprising at least one amino acid substitution of at least one furin recognition site of SEQ ID NO: 1 or SEQ ID NO: 2 and optionally further comprises at least one amino acid substitution of at least one meprin recognition site of SEQ ID NO: 1 or SEQ ID NO: 2, and combinations thereof, such that the ADAM10 modulating peptide is less sensitive to cleavage by furin and optionally by both furin and meprin.
  • cysteine 151 of SEQ ID NO: 1 or SEQ ID NO: 2 is substituted with alanine, serine, glycine, or threonine, or is pegylated.
  • the ADAM10 modulating peptide comprises, consists essentially of, or consists of an amino acid sequence as set forth in either of SEQ ID NOs: 3 and 4.
  • cysteine 151 of SEQ ID NO: 3 or SEQ ID NO: 4 is substituted with alanine, serine, glycine, or threonine, or is pegylated.
  • the ADAM10 modulating peptide comprises, consists essentially of, or consists of an amino acid sequence that comprises an alanine at one or more of positions 26, 28 or 29 of SEQ ID NO: 3 or SEQ ID NO: 4, an alanine at one or more of positions 52, 54 or 55 of SEQ ID NO: 3 or SEQ ID NO: 4, or any combination thereof.
  • one or both of amino acid positions 29 and 55 are independently selected from the group consisting of alanine and lysine.
  • the ADAM10 modulating peptide further comprises an alanine at one or more of positions 88, 89, 177, and 178 of SEQ ID NO: 3 and/or at positions 88 and 89 of SEQ ID NO: 4, or any combination thereof.
  • ADAM10 modulating peptide further comprises a modification at the cysteine at amino acid 151 selected from the group consisting of substitution of the cysteine with serine, glycine, alanine, or threonine and pegylation of the cysteine.
  • amino acid 151 is serine or cysteine151 is peglyated.
  • the ADAM10 modulating peptide further comprises a modification at the cysteine at amino acid 151 selected from the group consisting of substitution of the cysteine with serine, glycine, alanine, or threonine and pegylation of the cysteine.
  • amino acid 151 is serine and in some embodiments amino acid 151 is pegylated.
  • amino acid positions 29 and 55 are independently selected from the group consisting of serine, glycine, and threonine.
  • amino acid position 151 of SEQ ID NO: 3 or SEQ ID NO: 4 is a serine, glycine, alanine, or threonine.
  • amino acid position 151 of SEQ ID NO: 3 or SEQ ID NO: 4 is pegylated.
  • the N-terminus, the C-terminus, or both are pegylated.
  • amino acid position 26 is not arginine, amino acid position 27 is not alanine, amino acid position 28 is not lysine, and/or amino acid position 29 is not arginine; and/or amino acid position 52 is not arginine, amino acid position 53 is not methionine, amino acid position 54 is not lysine, and/or amino acid position 55 is not arginine; and/or amino acid position 34 is not glutamic acid, amino acid position 35 is not aspartic acid, and/or amino acid position 36 is not glutamine; amino acid position 62 is not aspartic acid and/or amino acid position 63 is not glutamic acid; and/or amino acid position 88 is selected from the group consisting of alanine, serine, glycine, threonine, and asparagine and/or amino acid position 89 is selected from the group consisting of alanine, serine, glycine, threonine
  • the amino acid at position 151 is selected from the group consisting of serine, glycine, alanine, and threonine.
  • the N-terminus, the C-terminus, the cysteine at position 151, or any combination thereof is pegylated.
  • the ADAM10 modulating peptide comprises, consists essentially of, or consists of one or more modifications that inactivate a furin recognition site at amino acid positions 26-28 of SEQ ID NOs: 1 or SEQ ID NO: 2, a furin recognition site at amino acid positions 52-54 of SEQ ID NOs: 1 or SEQ ID NO: 2, or both.
  • the ADAM10 modulating peptide comprises, consists essentially of, or consists of one or more modifications that inactivate a meprin recognition site at amino acid positions amino acid positions 34-36 of SEQ ID NO: 1 or SEQ ID NO: 2, amino acid positions 62 and 63 of SEQ ID NO: 1 or SEQ ID NO: 2, amino acid positions 88 and 89 of SEQ ID NO: 1 or SEQ ID NO: 2, amino acid positions 136-138 of SEQ ID NO: 1 or SEQ ID NO: 2, amino acid positions 169 and 170 of SEQ ID NO: 1 or SEQ ID NO: 2, amino acid positions 176-178 of SEQ ID NO: 1, or any combination thereof.
  • the ADAM10 modulating peptide comprises, consists essentially of, or consists of at least two modifications that inactivate the meprin recognition site at amino acid positions 88 and 89 of SEQ ID NO: 1 and amino acid positions 176-178 of SEQ ID NO: 1.
  • the ADAM10 modulating peptide comprises, consists essentially of, or consists of a plurality of modifications that inactivate the furin recognition sites at amino acid positions 26-28 and 52-54 of SEQ ID NOs: 1 or SEQ ID NO: 2; and the meprin recognition sites at amino acid positions 34-36, 62 and 63, 88 and 89, 136-138, and 169 and 170 of SEQ ID NO: 1 or SEQ ID NO: 2; and optionally further inactivate the meprin recognition site at amino acid positions 176-178 of SEQ ID NO: 1.
  • the ADAM10 modulating peptide comprises, consists essentially of, or consists of a plurality of modifications that inactivate the furin recognition sites at amino acid positions 26-28 and 52-54 of SEQ ID NOs: 1 or SEQ ID NO: 2; and the meprin recognition sites at amino acid positions 88 and 89 and 176-178 of SEQ ID NO: 1.
  • the ADAM10 modulating peptide comprises, consists essentially of, or consists of an amino acid sequence that with reference to SEQ ID NO: 1, comprises amino acid substitutions as defined in SEQ ID NO: 3 at one or more of amino acid positions 26-29; and one or more of amino acid positions 52-55; and amino acid positions 88/89; and amino acid positions 176-178.
  • amino acid position 151 is selected from the group consisting of serine, glycine, alanine, and threonine or the cysteine at amino acid position 151 is pegylated.
  • the ADAM10 modulating peptide further comprises a PEG moiety at the N-terminus, the C-terminus, amino acid position 151, or any combination thereof. In some embodiments, the ADAM10 modulating peptide further comprises a PEG moiety at the N-terminus, the C-terminus, amino acid position 151, or any combination thereof.
  • the ADAM10 modulating peptide further comprises one or more amino acid substitutions as defined in SEQ ID NO: 3 at one or more of the meprin recognition sites at amino acid positions 34-36, 62/63, 136-138, and 169/170 of SEQ ID NO: 1 or SEQ ID NO: 2 and optionally 176-178 of SEQ ID NO: 1.
  • at least one of the amino acid substitutions is an alanine substitution.
  • the ADAM10 modulating peptide further comprises a PEG moiety at the N-terminus, the C-terminus, amino acid position 151, or any combination thereof.
  • the ADAM10 modulating peptide further comprises a PEG moiety at the N-terminus, the C-terminus, amino acid position 151, or any combination thereof.
  • the ADAM10 modulating peptide comprises an amino acid sequence as set forth in any of SEQ ID NOs: 5-71,814.
  • each of amino acid positions 29 and 55 are substituted with conservative amino acid changes serine, glycine, threonine or serine; at least one of each of amino acid positions 34-36 are substituted are substituted with conservative amino acid changes asparagine, serine, glycine, threonine or serine; at least one of amino acid positions 62 and 63 are substituted with conservative amino acid changes asparagine, serine, glycine, threonine or serine; at least one of amino acid positions 88 and 89 are substituted with conservative amino acid changes asparagine, serine, glycine, threonine or serine; at least one of amino acid positions 136-138 are substituted are substituted with conservative amino acid changes asparagine, serine, glycine, threonine or serine;
  • the ADAM10 modulating peptide comprises an amino acid sequence at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 5-71,814.
  • the amino acid sequence comprises one or more amino acid substitutions of SEQ ID NOs: 5-6,281 at an amino acid position selected from the group consisting of amino acid positions 26, 28, 29, 34-36, 52, 54,55, 62, 63, 88, 89, 136-138, 151, 169-178, 182, and 183 as defined in SEQ ID NO: 3 for these amino acid positions.
  • percent identity and “percent identical,” in the context of two protein sequences refer to two or more sequences or subsequences that have in some embodiments at least 60%, in some embodiments at least 70%, in some embodiments at least 80%, in some embodiments at least 85%, in some embodiments at least 87%, in some embodiments at least 88%, in some embodiments at least 89%, in some embodiments at least 90%, in some embodiments at least 91%, in some embodiments at least 92%, in some embodiments at least 93%, in some embodiments at least 94%, in some embodiments at least 95%, in some embodiments at least 96%, in some embodiments at least 97%, in some embodiments at least 98%, and in some embodiments at least 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • the percent identity exists in some embodiments over a region of the sequences that is at least about 50 residues in length, in some embodiments over a region of at least about 100 residues, and in some embodiments the percent identity exists over at least about 150 residues. In some embodiments, the percent identity exists over the entire length of one of sequences being compared. In some embodiments, the percent identity is calculated over the entire length of the amino acid sequence of the ADAM10 modulating peptide.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm described in Smith & Waterman, 1981, by the homology alignment algorithm described in Needleman & Wunsch, 1970, by the search for similarity method described in Pearson & Lipman, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG® WISCONSIN PACKAGE®, available from Accelrys, Inc., San Diego, Calif., United States of America), or by visual inspection. See generally, Ausubel et al., 1989.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always >0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative scoring residue alignments, or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • E expectation
  • BLOSUM62 a wordlength of 3
  • W wordlength of 3
  • E expectation
  • BLOSUM62 scoring matrix See Henikoff & Henikoff, 1992.
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences. See e.g., Karlin & Altschul. 1993.
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is in some embodiments less than about 0.1, in some embodiments less than about 0.01, and in some embodiments less than about 0.001.
  • amino acid position 151 is selected from the group consisting of serine, glycine, alanine, and threonine or the cysteine at amino acid position 151 is pegylated.
  • at least one of amino acids 26, 28 and 29 and/or at least one of amino acids 52, 54 and 55 are substituted are substituted with conservative amino acid changes serine, glycine, threonine or serine; and at least one amino acids of position 88/89 and/or 176-178 are substituted with conservative amino acid changes asparagine, serine, glycine, threonine or serine.
  • the ADAM10 modulating peptide further comprises a PEG moiety at the N-terminus, the C-terminus, amino acid position 151 of SEQ ID NO: 1, or any combination thereof.
  • ADAM10 modulating peptides comprising, consisting essentially of, or consisting of SEQ ID NO: 1 or SEQ ID NO: 2 conjugated to a PEG moiety.
  • the PEG moiety is conjugated to the cysteine at amino acid position 151 of SEQ ID NO: 1 or SEQ ID NO: 2.
  • ADAM10 modulating peptides comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in any one of SEQ ID NOs: 5, 6, 9, 34, 36, and 62-71.
  • the peptide comprises, consists essentially of, or consists of an amino acid sequence as set forth in SEQ ID NO: 62 or SEQ ID NO: 63.
  • ADAM10 modulating peptides comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 5, 6, 9, 34, 36, and 62-71.
  • the amino acid sequence is at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 62 or SEQ ID NO: 63.
  • ADAM10 modulating peptides comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3 of SEQ ID NO: 4.
  • the amino acid sequence comprises one or more conservative amino acid substitutions at one or more positions selected from the group consisting of positions 39, 48, 73, 107, 115, 126, 135, 171-175, 182, and 183 of SEQ ID NO: 3 or the corresponding positions in SEQ ID NO: 4.
  • the amino acid sequence comprises a leucine substitution at amino acid position 39, an alanine substitution at amino acid position 48, a proline substitution at amino acid position 73, a lysine substitution at amino acid position 107, an isoleucine substitution at amino acid position 115, an isoleucine substitution at amino acid position 126, or any combination thereof.
  • the amino acid sequence is selected from the group consisting of SEQ ID NOs: 64-66.
  • the amino acid sequence is at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 5, 6, 9, 34, 36, and 62-71.
  • the amino acid sequence comprises at least one introduction of a cysteine into any one of SEQ ID NOs: 5, 6, 9, 34, 36, and 62-71 internally, at the N-terminus, at the C-terminus, or any combination thereof, and further wherein the at least one introduced cysteine results from a substitution of one or more amino acids of SEQ ID NOs: 5, 6, 9, 34, 36, and 62-71, an insertion of a cysteine into any one of SEQ ID NOs: 5, 6, 9, 34, 36, and 62-71, or any combination thereof.
  • the introduced cysteine is pegylated.
  • the pegylated introduced cysteine is located within the first 20 amino acids of any one of SEQ ID NOs: 5, 6, 9, 34, 36, and 62-71; within the last 20 amino acids of any one of SEQ ID NOs: 5, 6, 9, 34, 36, and 62-71.
  • ADAM10 modulating peptides that have 87-100% identity (e.g., at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to one of SEQ ID NOs: 1-71,814, in some embodiments an amino acid substitution occurs at a position other than one of the furin recognition sites, one of the meprin recognition sites, or Cys151 of SEQ ID NO: 1 or SEQ ID NO: 2. As such, in some embodiments an ADAM10 modulating peptide of the presently disclosed subject matter includes an amino acid substitution, optionally a conservative amino acid substitution, at another amino acid position of SEQ ID NOs: 1-4.
  • conservative amino acid substitution and “conservative amino acid change” are used interchangeably to refer to amino acid substitutions that are contemplated to impact a biological activity of the ADAM10 modulating peptide as little as possible.
  • Table 4 lists exemplary conservative amino acid substitutions.
  • Amino acid substitutions such as those that might be employed in modifying a furin and/or a meprin recognition site of an ADAM10 modulating peptide of the presently claimed subject matter are generally, but not necessarily, based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • An analysis of the size, shape, and type of the amino acid side-chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; that alanine, glycine, and serine are all of similar size; and that phenylalanine, tryptophan, and tyrosine all have a generally similar shape.
  • arginine, lysine, and histidine are defined herein as biologically functional equivalents.
  • Other biologically functionally equivalent changes will be appreciated by those of ordinary skill in the art. It is implicit in the above discussion, however, that one of skill in the art can appreciate that a radical, rather than a conservative substitution can be warranted in a given situation.
  • Non-conservative substitutions at any position of any of SEQ ID NOs: 5-71,814 of the presently claimed subject matter are also an aspect of the presently claimed subject matter.
  • the hydropathic index of amino acids can be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics. The assigned hydropathic indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine ( ⁇ 0.4); threonine ( ⁇ 0.7); serine ( ⁇ 0.8); tryptophan ( ⁇ 0.9); tyrosine ( ⁇ 1.3); proline ( ⁇ 1.6); histidine ( ⁇ 3.2); glutamic acid ( ⁇ 3.5); glutamine ( ⁇ 3.5); aspartic acid ( ⁇ 3.5); asparagine ( ⁇ 3.5); lysine ( ⁇ 3.9); and arginine ( ⁇ 4.5).
  • hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, 1982). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices can be in one embodiment within ⁇ 2 of the original value, in another embodiment within ⁇ 1 of the original value, and in still another embodiment within ⁇ 0.5 of the original value.
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ⁇ 1); glutamic acid (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine ( ⁇ 0.4); proline ( ⁇ 0.5 ⁇ 1); alanine ( ⁇ 0.5); histidine ( ⁇ 0.5); cysteine ( ⁇ 1.0); methionine ( ⁇ 1.3); valine ( ⁇ 1.5); leucine ( ⁇ 1.8); isoleucine ( ⁇ 1.8); tyrosine ( ⁇ 2.3); phenylalanine ( ⁇ 2.5); and tryptophan ( ⁇ 3.4).
  • the substitution of amino acids whose hydrophilicity values can be in one embodiment within ⁇ 2 of the original value, in another embodiment within ⁇ 1 of the original value, and in still another embodiment within ⁇ 0.5 of the original value.
  • the amino acid sequence comprises at least one introduction of a cysteine into any one of SEQ ID NOs: 5-6,281 internally, at the N-terminus, at the C-terminus, or any combination thereof, and further wherein the at least one introduced cysteine results from a substitution of one or more amino acids of SEQ ID Nos: 5-6281, or any combination thereof.
  • the introduced cysteine is pegylated. In some embodiments, the pegylated introduced cysteine is located within the first 20 amino acids of any one of SEQ ID NOs: 5-6,281; within the last 20 amino acids of any one of SEQ ID NOs: 5-6281.
  • the amino acid sequence comprises at least one introduction of a cysteine into any one of SEQ ID NOs: 6,282-71,814 internally, at the N-terminus, at the C-terminus, or any combination thereof, and further wherein the at least one introduced cysteine results from a substitution of one or more amino acids of SEQ ID Nos: 6,282-71,814, or any combination thereof.
  • the introduced cysteine is pegylated. In some embodiments, the pegylated introduced cysteine is located within the first 20 amino acids of any one of SEQ ID NOs: 6,282-71,814; within the last 20 amino acids of any one of SEQ ID NOs: 5-6281.
  • the amino acid sequence comprises at least one introduction of a cysteine into any one of SEQ ID NOs: 3 and 4 internally, at the N-terminus, at the C-terminus, or any combination thereof, and further wherein the at least one introduced cysteine results from a substitution of one or more amino acids of SEQ ID Nos: 3 and 4, or any combination thereof.
  • the introduced cysteine is pegylated. In some embodiments, the pegylated introduced cysteine is located within the first 20 amino acids of any one of SEQ ID NOs: 3 and 4; within the last 20 amino acids of any one of SEQ ID NOs: 3 and 4.
  • compositions optionally pharmaceutical compositions, comprising one or more ADAM10 modulating peptides as disclosed herein.
  • the ADAM10 modulating peptide comprises one or more modifications selected from the group consisting of conservative amino acid substitutions, non-natural amino acid substitutions, D- or D,L-racemic mixture isomer form amino acid substitutions, amino acid chemical substitutions, carboxy- or amino-terminal modifications, addition of one or more glycosyl groups, and conjugation to a molecule selected from the group consisting of a fatty acid, a PEG, a sugar, a fluorescent molecule, a chromophore, a radionuclide, a bioconjugate, a tag that can be employed for purification and/or isolation of the ADAM10 modulating peptide, and an antibody or a paratope-containing fragment or derivative thereof.
  • the ADAM10 modulating peptides of the presently disclosed subject matter are present singly or in combination with each other in a composition or a pharmaceutical composition.
  • a pharmaceutical composition is a composition that comprises one or more ADAM10 modulating peptides as set forth herein and a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutically composition is pharmaceutically acceptable for use in a human.
  • Suitable formulations include aqueous and non-aqueous sterile injection solutions which can contain anti-oxidants, buffers, bacteriostats, bactericidal antibiotics, and solutes which render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents.
  • the formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a frozen or freeze-dried (lyophilized) condition requiring only the addition of sterile liquid carrier, for example water for injections, immediately prior to use.
  • Some exemplary ingredients are sodium dodecyl sulfate (SDS) in the range of in some embodiments 0.1 to 10 mg/ml, in some embodiments about 2.0 mg/ml; and/or mannitol or another sugar in the range of in some embodiments 10 to 100 mg/ml, in some embodiments about 30 mg/ml; and/or phosphate-buffered saline (PBS). Any other agents conventional in the art having regard to the type of formulation in question can be used.
  • SDS sodium dodecyl sulfate
  • PBS phosphate-buffered saline
  • the presently disclosed subject matter also provides methods for using the presently disclosed ADAM10 modulting peptides.
  • the presently disclosed subject matter relates to methods for modulating ADAM10 biological activities in vitro.
  • the methods comprise contacting a solution or a cell comprising an ADAM10 polypeptide with one or more ADAM10 modulating peptides as disclosed herein, wherein the contacting comprises contacting the ADAM10 polypeptide with an amount of the ADAM10 modulating peptide sufficient to modulate a biological activity of the ADAM10 polypeptide.
  • the presently disclosed subject matter relates to methods for inhibiting ADAM10 biological activities in vivo.
  • the methods comprise administering to a subject a composition comprising, consisting essentially of, or consisting of one or more ADAM10 modulating peptides of the presently disclosed subject matter via a route and in an amount sufficient to inhibit a biological activity of an ADAM10 polypeptide in the subject.
  • the presently disclosed subject matter relates to methods for inhibiting ADAM10 biological activities associated with a disease, disorder, or condition in a subject.
  • the methods comprise contacting an ADAM10 polypeptide present in the subject with an effective amount of one or more of the ADAM10 modulating peptides disclosed herein, wherein the disease or disorder is selected from the group consisting of cancer, inflammation, an allergic response, lupus, asthma, an infectious disease, and fibrosis, and further wherein the subject has the disease, disorder, or condition or is predisposed thereto.
  • the disease, disorder, or condition results at least in part from excess cell proliferation associated with an ADAM10 biological activity.
  • the disease, disorder, or condition is characterized at least in part by presence of an excess of ADAM10 biological activity or protein.
  • the presently disclosed subject matter relates to methods for inhibiting release of ADAM10 substrates from cells.
  • the methods comprise contacting the cell with one or more ADAM10 modulating peptides as disclosed herein in an amount sufficient to inhibit release of the ADAM10 substrate from the cell.
  • ADAM10 substrate refers to any substrate upon which an ADAM10 polypeptide can act and that results in release of the substrate from a cell.
  • ADAM10 substrates include CD23, IL6-R, EGF, Her-2, HB-EGf, betacellulin, jagged-1, Notch receptor 1, Notch receptor 3, RAGE, fractalkine, MICA A, I-TAC, HGFR, GITR, GM-CSF, an IGF soluble receptor, and TGF beta.
  • the cell is present in a subject and the release of the ADAM10 substrate is into the subject's circulation.
  • ADAM10 prodomain peptides were prepared by cloning a nucleotide sequence encoding amino acids 23-211 of the human ADAM10 polypeptide as set forth in Accession No. NP_001101.1 of the GENBANK® biosequence database (SEQ ID NO: 1) along with a His tag into a plasmid vector.
  • the prodomain peptides were prepared by transformation of BL21DE3 cells followed by expression at 16 ° C. This yielded both a soluble prodomain peptide and a prodomain peptide in inclusion bodies.
  • the soluble prodomain peptide was purified by breaking the bacteria in 50 mM Tris pH 8 and 5 mM with or without Tris(2-carboxyethyl)phosphine (TCEP; Gold Biotechnology, Inc., St. Louis, Mo., United States of America) with 1 mg/ml lysozyme (Sigma-Aldrich Corp., St. Louis, Mo., United States of America), benzonase, (Sigma-Aldrich) protease inhibitor cocktail (Gold Biotechnology), and 1 ⁇ CELLLYTIC® solution (Sigma-Aldrich) for 15-20 minutes at room temperature. The pellet after centrifugation was again treated with the same buffer and centrifuged once more.
  • TCEP Tris(2-carboxyethyl)phosphine
  • 1 mg/ml lysozyme Sigma-Aldrich Corp., St. Louis, Mo., United States of America
  • benzonase Sigma-Aldrich
  • Both supernatants were applied to a Ni-NTA column. After washing, the prodomain peptide was eluted with 1M imidazole, 20 mM Tris, pH 8 with or without 5 mM TCEP. The material was then passed over a SUPERDEX® 200 brand column (GE Healthcare Bio-Sciences, Pittsburgh, Pa., United States of America) equilibrated in 20 mM Tris pH 8 and 40 mM NaCl. After concentration and endotoxin removal, glycerol was added to 10% and the material was stored at ⁇ 20° C.
  • SUPERDEX® 200 brand column GE Healthcare Bio-Sciences, Pittsburgh, Pa., United States of America
  • Insoluble prodomains were prepared from inclusion bodies after solubilization with urea and then refolding on the Ni-NTA beads. They were then either passed through the SUPERDEX® 200 brand column as described above or put directly through ZEBATM brand spin columns equilibrated in 20 mM Tris, pH 8 and 40mM NaCl buffer (7K molecular weight cutoff). Table 5 below presents the IC 50 values for the ADAM10 peptides and selectivity profiles for some of the inhibitors.
  • the prodomain peptides were passed through a ZEBATM brand spin column equilibrated in 20 mM phosphate, pH 7.2, with or without 4 mM TCEP. A 1.2-fold to five-fold excess of 10 kDa maleimide PEG (Nanocs Inc. Boston, Mass., United States of America) was added. After 2-8 hours, the reaction progress was monitored by SDS PAGE gel electrophoresis. Sometimes more maleimide PEG was added and the reaction was allowed to proceed overnight in the cold.
  • the material was then passed twice over a SUPERDEX® 200 brand G200 column equilibrated in 20 mM Tris, pH 8, and 40 mM NaCl, fractions containing the pure pegylated material were concentrated and reacted with Endotoxin removal columns and frozen at ⁇ 20° C. after addition of glycerol to 10%.
  • Prodomain peptides were prepared as described above and pegylation was performed with 10 kDa maleimide PEG.
  • the human prodomain pegylated or non-pegylated peptides were reacted with ADAM17, ADAM9, or ADAM8 from R & D Systems (Minneapolis, Minn., United States of America) with the same substrate buffer mix as described for ADAM10. There was only slight inhibition for the mutants and pegylated versions. The mutants and pegylated versions were also tested against ADAM8 and ADAM9 from R&D Systems using 15 ⁇ M PEPDAB064 with the same Tris/BRIJ® brand buffer except that NaCl was added to 150 mM and CaCl 2 to 10 mM. There was either no inhibition of these enzymes or slight inhibition, at 4-10 ⁇ M.
  • ADAM17 ADAM9 (2, 3, 9, 12, 14) SEQ ID NO: 1 28% at 8 ⁇ M 0% at 8 ⁇ M 0% at 8 ⁇ M (wild type) PEG-SEQ ID NO: 1 33% at 6.7 ⁇ M (wild type) SEQ ID NO: 6 43% at 10 ⁇ M SEQ ID NO: 34 13% at 6.2 ⁇ M 0% at 3.2 ⁇ M SEQ ID NO: 62 13% at 4 ⁇ M 0% at 6.2 ⁇ M 0% at 8 ⁇ M SEQ ID NO: 64 0% at 7.7 ⁇ M 56% at 7.5 ⁇ M
  • Meprin was also tested as ADAM10 prodomain is a substrate for this enzyme. Meprin was purchased from R & D Systems and was activated according the manufacturer's instructions. To 20 mM Tris buffer, pH 8 and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) (Gold Biotechnology) was added PEPDAB022 (BioZyme Inc.) to a final concentration of 15 ⁇ M. Mutants or wild-type (i.e., SEQ ID NO: 1) ADAM10 modulating peptides or pegylated versions thereof were then added.
  • AEBSF 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride
  • PEPDAB022 BioZyme Inc.
  • the ADAM10 modulating peptide such as SEQ ID NO: 63 was pegylated at the N- or C-terminus or at C151, the IC 50 increased to about 5-7 ⁇ M, indicating that pegylation decreased potency against meprin.
  • SEQ ID NO: 64 and SEQ ID NO: 67 have mouse ADAM10 prodomain sequences where the C-terminal tail has no meprin site at 176-178 (compare SEQ ID NO: 1 to SEQ ID NO: 2).
  • SEQ ID NO: 67 is a mouse/human hybrid, where the human prodomain has its C-terminal tail replaced with the corresponding mouse prodomain sequence. These sequences were also much more resistant to meprin cleavage as compared to the WT (SEQ ID NO: 1), Mutt (SEQ ID NO: 6), and SEQ ID NO: 34 (see FIGS. 9A and 9B ).
  • Mut3/SEQ ID NO: 9 is a double meprin double furin mutant (K28A; K54A; E88A; E177A;). It was completely resistant to meprin cleavage under the experimental conditions tested (see FIG. 4 ). Pegylated SEQ ID NOs: 69-71, which were not very inhibitory against meprin in the fluorescence assay described above (see Table 7), were very resistant to meprin cleavage (see FIG. 9C ) and furin cleavage (see FIG. 8C ).
  • ADAM10 Modulating Peptides Inhibit Cellular Shedding Events in vitro and in vivo
  • Table 8 presents the IC 50 values for inhibition of shedding of Notch1, Notch3, Jagged 1, betacellulin, and Mica A.
  • SEQ ID NO: 62 which is much more resistant to furin and meprin cleavage as compared to Mut2/SEQ ID NO: 6 and all the other constructs, improved the IC 50 of Notch 3 over 10-fold (890 vs. 76 nM).
  • Hollow fiber model On day -1, MDA-MB-468, BT-20, and A549 cells were loaded into Hollow Fibers, placed in cell culture dishes containing medium, and were equilibrated in an incubator at 37° C., 5% CO 2 overnight. Tumor cells were then loaded using medium into KROSFLO® brand hollow fiber membranes. 1-2 hours before surgery mice received Meloxicam (1 mg/kg in 5 ml/kg) subcutaneously. Implantation was performed while mice were under inhalational isoflurane anesthesia. Three fibers containing either MDA-MB-468, A549, or BT-20 cells were implanted into two different compartments: subcutaneous and intraperitoneal. Therefore, every mouse received 6 fibers.
  • CELLTITER-GLO® brand luminescent cell viability assay Each fiber was cut into 4 pieces and directly placed into 2 ml Lysing Matrix A vials (Catalogue No. 6910, MP Biomedicals, Santa Ana, Calif., United States of America) containing 1 ml ex vivo Luciferase buffer (25 mM Tris-Phosphate pH 7.8, 2 mM EDTA, 2 mM DTT, 0.1% TRITONTM X-100) at 4° C.
  • 1 ml ex vivo Luciferase buffer 25 mM Tris-Phosphate pH 7.8, 2 mM EDTA, 2 mM DTT, 0.1% TRITONTM X-100
  • Body weights and tumors are measured twice weekly, and tumor growth and regression rates are determined Animals are euthanized at the end of the experiment, and the liver, heart, visceral fat, kidneys, and brain are retained and examined for tissue damage and toxicity.
  • ADAM10 Modulating Peptides Reduce Allergic Responses to Dust Mite Antigens
  • BALF supernatants are analyzed for MUC5AC protein by ELISA as described and lung morphology is assessed for peribronchiolar perivascular inflammatory cellular infiltration.
  • TACE TNFalpha Converting Enzyme

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